Immune composition and use thereof

Abstract

An immune composition, a vaccine comprising the immune composition, and a preparation method therefor and use thereof in the prevention and / or treatment of diseases or conditions, particularly tumors. The immune composition comprises: (1) a cell membrane derived from pluripotent stem cells, (2) a first adjuvant inserted into the cell membrane, and (3) a second adjuvant encapsulated within the cell membrane.

Description

An immune composition and its application Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to an immune composition and its application in preparing vaccines and preventing tumors. Background Art

[0002] Currently, cancer preventive vaccines are considered the primary strategy for cancer prevention, but the types of vaccines used to prevent tumor occurrence, treat tumors, and delay the progression of tumor diseases are relatively limited.

[0003] For virus-induced cancers, currently approved preventive vaccines include HPV vaccine and HBV vaccine; for non-viral-induced cancer preventive vaccines, the tumor-associated antigens that can be used for cancer preventive vaccines include MUC1 and HER2, and they are only suitable for tumor types with high expression of specific antigens.

[0004] Therefore, there is a need in the art for a tumor vaccine that is more effective, safer, easier to prepare, and has a wider range of applications. Summary of the Invention

[0005] The inventors of this application analyzed the common upregulated proteins of more than 6,000 tumor cell membranes and induced pluripotent stem cell membranes, creatively designed a stem cell membrane vaccine, used pluripotent stem cells (e.g., induced pluripotent stem cells) as a tumor vaccine antigen library, and developed a universal tumor vaccine. The vaccine or immune composition of this application can be implemented in the form of stem cell membranes including adjuvant nanospheres, and the stem cell membrane is selected as a tumor prevention antigen to avoid the safety risks brought by iPSC whole cells; in addition, the functional integration characteristics of nanomaterials overcome the challenges of antigens, adjuvants and carriers. The vaccine or immune composition of this application can be used as a universal cancer vaccine to effectively prevent the occurrence of various cancers or delay the progression of various cancers. The immune composition or vaccine of this application has one or more of the following characteristics: (1) can be rapidly taken up by dendritic cells (DCs), (2) promote DC maturation and release inflammatory factors, (3) can be effectively enriched in lymph nodes, (4) effectively activate T cells and release cytokines, such as IFN-γ, (5) activate B cells to produce antibodies, (6) produce long-term T cell memory effects, and (7) no safety risks.

[0006] The present application provides an immune composition comprising a cell membrane derived from a pluripotent stem cell and an adjuvant. The immune composition may comprise a cell membrane derived from a pluripotent stem cell and a second adjuvant encapsulated within the cell membrane, and may further comprise a first adjuvant inserted into the lipid bilayer of the cell membrane.

[0007] In certain embodiments, the first adjuvant may be selected from the group consisting of an agent capable of enhancing antigen presentation, an agent capable of stimulating and / or boosting an immune response, and combinations thereof.

[0008] In certain embodiments, the first adjuvant can be selected from the group consisting of aluminum salts, water-oil emulsions, liposomes, cytokines, antibodies, polypeptides, DNA, RNA, and small molecules.

[0009] In certain embodiments, the first adjuvant is nanoparticle sized.

[0010] In certain embodiments, the first adjuvant has a size of 200 nm to 1000 nm.

[0011] In certain embodiments, the first adjuvant can be selected from the group consisting of a TLR agonist, CpG, R848, QS21, AS02, AS04, poly(di(carboxyphenoxy)phosphazene), a lipopolysaccharide derivative, monophosphoryl lipid A (MPLA) or an analog thereof, DDA, muramyl dipeptide, threonyl-muramyl dipeptide, OM-174, cholera toxin, and Leishmania elongation factor.

[0012] In certain embodiments, the first adjuvant can be selected from the following groups: aluminum salts, water-oil emulsions, manganese salts, liposomes, cytokines, antibodies, polypeptides, DNA, chitosan, polysaccharide derivatives, Poly (I: C), RNA, virus-like particles, flagellin, Freund's adjuvant, MF59, emulsions, AS03, viral particles, small molecules, TLR1 / 2, TLR4, TLR5, TLR7 / 8, TLR9, monophosphoryl lipid A (MPLA) or its analogs, DDA, muramyl dipeptide, threonyl-muramyl dipeptide, OM-174, cholera toxin and inorganic salt nanoparticles that can induce an immune response, etc.

[0013] In certain embodiments, the first adjuvant may comprise a combination of two or more adjuvants.

[0014] In certain embodiments, the first adjuvant may be a TLR receptor agonist.

[0015] In certain embodiments, the first adjuvant may be MPLA or an analog thereof.

[0016] In certain embodiments, the first adjuvant may be CpG or an analog thereof or R848. In certain embodiments, the second adjuvant may be a combination of CpG and R848.

[0017] In certain embodiments, the first adjuvant may be an aluminum salt.

[0018] In certain embodiments, the first adjuvant may be alum aluminum hydroxide.

[0019] In certain embodiments, the first adjuvant may be a glycan.

[0020] In certain embodiments, the first adjuvant may be mannan.

[0021] In certain embodiments, the second adjuvant may be selected from the group consisting of an agent capable of enhancing antigen presentation, an agent capable of stimulating and / or boosting an immune response, and combinations thereof.

[0022] In certain embodiments, the second adjuvant may be selected from the group consisting of aluminum salts, water-oil emulsions, liposomes, cytokines, antibodies, polypeptides, DNA, RNA, and small molecules.

[0023] In certain embodiments, the second adjuvant is nanoparticle sized.

[0024] In certain embodiments, the second adjuvant has a size of 200 nm to 1000 nm.

[0025] In certain embodiments, the second adjuvant can be selected from the group consisting of a TLR agonist, CpG, R848, QS21, AS02, AS04, poly(di(carboxyphenoxy)phosphazene), a lipopolysaccharide derivative, monophosphoryl lipid A (MPLA) or an analog thereof, DDA, muramyl dipeptide, threonyl-muramyl dipeptide, OM-174, cholera toxin, and Leishmania elongation factor.

[0026] In certain embodiments, the second adjuvant can be selected from the following groups: aluminum salts, water-oil emulsions, manganese salts, liposomes, cytokines, antibodies, polypeptides, DNA, chitosan, polysaccharide derivatives, Poly (I: C), RNA, virus-like particles, flagellin, Freund's adjuvant, MF59, emulsions, AS03, viral particles, small molecules, TLR1 / 2, TLR4, TLR5, TLR7 / 8, TLR9, monophosphoryl lipid A (MPLA) or its analogs, DDA, muramyl dipeptide, threonyl-muramyl dipeptide, OM-174, cholera toxin and inorganic salt nanoparticles that can induce an immune response, etc.

[0027] In certain embodiments, the second adjuvant may comprise a combination of two or more adjuvants.

[0028] In certain embodiments, the second adjuvant may be a TLR receptor agonist.

[0029] In certain embodiments, the second adjuvant may comprise CpG or R848. In certain embodiments, the second adjuvant may be a combination of CpG and R848.

[0030] In certain embodiments, the second adjuvant may be CpG or an analog thereof.

[0031] In certain embodiments, the second adjuvant may be an aluminum salt.

[0032] In certain embodiments, the second adjuvant may be alum aluminum hydroxide.

[0033] In certain embodiments, the second adjuvant may be a polysaccharide.

[0034] In certain embodiments, the second adjuvant may be mannan.

[0035] In certain embodiments, the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

[0036] In some embodiments, the cell membrane is a cell membrane of a pluripotent stem cell. In some embodiments, the cell membrane comprises all components of a cell membrane of a pluripotent stem cell. In some embodiments, the cell membrane comprises surface proteins of a pluripotent stem cell, such as SSEA-1, Na+-K+ ATPase, and / or E-cadherin.

[0037] In certain embodiments, the surface of the cell membrane comprises a tumor antigen.

[0038] In certain embodiments, the surface of the cell membrane comprises a tumor-specific antigen.

[0039] In certain embodiments, the surface of the cell membrane comprises a tumor-associated antigen.

[0040] In certain embodiments, the tumor antigen comprises a protein or a functionally active fragment thereof selected from the following group: antigen peptide transporter 1, H-2 class II histocompatibility antigen γ chain, tyrosine protein kinase SYK, high affinity immunoglobulin epsilon receptor subunit γ, Ras-related C3 botulinum toxin substrate 2, tyrosine protein kinase BTK, receptor type tyrosine protein phosphatase C, Na + / K +-ATPase, ATP5A (IM CVa), ubiquinone cytochrome c reductase core protein I (IM Core I), VDAC1 / porin (OM Porin), matrix cyclophilin D (Matrix CypD), intermembrane space cytochrome c (IMS Cytc), basement membrane-specific heparan sulfate proteoglycan core protein (HSPG2), plasma membrane calcium-transporting ATPase 1 (ATP2b1), endoplasmic reticulum membrane protein complex subunit 1 (EMC1), transmembrane protein 43 (TMEM43), vesicle membrane protein VIP36 (LMAN2), vesicle-associated membrane protein-associated protein A (VAPA), transmembrane 9 superfamily member 2 (TM9SF2), transmembrane emp24 domain-containing protein 10 (TMED10), adipocyte plasma membrane-associated protein (APAMP), synaptic vesicle membrane protein VAT (VAT1), nuclear pore membrane glycoprotein 210 (NUP210), plasma membrane calcium-transporting ATPase 4 (ATP2b4), plasma membrane calcium-transporting ATPase 2 (ATP2b2), erythrocyte band 7 integral membrane protein (STOM), transmembrane emp24 domain-containing protein 9 (TMED9), mitochondrial import inner membrane transposase subunit TIM44 (TIMM44), membrane-associated progesterone receptor component 1 (PGRMC1), ER membrane protein complex subunit 3 (EMC3), vesicle-associated membrane protein-associated protein B (VAPB), membrane primary amine oxidase (AOC3), vesicle-associated membrane protein 7 (VAMP7), Golgi membrane protein 4 (GOLIM4), membrane-associated progesterone receptor component 2 (PGRMC2), transmembrane 9 superfamily member 3 (TM9SF3), transmembrane 9 superfamily member 4 (TM9SF4), endoplasmic reticulum membrane protein complex subunit 2 (EMC2), mitochondrial import inner membrane transposase subunit TIM50 (TIMM50), peroxide Proteosomal membrane protein 11B (PEX11b), transmembrane emp24 domain-containing protein 2 (TMED2), secretory carrier-associated membrane protein 3 (SCAMP3), thioredoxin-related transmembrane protein 4 (TMX4), peroxisomal membrane protein PMP34 (SLC25a17), peroxisomal membrane protein PEX14 (PEX14), endoplasmic reticulum membrane protein complex subunit 8 (EMC8), interferon-induced transmembrane protein 3 (IFITM3), lysosomal-associated membrane glycoprotein 2 (LAMP2), thioredoxin-related transmembrane protein 2 (TMX2), vesicle-associated membrane protein 3 (VAMP3), lysosomal-associated membrane glycoprotein 1 (LAMP1), mitochondrial import inner membrane transposase subunit Tim8A (TIMM8a1), lysosomal membrane protein 2 (SCARB2), Iggamma-2A chain C region (IGHG2a), transmembrane 9 superfamily member 1 (TM9SF1), inner nuclear membrane protein Man1 (LEMD3), transmembrane emp24 domain-containing protein 4 (TMED4), Thy-1 membrane glycoprotein (Thy1), mitochondrial import inner membrane transposase subunit Tim23 (TIMM23), mitochondrial import inner membrane transposase subunit Tim9 (TIMM9), mitochondrial import inner membrane transposase subunit Tim13 (TIMM13), secretory carrier-associated membrane protein 2 (SCAMP2), secretory carrier-associated membrane protein 1 (SCAMP1), integral membrane protein 2B (ITM2b), mitochondrial import inner membrane transposase subunit Ti m10 (TIMM10), vacuole membrane protein 1 (VMP1), growth hormone-induced transmembrane protein (GHITM), death domain-containing membrane protein (NRADD), vesicle-associated membrane protein 8 (VAMP8), transmembrane anterior-posterior transition protein 1 (TAPT1), mitochondrial intermembrane space import and assembly protein 40 (CHCHD4), glycosylated lysosomal membrane protein (GLMP), transmembrane 4 domain superfamily A member 6D (MS4A6D), translocation chain-associated membrane protein 1 (TRAM1), interferon-induced transmembrane protein 2 (IFITM2), sarcolemma-associated protein (SLMAP), membrane magnesium transporter 1 (MMGT1), nuclear envelope pore protein POM121 (POM121), transmembrane protein C16orf54 homologous protein (AI467606), vacuolar ATPase assembly integral membrane protein Vma21 (VMA21), epithelial membrane protein 1 (EMP1), membrane-associated phosphatidylinositol transfer protein 1 (PITPNM1), small integral membrane protein 15 (SMIM15), nuclear integral membrane protein 1 (NEMP1), integral membrane protein GPR180 (GPR180), secretory carrier-associated membrane protein 4 (SCAMP4), translocation chain-associated membrane protein 2 (TRAM2), transmembrane and coiled-coil domain protein 3 (TMCC3), matrix membrane-associated protein 1 (SMAP1), neuronal membrane glycoprotein M6-b (GPM6b), epithelial membrane protein 2 (EMP2), Golgi membrane protein 1 (GOLM1), SI D1 transmembrane family member 2 (SIDT2), trans-Golgi network integral membrane protein 2 (TGOLN2), Golgi membrane protein TVP23 homolog B (FAM18b), mitochondrial inner membrane protein OXA1L (OXA1L), ossification-related transmembrane protein 1 (OSTM1), peroxisomal membrane protein PEX13 (PEX13), unilaterally passing membrane and coiled-coil domain protein 1 (SMCO1), distal membrane arm assembly complex protein 1 (DMAC1), secretory carrier-associated membrane protein 5 (Scamp5), cystic fibrosis transmembrane conductance regulator (CFTR), osteoclast-stimulating transmembrane protein (OCSTAMP), fat storage-inducing transmembrane protein 2 (FITM2) and transmembrane channel-like protein 5 (TMC5), and any combination thereof.

[0041] In some embodiments, the tumor antigen on the surface of the cell membrane is not artificially introduced. In some embodiments, the tumor antigen on the surface of the cell membrane is naturally present.

[0042] In certain embodiments, the surface of the cell membrane is capable of expressing proteins having one or more functions selected from the group consisting of nutrient transport, cell proliferation, and cell division.

[0043] In certain embodiments, the mass ratio between the cell membrane and the second adjuvant is 1:20 to 20:1. In certain embodiments, the mass ratio between the cell membrane and the second adjuvant is 1:18 to 18:1. In certain embodiments, the mass ratio between the cell membrane and the second adjuvant is 1:16 to 16:1. In certain embodiments, the mass ratio between the cell membrane and the second adjuvant is 1:14 to 14:1. In certain embodiments, the mass ratio between the cell membrane and the second adjuvant is 1:12 to 12:1. In certain embodiments, the mass ratio between the cell membrane and the second adjuvant is 1:10 to 10:1. In certain embodiments, the mass ratio between the cell membrane and the second adjuvant is 1:8 to 8:1. In certain embodiments, the mass ratio between the cell membrane and the second adjuvant is 1:6 to 6:1. In certain embodiments, the mass ratio between the cell membrane and the second adjuvant is 1:4 to 4:1. In certain embodiments, the mass ratio between the cell membrane and the second adjuvant is 1:2 to 2:1.

[0044] In certain embodiments, the mass ratio between the cell membrane and the second adjuvant is 1:50 to 1:20. In certain embodiments, the mass ratio between the cell membrane and the second adjuvant is 1:40 to 1:20. In certain embodiments, the mass ratio between the cell membrane and the second adjuvant is 1:30 to 1:20.

[0045] In certain embodiments, the mass ratio between the cell membrane and the second adjuvant is 1:30 to 1:10. In certain embodiments, the mass ratio between the cell membrane and the second adjuvant is 1:25 to 1:15.

[0046] In certain embodiments, the mass ratio between the cell membrane and the second adjuvant is 1:20.

[0047] In certain embodiments, the particle size of the immune composition is between 1 and 1000 nm. In certain embodiments, the particle size of the immune composition is between 1 and 900 nm. In certain embodiments, the particle size of the immune composition is between 1 and 800 nm. In certain embodiments, the particle size of the immune composition is between 1 and 700 nm. In certain embodiments, the particle size of the immune composition is between 1 and 600 nm. In certain embodiments, the particle size of the immune composition is between 1 and 500 nm.

[0048] In certain embodiments, the particle size of the immune composition is between 900-1000 nm. In certain embodiments, the particle size of the immune composition is between 800-1000 nm. In certain embodiments, the particle size of the immune composition is between 700-1000 nm. In certain embodiments, the particle size of the immune composition is between 600-1000 nm. In certain embodiments, the particle size of the immune composition is between 500-1000 nm. In certain embodiments, the particle size of the immune composition is between 400-1000 nm. In certain embodiments, the particle size of the immune composition is between 300-1000 nm. In certain embodiments, the particle size of the immune composition is between 200-500 nm. In certain embodiments, the particle size of the immune composition is between 400-800 nm.

[0049] In certain embodiments, the immune composition comprises an outer shell and an inner core, wherein the outer shell comprises the cell membrane derived from pluripotent stem cells and the first adjuvant inserted into the lipid bilayer of the cell membrane, and the inner core comprises the second adjuvant.

[0050] In certain embodiments, the immune composition does not comprise the cytoplasm and / or nucleus of the pluripotent stem cell.

[0051] On the other hand, the present application provides a vaccine comprising the immune composition.

[0052] In another aspect, the present application provides a method for preparing the immune composition or vaccine. In certain embodiments, the method comprises: a) obtaining a cell membrane derived from a pluripotent stem cell, b) inserting a first adjuvant into the cell membrane, and c) encapsulating a second adjuvant inside the cell membrane.

[0053] In certain embodiments, the inserting in b) comprises using sonication.

[0054] In certain embodiments, the encapsulation in c) comprises using a method selected from the group consisting of porous membrane co-extrusion, sonication, electroporation, and microfluidics combined with in situ packaging of living cells.

[0055] On the other hand, the present application provides a pharmaceutical composition comprising the immune composition and / or the vaccine, and a pharmaceutically acceptable carrier.

[0056] On the other hand, the present application provides the use of the immune composition, the vaccine and / or the pharmaceutical composition in the preparation of a medicament for preventing or treating a disease and / or condition.

[0057] On the other hand, the present application provides a method for treating, preventing or treating a disease and / or condition, comprising administering an effective amount of the immune composition, the vaccine and / or the pharmaceutical composition to a subject in need thereof.

[0058] In another aspect, the present application provides the immune composition, the vaccine and / or the pharmaceutical composition for use in treating, preventing or treating a disease and / or disorder.

[0059] In certain embodiments, the disease and / or condition comprises a tumor and / or an infectious disease.

[0060] In certain embodiments, the disease and / or condition comprises colon cancer, melanoma, and / or breast cancer.

[0061] In certain embodiments, the disease and / or condition comprises an infectious disease having a viral or bacterial pathogen.

[0062] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0064] Figure 1A shows the shared proteins in two iPS cell lines, two MEF cell lines, and four tumor cell lines.

[0065] Figure 1B shows the up-regulated proteins ranked by Log2 (fold change). FC is the protein abundance ratio between tumor cells and MEF cells, and FC > 1.5 indicates up-regulation.

[0066] Figure 1C shows a volcano plot of protein expression in iPS cell lines compared to MEF cell lines. Red indicates significant upregulation (FC > 1.5), blue indicates significant downregulation, and gray indicates non-significant.

[0067] Figure 1D shows a Venn diagram of the number of unique highly expressed proteins identified in tumor cell lines (left) and iPS cell lines (right).

[0068] Figure 1E shows a bubble plot of differentially enriched genes (GO terms). The size of the bubble is proportional to the number of differentially expressed genes assigned to the GO term.

[0069] Figure 1F shows a TEM image of the iPM nanovaccine. Scale bar, 200 nm.

[0070] Figure 1G shows the average hydrodynamic diameter and surface zeta potential of Lipo-adjuvant, iPM nanovax, and iPM vesicles. n = 3, data are ± sd.

[0071] Figure 1H shows the flow cytometric analysis of the iPM-nanovax fluorescence signal when the mass ratios of iPM vesicles and CR-NPs were 20:1 and 1:20, respectively. iPM vesicles and CR NPs were stained with DID and rhodamine BN.

[0072] Figure 1I shows a super-resolution fluorescence microscopy image of iPM nanovax. DiD-labeled iPS cell membranes are shown in red; rhodamine B-labeled CR NPs are in green; and FITC-labeled MPLA analogue LPS is in blue. Scale bar, 100 nm.

[0073] Figure 1J and Figure 1K show the protein expression profiles of Lipoadjuvant, iPSC cell lysates, iPM nanovax (iPM nanovaccine), and iiPMvesicles analyzed by SDS-PAGE after Coomassie Brilliant Blue staining.

[0074] Figure 2A shows in vivo lymph node drainage. C57BL6N mice received a subcutaneous injection of 50 μg of DiD-labeled iPM nanovax (red). DID-labeled iPM nanovax (iPM nanovax), Lipo adjuvant, and iPM vesicles were subcutaneously injected into the mice. Ex vivo imaging of inguinal lymph nodes was performed 24 hours later.

[0075] Figures 2B-2D show the uptake of DiD-labeled iPM nanovaccines in different types of DCs, including cDCs, migrating DCs, and resident DCs at 3, 12, and 24 hours after inoculation. + (2B), CD11c int -MHC-II high (2C) and CD11c high -MHC-II int (2D) Differentiation was performed. Data are presented as mean ± sd (n = 6).

[0076] Figures 2E-2F show the expression of MHC-II (2E), CD86, and CD80 (2F) by cDCs in LNs 24 hours after treatment in different groups by flow cytometry analysis. n = 6, data ± sd.

[0077] Figure 2G shows the expression of the inflammatory cytokine IL-1β in the serum of mice in different groups 72 hours after injection, as measured by ELISA. n = 6, data ± sd.

[0078] Figures 2H-2I show the C57BL6N CD8 in draining LN of mice four days after injection of different nanoparticles. + T cells, CD4 + Representative images (2H) and quantitative analysis (2I) of T cell activation.

[0079] Figure 2J shows the analysis of activated B cells in the germinal centers of lymph nodes four days after immunization. n = 6, data ± SD.

[0080] Figure 2K shows the analysis of helper T cell activation in lymph node germinal centers four days after immunization. n = 6, data ± SD.

[0081] Figure 2L shows the percentage of effector / memory CD4+CD44+ T cells in PBMCs. n = 6, data ± sd.

[0082] Figure 3A shows a schematic diagram of the immunization protocol. C57BL6N mice were subcutaneously immunized with PBS, Lipo adjuvant, and iPM nanovaccine on days -22, -15, -8, and -1, respectively. 5×10 4 MC38 cells were subcutaneously incubated on day 0, and tumor curves were recorded weekly. After 30 days, animals were euthanized, and the immune responses of lymphoid organs were tested in vitro.

[0083] Figure 3B shows the average tumor growth of different groups in the MC38 colon cancer model during the prevention experiment.

[0084] Figure 3C shows the growth curves of individual tumors for each group of mice.

[0085] Figure 3D shows that when PMA (10 μg / 10 6 When MC38 tumor tissue cell suspension was stimulated with IFN-γ + , CD8 + CD3 + Expression in T cells. n = 5, data are ± SD.

[0086] Figure 3E shows the expression of cytotoxic CD8 + CD3+ Flow cytometric analysis of GranzymB+ in T cells.

[0087] Figure 3F shows the infiltrating CD8 + CD3 + Representative flow cytometric analysis of T cell percentages.

[0088] Figure 3G shows representative confocal microscopy images of tumor sections stained with CD4 and CD8 antibodies (blue: cell nuclei, green: CD8, red: CD4). Scale bars: 200 μm (left) and 50 μm (right).

[0089] Figure 3H shows the ratio of memory / effector CD4 T cells in PBMCs analyzed by flow cytometry. n = 5, data are ± SD.

[0090] Figures 3I-3J show the percentage of memory / effector CD8 T cells in the spleen (3I) and LN (3J) of immunized animals (PBS). n = 5, data are ± sd.

[0091] Figure 3K shows the specific immune cell subsets (CD4 + T cells, CD8 + T cells and B cells were depleted to explore their relative contribution to the observed therapeutic effects. n = 5, data are ± SD.

[0092] Figures 3L-3M show ELISPOT images (3L) and quantification of IFN-γ spot counts (3M) when splenocytes were stimulated with iPSC membrane antigens and MC38 tumor antigens. n = 5, data are ± sd.

[0093] Figure 4A shows the results of nanovaccine enrichment in lymph nodes.

[0094] Figure 4B shows that the nanovaccine induced antibody responses against iPSC cells.

[0095] Figure 4C shows that the nanovaccine can inhibit tumor progression. DETAILED DESCRIPTION

[0096] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0097] Definition of terms

[0098] In this application, the term "CpG" generally refers to a synthetic oligodeoxynucleotide containing an unmethylated CpG motif that can serve as an agonist for Toll-like receptor 9 (TLR9). The CpGs of this application include CpGs of any length or sequence, and may be modified or unmodified. The CpGs of this application include D-type, K-type, C-type, and / or P-type CpGs. CpG adjuvants can be obtained commercially. For example, CpG7909 and CpG1018 are both common commercial CpGs. For more information about CpG, please refer to Bode C, Zhao G, Steinhagen F, Kinjo T, Klinman DM. CpG DNA as a vaccine adjuvant. Expert Rev Vaccines. 2011 Apr; 10(4): 499-511. doi: 10.1586 / erv.10.174. PMID: 21506647; PMCID: PMC3108434.

[0099] In this application, the term "MPLA" generally refers to monophosphoryl lipid A, a lipid A analog that can act as a TLR receptor 4 agonist. The term includes MPLA and its functional derivatives, analogs, and combination adjuvants containing MPLA. Commercially available MPLA may include MPL (GlaxoSmithKline) and GLA (Immune Design).

[0100] In this application, the term "R848" generally refers to an imidazoquinoline that acts as an agonist for TLR7 and TLR8. The term includes R848 and its functional derivatives, analogs, and combination adjuvants containing R848. R848 is commercially available.

[0101] In this application, the term "induced pluripotent stem cells" generally refers to mature cells that are unable to differentiate into somatic cells and, under certain conditions, return to a state of totipotency. The totipotency refers to the ability to differentiate into all cell types in the body and form a complete embryo or further develop into a new individual.

[0102] In this application, the term "surface potential" generally refers to the zeta potential of a particle surface. For example, surface potential can be correlated with the stability of a dispersion. For example, surface potential can be measured using dynamic light scattering.

[0103] In this application, the term "hydrated particle size" generally refers to the diameter of a substance in fluid dynamics. For example, the hydrated particle size of a substance can be measured by a dynamic light scattering instrument.

[0104] In this application, the term "pharmaceutical composition" generally refers to a composition that is administered to a patient. In certain embodiments, the pharmaceutical composition may comprise a composition for parenteral, transdermal, intracavitary, intraarterial, intrathecal and / or intranasal administration or direct injection into a tissue. For example, the pharmaceutical composition is administered to a patient by infusion or injection. Administration of suitable compositions is achieved in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. The pharmaceutical composition of the present application may further comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known and include phosphate buffered saline, water, emulsions (such as oil / water emulsions), various moisturizers, sterile solutions, liposomes, and the like. Compositions comprising these carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to a subject in a suitable dose. The dosage regimen can be determined by the participating physician and clinical factors.

[0105] In this application, the terms "tumor" and "cancer" generally refer to cells that exhibit at least partial loss of control in normal growth and / or development. For example, common tumors or cancer cells have typically lost contact inhibition and may be invasive and / or have the ability to metastasize.

[0106] The term "tumor antigen" as used herein is understood to mean those antigens that are presented on tumor cells. These antigens may be presented on the cell surface, with an extracellular portion generally associated with the transmembrane and cytoplasmic portions of the molecule. These antigens may sometimes be presented only by tumor cells and not by normal cells. Tumor antigens may be expressed exclusively on tumor cells or may represent tumor-specific mutations compared to normal cells. In this case, they are referred to as tumor-specific antigens. More commonly, antigens are presented by both tumor cells and normal cells, and they are referred to as tumor-associated antigens. These tumor-associated antigens may be overexpressed compared to normal cells, or may be readily available for antibody binding in tumor cells because tumor tissue has a less compact structure than normal tissue.

[0107] As used herein, the term "adjuvant" generally refers to a compound that, when used in combination with an antigen, enhances the immune response to the antigen, but does not produce an immune response to the antigen when used alone. Adjuvants enhance the immune response through several mechanisms, including recruitment of lymphocytes, stimulation of B cells and / or T cells, and stimulation of macrophages.

[0108] Detailed Description of the Invention

[0109] In one aspect, the present application provides an immune composition that can stimulate an innate immune response and / or an adaptive immune response. Structurally, the immune composition of the present application comprises a cell membrane derived from a pluripotent stem cell and an adjuvant encapsulated within the cell membrane.

[0110] cell membrane

[0111] In the present application, the cell membrane in the immune composition and / or vaccine can be derived from induced pluripotent stem cells and contain multiple upregulated proteins on the cell membrane surface that are shared with tumor cells. These proteins may not be expressed or are expressed at low levels on the surface of non-tumor cells. In certain embodiments, the cell membrane surface contains multiple proteins significantly associated with cancer physiological processes, for example, proteins related to nutrient transport, cell proliferation, and cell division.

[0112] In some embodiments, the cell membrane is formed by physical extrusion of a cell membrane of a pluripotent stem cell. In some embodiments, the cell membrane comprises all components of a cell membrane of a pluripotent stem cell, such as proteins, sugars, lipids, etc.

[0113] In certain embodiments, the surface of the cell membrane contains tumor antigens, for example, tumor-associated antigens. These tumor-associated antigens may be known or unknown.

[0114] In certain embodiments, the tumor antigen on the surface of the cell membrane is not artificially introduced. In certain embodiments, the tumor antigen on the surface of the cell membrane is not expressed on the cell membrane surface through transgenic technology.

[0115] In certain embodiments, the desired antigen protein can be expressed on the cell membrane surface through transgenic technology.

[0116] In certain embodiments, the tumor antigen on the surface of the cell membrane is naturally occurring. In certain embodiments, the tumor antigen on the surface of the cell membrane is expressed by an endogenous gene. In certain embodiments, the tumor antigen on the surface of the cell membrane can express different antigen proteins under different culture conditions. In certain embodiments, the culture conditions of pluripotent stem cells can be adjusted to cause the cell membrane to express different antigen proteins.

[0117] In certain embodiments, the cell membrane is derived from human pluripotent stem cells, e.g., human induced pluripotent stem cells. In certain embodiments, the cell membrane is derived from mouse pluripotent stem cells, e.g., mouse induced pluripotent stem cells.

[0118] In certain embodiments, the cell membrane may comprise a protein or a functionally active fragment thereof selected from the following group: stage-specific mouse embryonic antigen SSEA-1, Na+-K+ ATPase, E-cadherin, MHC-I class II antigen peptide molecule complex, tyrosine protein kinase SYK, high affinity immunoglobulin epsilon receptor subunit γ, Ras-related C3 botulinum toxin substrate 2, tyrosine protein kinase BTK, receptor type tyrosine protein phosphatase C, Na + / K +-ATPase, ATP5A (IM CVa), ubiquinone cytochrome c reductase core protein I (IM Core I), VDAC1 / porin (OM Porin), matrix cyclophilin D (Matrix CypD), stage-specific embryonic antigen 1 (SSEA-1), E-cadherin and intermembrane space cytochrome c (IMS Cytc), proteins related to cell functions such as cell proliferation, invasion, and nutrient transport, and any combination of the above.

[0119] adjuvant

[0120] In some embodiments, the adjuvant is encapsulated in the cell membrane of pluripotent stem cells in the form of nanospheres, and this adjuvant is referred to as the second adjuvant in this application. In other embodiments, another adjuvant is further inserted into the cell membrane of the pluripotent stem cells, and this adjuvant is referred to as the first adjuvant in this application. The difference between the first adjuvant and the second adjuvant lies in the difference in their location on the cell membrane in the immune composition, and does not represent a difference in the actual molecular species, that is, the first adjuvant and the second adjuvant can be the same one or more adjuvant molecules.

[0121] In the present application, the first adjuvant and the second adjuvant can be independently selected from any one or more agents capable of activating an immune response, and substances having one or more of the following properties can be selected: (1) sustained release of antigens; (2) increased phagocytosis of antigens; (3) stimulation of cytokine and chemokine release; (4) enhanced antigen presentation; (5) activation of inflammasomes; and (6) delayed digestion of antigens.

[0122] In certain embodiments, the first adjuvant may comprise a Toll-like receptor (TLR) agonist. In certain embodiments, the first adjuvant may comprise a TLR7 agonist. In certain embodiments, the first adjuvant may comprise a TLR8 agonist. In certain embodiments, the first adjuvant may comprise a TLR9 agonist. In certain embodiments, the first adjuvant may comprise a TLR2 agonist. In certain embodiments, the first adjuvant may comprise a TLR4 agonist. In certain embodiments, the first adjuvant may comprise a TLR5 agonist. In certain embodiments, the first adjuvant may comprise a TLR1 agonist. In certain embodiments, the first adjuvant may comprise a TLR3 agonist. In certain embodiments, the first adjuvant may comprise a TLR6 agonist. In certain embodiments, the first adjuvant may comprise a TLR10 agonist. In certain embodiments, the first adjuvant may comprise an adjuvant selected from the group consisting of a TLR3 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist. In certain embodiments, the first adjuvant may be a TLR4 agonist. In certain embodiments, the first adjuvant may be MPLA, GLA, or an analog thereof.

[0123] In certain embodiments, the second adjuvant may comprise a Toll-like receptor (TLR) agonist. In certain embodiments, the second adjuvant may comprise a TLR7 agonist. In certain embodiments, the second adjuvant may comprise a TLR8 agonist. In certain embodiments, the second adjuvant may comprise a TLR9 agonist. In certain embodiments, the second adjuvant may comprise a TLR2 agonist. In certain embodiments, the second adjuvant may comprise a TLR4 agonist. In certain embodiments, the second adjuvant may comprise a TLR5 agonist. In certain embodiments, the second adjuvant may comprise a TLR1 agonist. In certain embodiments, the second adjuvant may comprise a TLR3 agonist. In certain embodiments, the second adjuvant may comprise a TLR6 agonist. In certain embodiments, the second adjuvant may comprise a TLR10 agonist. In certain embodiments, the second adjuvant may comprise an adjuvant selected from the group consisting of a TLR3 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist.

[0124] In certain embodiments, the second adjuvant may comprise a TLR7 agonist, a TLR8 and / or a TLR9 agonist.

[0125] In certain embodiments, the second adjuvant may comprise CpG, CpG-ODN, imiquimod and / or R848. In certain embodiments, the second adjuvant may be a combination of CpG and R848.

[0126] In addition to the above, other adjuvants that can help the immune system recognize the cell membrane antigens of the present application or activate antigen presenting cells through multiple Toll-like receptors (TLRs) can also be used as the first adjuvant or the second adjuvant, including but not limited to substances selected from the following groups: aluminum salt adjuvants, emulsions (such as MF59, AS03), dsRNA analogs (such as poly (I: C)), liposome A analogs (such as MPLA or its analogs, GLA or its analogs), flagellin , imiquimod, R848, CpG-ODN, CpG, saponins (such as QS21), C-type lectin ligands (such as TDB), CD1d ligands, virosomes or fragments thereof, AS01 (a combination of MPL, QS21, and liposomes), AS02 (a combination of MPL, QS21, and an emulsion), AS04 (a combination of MPLA and an aluminum salt adjuvant), AS15 (a combination of MPLA, QS21, CPG, and liposomes) and GLA-SE (a combination of GLA and an emulsion).

[0127] In the present application, the second adjuvant can be prepared in the form of nanoparticles. Nanoparticles (or nanospheres) containing the second adjuvant can be prepared by any method in the prior art.

[0128] The content of the first adjuvant and / or the second adjuvant in the immune composition or vaccine can be adjusted according to the intensity of the immune response desired to be induced.

[0129] In the present application, in the immune composition or vaccine, the cell membrane derived from pluripotent stem cells forms an outer shell, and the outer shell contains the first adjuvant, that is, the cell membrane derived from pluripotent stem cells and the first adjuvant together form an outer shell (for example, by ultrasonic mixing), and the second adjuvant forms an inner core in the form of nanoparticles, and the outer shell contains the inner core, together forming the immune composition or vaccine of the present application.

[0130] Immunological compositions and vaccines

[0131] In the present application, the cell membrane encapsulates nanoparticles of the second adjuvant to form the immune composition, wherein the cell membrane serves as an immunogen that induces cellular immunity and / or humoral immunity of the body. Furthermore, the immune composition also inserts the first adjuvant on the lipid bilayer of the cell membrane. The first adjuvant and the second adjuvant can jointly improve the body's response ability to immunogen stimulation.

[0132] In certain embodiments, the mass ratio between the cell membrane and the second adjuvant is 1:30 to 1:10. In certain embodiments, the mass ratio between the cell membrane and the second adjuvant is 1:25 to 1:15. In certain embodiments, the mass ratio between the cell membrane and the second adjuvant is less than 1:20. In certain embodiments, the mass ratio between the cell membrane and the second adjuvant is 1:20.

[0133] In the present application, the immune composition or vaccine is in the form of particles. In certain embodiments, the particle size of the immune composition is between about 1 nm and about 1000 nm. The particle size can be a hydrated size. In certain embodiments, the surface potential of the immune composition is between about +50 mV and about -50 mV. For example, the hydrated particle size and / or surface potential of the particles can be measured by a nanoparticle size potentiometer.

[0134] In the present application, the immune composition or vaccine can maintain stability in solution. For example, the stability of the particles can include that the hydrated particle size and / or surface potential of the particles after storage for a period of time are not significantly different from those before the period of time.

[0135] In the present application, the immune composition or vaccine may have no significant difference in hydrated particle size and / or surface potential after being stored in phosphate buffered saline (PBS) at 4 degrees Celsius for a period of time compared to that before the period of time.

[0136] In the present application, the stability of the immune composition or vaccine can be determined by showing that the hydrated particle size and / or surface potential after storage in phosphate buffered saline (PBS) at 4 degrees Celsius for a period of time is not significantly different from that before the period of time. For example, the period of time can be no less than about 21 days. For example, the period of time can be no less than about 21 days, no less than about 20 days, no less than about 15 days, no less than about 10 days, no less than about 5 days, no less than about 4 days, no less than about 3 days, no less than about 2 days, or no less than about 1 day.

[0137] In the present application, the immune composition or vaccine does not contain whole pluripotent stem cells. In certain embodiments, the immune composition or vaccine does not contain the cytoplasmic portion of the pluripotent stem cells. In certain embodiments, the immune composition or vaccine does not contain the nuclear portion of the pluripotent stem cells. This can avoid the safety risks associated with whole iPSC cells as vaccines, such as eliciting an undesirable immune response.

[0138] In addition, the immune composition or vaccine of the present application may further comprise a third adjuvant that is different from the first adjuvant and the second adjuvant in the form of enhanced immune stimulation. The third adjuvant may exist in a form independent of the nanoparticles, or in the form of a mixture with the nanoparticles.

[0139] On the other hand, the present application provides a kit that may contain the immune composition of the present application and / or the vaccine of the present application.

[0140] On the other hand, the present application provides a pharmaceutical composition, which may include the immune composition of the present application and / or the vaccine of the present application, and an optional pharmaceutically acceptable carrier.

[0141] The immune composition of this application, a system for co-delivering membrane antigens and adjuvants, effectively activates DC maturation and antigen presentation, produces long-term memory effects, induces tumor-specific T cells, and significantly delays tumor growth in multiple mouse models. By stimulating the immune system to kill tumors, this immune composition, as a universal vaccine, offers versatility not only in terms of vaccine antigen iPSC membrane preparation but also in the prevention or treatment of multiple cancer types.

[0142] method

[0143] In another aspect, the present application provides a method for enhancing the uptake of a target antigen by immune cells, the method comprising providing the immune composition of the present application. For example, the method of the present application can be in vitro or ex vivo. For example, the method of the present application can be for non-preventive, non-therapeutic and / or non-diagnostic purposes.

[0144] In another aspect, the present application provides a method for activating immune cells, which may comprise administering the immune composition, vaccine, kit, or pharmaceutical composition of the present application. For example, the method of the present application may be in vitro or ex vivo. For example, the method of the present application may be for non-preventive, non-therapeutic, and / or non-diagnostic purposes.

[0145] For example, the immune cells may comprise immune presenting cells.

[0146] For example, the immune cells may include the following group: dendritic cells (DC), T lymphocytes, macrophages and natural killer (NK) cells, and any combination thereof.

[0147] For example, the immune cell may comprise bone marrow-derived dendritic cells (BMDCs). For example, the immune cell may comprise CD8 positive cells and / or CD4 positive cells. For example, the immune cell may comprise T lymphocytes. For example, the immune cell may comprise the immune cells in lymph nodes and / or spleen.

[0148] For example, compared to immune cells to which the immune composition, vaccine, kit or pharmaceutical composition of the present application is not administered, the activation effect of the immune cells administered with the immune composition, vaccine, kit or pharmaceutical composition of the present application can be selected from the following groups: increasing the expression level of the antigen recognition receptor of the immune cells, increasing the expression level of the nuclear factor κB (NF-κB) protein of the immune cells, increasing the expression and / or secretion level of the cytokines of the immune cells and increasing the proportion of mature immune cells, and any combination thereof. For example, the increase comprises an increase of about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% in the immune cells administered with the immune composition, vaccine, kit or pharmaceutical composition of the present application compared to immune cells to which the immune composition, vaccine, kit or pharmaceutical composition of the present application is not administered. For example, the cytokines can include proinflammatory cytokines. For example, the cytokines may include interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1β and / or interferon (IFN)-γ. For example, the mature immune cells may include CD80-positive cells, CD86-positive cells and / or effector memory cells.

[0149] For example, the ratio of mature immune cells may include the ratio of CD80-positive and / or CD86-positive immune cells to CD11c-positive immune cells.

[0150] For example, the ratio of mature immune cells may include the ratio of immune cells with high CD44 expression and low CD62L expression to CD8-positive immune cells.

[0151] On the other hand, the present application provides a method for enhancing innate immunity and / or specific immune response, which may comprise administering the immune composition, vaccine, kit or pharmaceutical composition of the present application to a subject in need thereof.

[0152] For example, administration of the immune composition, vaccine, kit or pharmaceutical composition of the present application can promote dendritic cell maturation and / or increase cytokine secretion by lymphocytes compared to not administering the immune composition, vaccine, kit or pharmaceutical composition of the present application.

[0153] For example, the methods can be performed without substantially inducing a systemic inflammatory response.

[0154] For example, the methods do not substantially increase the concentration of IFN-γ, TNF-α, macrophage chemoattractant protein-1 (MCP-1), IL-12p70, IL-10, IL-23, IL-27, IL-17A, IFN-β, granulocyte-macrophage colony stimulating factor (GM-CSF), and / or IL-1α in the serum of the subject.

[0155] For example, the method can result in substantially no hemolysis, heart damage, liver damage, spleen damage, lung damage, and / or kidney damage.

[0156] On the other hand, the present application also provides a method for preventing and / or treating a disease or condition, which may comprise administering the immune composition, vaccine, kit or pharmaceutical composition of the present application to a subject in need thereof.

[0157] For example, the disease or condition may comprise an infectious disease.

[0158] For example, the disease or disorder may comprise a tumor. For example, the prevention and / or treatment of a tumor may comprise slowing down the rate of increase in the volume of the tumor and / or reducing the volume of the tumor.

[0159] For example, the tumor may include the tumor that was not completely removed after tumor resection. For example, after a patient's tumor is removed by surgery, the tumor cells and / or tumor tissue that were not completely removed may develop into a tumor again, and this tumor may also be prevented and / or treated by the method of the present application. For example, in the method of the present application, the tumor may include a tumor that is regenerated after the tumor is cleared. For example, after a patient's tumor is removed by surgery, even if the tumor is completely removed, the patient may regenerate a tumor, such as if the patient is prone to tumors. This newly regenerated tumor may also be prevented and / or treated by the method of the present application. For example, this newly regenerated tumor has at least one identical antigen with the original tumor.

[0160] For example, the tumor may include a virus-induced tumor. For example, the tumor may include a non-viral-induced tumor. For example, the tumor may include a tumor that highly expresses a specific antigen.

[0161] For example, the tumor may comprise a solid tumor.

[0162] For example, the tumor may be selected from the group consisting of colon cancer, melanoma, and breast cancer.

[0163] In another aspect, the present application provides a method for preparing the immune composition or vaccine. In certain embodiments, the method comprises: a) obtaining a cell membrane derived from a pluripotent stem cell, b) inserting a first adjuvant into the cell membrane, and c) encapsulating a second adjuvant within the cell membrane. For example, the insertion in b) may include sonication. Alternatively, the encapsulation in c) may include a method selected from the group consisting of porous membrane coextrusion, sonication, electroporation, a combination of microfluidics, and in situ packaging of living cells.

[0164] On the other hand, the present application provides the following implementation methods:

[0165] An immune composition comprising a cell membrane derived from pluripotent stem cells and an adjuvant.

[0166] 2. According to the immune composition of embodiment 1, the adjuvant includes a first adjuvant that is inserted into the lipid bilayer of the cell membrane.

[0167] 3. According to the immune composition described in any one of embodiments 1-2, the adjuvant includes a second adjuvant encapsulated inside the cell membrane.

[0168] 4. The immunogenic composition of any one of embodiments 1-3, wherein the first adjuvant and the second adjuvant are each independently selected from the group consisting of agents capable of enhancing antigen presentation, agents capable of stimulating and / or strengthening an immune response, and combinations thereof.

[0169] 5. The immunological composition according to any one of embodiments 1-4, wherein the size of the first adjuvant and / or the second adjuvant is 200 nm to 1000 nm.

[0170] 6. An immune composition according to any one of embodiments 1-5, wherein the first adjuvant and the second adjuvant are each independently selected from the group consisting of aluminum salts, water-oil emulsions, manganese salts, liposomes, cytokines, antibodies, polypeptides, DNA, chitosan, polysaccharide derivatives, Poly (I: C), RNA, virus-like particles, flagellin, Freund's adjuvant, MF59, emulsions, AS03, viral particles, small molecules, TLR1 / 2, TLR4, TLR5, TLR7 / 8, TLR9, monophosphoryl lipid A (MPLA) or its analogs, DDA, muramyl dipeptide, threonyl-muramyl dipeptide, OM-174, cholera toxin, and inorganic salt nanoparticles that can induce an immune response.

[0171] 7. The immunogenic composition according to any one of embodiments 1-6, wherein the first adjuvant or the second adjuvant each independently comprises two or more agents.

[0172] 8. The immunological composition according to any one of embodiments 1-7, wherein the first adjuvant is a TLR receptor agonist MPLA or an analog thereof.

[0173] 9. The immunological composition according to any one of embodiments 1-8, wherein the second adjuvant is TLR receptor agonist CpG and / or R848.

[0174] 10. The immune composition of any one of embodiments 1-9, wherein the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

[0175] 11. An immune composition according to any one of embodiments 1-10, wherein the surface of the cell membrane comprises a tumor antigen.

[0176] 12. An immune composition according to any one of embodiments 1-11, wherein the surface of the cell membrane contains tumor-specific antigens and / or tumor-associated antigens.

[0177] 13. The immune composition according to any one of embodiments 1-12, wherein the surface of the cell membrane is capable of expressing proteins having one or more functions selected from the group consisting of nutrient transport, cell proliferation, and cell division.

[0178] 14. An immune composition according to any one of embodiments 1-13, wherein the tumor antigen on the surface of the cell membrane is not artificially introduced.

[0179] 15. The immunological composition according to any one of embodiments 1-14, wherein the mass ratio between the cell membrane and the second adjuvant is 1:20 to 20:1.

[0180] 16. The immune composition according to any one of embodiments 1-15, wherein the mass ratio between the cell membrane and the second adjuvant is 1:20.

[0181] 17. The immune composition according to any one of embodiments 1-16, comprising particles comprising the cell membrane and the second adjuvant.

[0182] 18. The immunological composition of any one of embodiments 1-17, wherein the hydrated particle size of the particles is between 80 nm and 200 nm.

[0183] 19. The immunological composition of any one of embodiments 1-18, wherein the hydrated particle size of the particles is between 100 nm and 400 nm.

[0184] 20. The immunological composition of any one of embodiments 1-19, wherein the surface potential of the particles is between +50 mV and -50 mV.

[0185] 21. The immunological composition of any one of embodiments 1-20, wherein the surface potential of the particles is -26 mV.

[0186] 22. The immune composition of any one of embodiments 1-21, which does not comprise the cytoplasm and / or nucleus of the pluripotent stem cell.

[0187] 23. A method for preparing the immune composition of any one of embodiments 1-22.

[0188] 24. A vaccine comprising the immunogenic composition of any one of embodiments 1-22.

[0189] 25. A pharmaceutical composition comprising the immune composition of any one of embodiments 1-22, the vaccine of embodiment 24, and a pharmaceutically acceptable carrier.

[0190] 26. Use of the immune composition of any one of embodiments 1-22, the vaccine of embodiment 24 and / or the pharmaceutical composition of embodiment 25 in the preparation of a medicament for preventing or treating a disease and / or condition.

[0191] 27. The use according to embodiment 26, wherein the disease and / or condition comprises a tumor and / or an infectious disease.

[0192] 28. The use according to embodiment 27, wherein the disease and / or condition comprises colon cancer, melanoma and / or breast cancer.

[0193] 29. A method for activating immune cells, comprising administering the immune composition of any one of embodiments 1-22, the vaccine of embodiment 24, and / or the pharmaceutical composition of embodiment 25. The immune cells may be T cells, B cells, and / or NK cells.

[0194] 30. A method for enhancing an immune response in an organism, comprising administering the immune composition of any one of embodiments 1-22, the vaccine of embodiment 24, and / or the pharmaceutical composition of embodiment 25.

[0195] Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion protein, preparation method, and use of the present application, and are not intended to limit the scope of the present invention.

[0196] Example

[0197] Example 1 Identification of Stem Cell Membrane Protein Profiles

[0198] method

[0199] Analysis of upregulated proteins shared by tumor cell membranes and iPSC (induced pluripotent stem cell) membranes. Membrane proteins (10 μg) from MC38 colon cancer cells, B16F0 melanoma cells, 4T1 breast cancer cells, CT26 colon cancer cells, mouse embryonic fibroblasts (MEFs) (derived from BALB / c and C57BL6N mice), and iPSCs (derived from BALB / c and C57BL6N mice) were dissolved in PBS. After hydrolysis with alkylating enzymes and desalting, membrane proteins were analyzed by liquid phase tandem mass spectrometry (TIMS / PASEF). All acquired raw data were searched against a database (PaSER 3.0). Proteins with adjusted P values ​​< 0.05 and log2-fold changes > 1.5 in iPSC and tumor cell membranes compared with MEF membranes were considered highly expressed. These highly expressed proteins were identified using the GEPIA2 online tool based on proteins that were highly expressed in tumor tissue compared with normal tissue across more than 20 tumor types in the TCGA dataset. NetMHCpan was then used to perform step-by-step prediction of 8-mer and 12-mer MHC-I epitopes. Epitopes that strongly bound to C57 mouse MHC (H-2Kb, H-2Db) and HLA, and had greater than 10% human population coverage (The Immune Epitope Database), were retained for further synthesis and validation. Furthermore, these epitopes were present in both C57 mice and humans. Venn diagrams, protein expression analysis, and gene enrichment analysis were performed using the clusterProfile package in R.

[0200] result

[0201] To explore the feasibility of iPSC cell membranes as a universal anti-tumor antigen library, we first performed quantitative proteomic analysis according to the above method to compare the surface protein expression profiles of cancer cell lines (melanoma B16F0, colon cancer cells MC38 and CT26, breast cancer cells 4T1), healthy mouse embryonic fibroblasts MEF, and mouse iPSCs. 6570 shared proteins were selected for analysis among all samples (Figure 1A). Compared with MEF cell lines, 1272 proteins and 2218 proteins were significantly overexpressed in multiple tumor cell lines and iPS cell lines, respectively (Figure 1B). 1B-Figure 1C). Among them, 618 proteins were shared by tumor cells and iPS cell lines (Figure 1D). In addition, membrane proteins shared by tumor cells and iPS cells were identified by manual screening. By enriching their genes, it was found that these shared differential proteins were significantly associated with many important cancer physiological processes, including nutrient transport, cell proliferation and cell division (Figure 1E). Among them, the unrestricted proliferation of cancer is the goal of distinguishing tumors from normal tissues. This means that iPS cell membranes can be used to guide the host to produce immunity against known or unknown tumor-associated antigens (TAAs).

[0202] Example 2 iPM vaccine preparation

[0203] The iPSC cell membrane extraction method is briefly described as follows: iPSC cells were resuspended in PBS and centrifuged at 300×g for 5 minutes. 8 The cells were homogenized in 25 mL of membrane separation buffer. The separation buffer was formulated with 225 mM mannitol (HARVEYBIO), 75 mM sucrose (YEASEN; Product No. 60350ES80), 0.5% BSA (BIOLEADER; Product No. B7203S), 0.5 mM tetrasodium etanate (EGTA; Bioss), and 30 mM Tris and a protease inhibitor cocktail (Beyotime; Product No. P1051). The cells were fully disrupted by sonication (30 W) for 3 minutes in an ice bath. The cell homogenate was then centrifuged at 3000 × g for 5 minutes at 4°C, and the supernatant was collected. The supernatant was further centrifuged at 10,000 × g for 10 minutes at 4°C, and the resulting supernatant was ultracentrifuged at 100,000 × g for 2 hours at 4°C. After measuring the protein concentration of the membrane extract using a BCA protein assay kit (Thermo Fisher; Cat. No. 23227), the iPSC cell membranes were resuspended in deionized water and stored at -80°C.

[0204] CpG-R848 nanoparticles (CR NPs) were constructed using a one-step self-assembly method. Briefly, 15 μL of FeCl2 (10 mM; TRC; Cat. No. 1775005) was added to a mixed solution of 150 μL of CpG (25 μM; HEMA biology) and 50 μL of R848 (1 mg / mL; Invivogen; Cat. No. tlrl-R848) in a 1 mL PCR tube. The resulting suspension was vortexed for 30 seconds and then quickly incubated at 95°C for 1.5 hours. The CR NPs were washed twice with deionized water and then centrifuged at 11,000 rpm for 15 minutes.

[0205] Next, 10 μL of MPLA DMSO solution (1 mg / mL; Invivogen; Cat. No. tlrl MPLA) was ultrasonically mixed with the iPSC membrane solution for 10 minutes. The mixed membrane solution was then added to an equal volume of CR NPs and ultrasonically (18W) for 3 minutes. The membrane solution was then extruded using an Avanti liposome extruder through 800 and then 400 nm polycarbonate porous membranes to produce the iPM nanovaccine (nanovax).

[0206] Other adjuvants can also be selected to replace MPLA, R848 and CpG to prepare iPM nanovaccines, as long as they can help the immune system recognize iPS cell membrane antigens and activate antigen-presenting cells through multiple Toll-like receptors (TLRs).

[0207] Example 3 Characterization of physical properties of iPM nanovaccine (nanovax)

[0208] method

[0209] During the collection and washing process of CR NPs, all supernatants were collected to calculate the amount of CpG and R848 loaded in CR NPs, respectively. CpG concentration was determined by using UV-based HPLC at 260 nm. The CpG loading efficiency was calculated using the following formula: Loading efficiency = ((A e -A t ) / A e )×100%, where A t is the total amount of CpG not encapsulated in CR NPs, A e is the initial amount of CpG. Similarly, R848 was measured using HPLC-UV at 328 nm. RP-HPLC was performed using a C4 analytical column (5 μm, 250 × 4.6 mm). A mixture of solvent a and solvent B (solvent a: water (0.1% v / v trifluoroacetic acid), solvent B: acetonitrile) was run from 0% to 98% solvent B over 4 minutes, followed by a 2-minute wash with 98% solvent B and a return to 10% solvent B at a flow rate of 1.0 mL / min. The loading efficiency of R848 is the same as the above formula.

[0210] The iPM nano vaccine is diluted in deionized water and dripped on a copper grid coated with a carbon film overnight. The morphology of different nanoparticles was examined by TEM at an accelerating voltage of 120 kV (HT7700, Hitachi, Japan). In addition, elemental mapping and line scanning were performed on a TEM (Tecnai G2F20U-TWIN, FEI, USA) equipped with an energy dispersive spectrometer (EDS) accessory. The hydrated size (diameter, nm) of the iPM nano vaccine was tested using a ZetaSizer Nano series Nano ZS (Malvern, UK) equipped with a 633 nm He-Ne laser.

[0211] To characterize the efficiency of nanoparticle membrane coating, dual-dye-labeled iPM nanovaccines were prepared by coextrusion of membranes labeled with DID (Beyotime; Cat. No. C1039-10 mg) and CR NPs labeled with rhodamine B (HARVEYBIO). The membrane to CR NP mass ratio ranged from 1:20 to 20:1. The dye-labeled iPM-nanovax was then measured by flow cytometry (Cytoflex S, Beckman-Colter, USA).

[0212] To further determine the colocalization of the various components of the iPM nanovaccine, a triple-fluorescent-labeled iPM nanovaccine was formed using DiD-labeled membranes, rhodamine B-labeled CR NPs, and FITC-labeled MPLA analog LPS (Sigma-Aldrich; Cat. No. F3665) (mass ratio, 20:1:10). Nanoparticle fluorescence colocalization was observed using a super-resolution laser confocal microscope (Letica SP8 STED 3X).

[0213] result

[0214] After synthesis, transmission electron microscopy (TEM) images of the iPM nanovaccine revealed a uniform spherical nanostructure with an inner core and an outer shell (Figure 1F). Furthermore, the hydrodynamic diameter of the iPM nanovaccine increased to 250 nm compared to that of the iPM vesicles, and the iPM nanovaccine exhibited a similar electrical potential of –26 mV as that of the iPM vesicles (Figure 1G). This also indicated that encapsulation of the iPM vesicles had been achieved. Furthermore, to further explore the efficiency of membrane antigen encapsulation in the iPM nanovaccine, when the mass ratio of iPM vesicles to CR NPs was 1:20, more aggregated communities of DiD representing iPM vesicles and Rhodamine B (RhB) representing CR NPs were observed compared to 20:1 (Figure 1H). Therefore, a mass ratio of iPM vesicles to CR NPs of 1:20 was used for subsequent studies of antitumor immune responses. Furthermore, to demonstrate that membrane antigens and adjuvants were localized on a single nanoparticle, super-resolution fluorescence microscopy revealed that iPS cell membrane antigens, the MPLA analog LPS, and CR NPs were colocalized within a single nanoparticle (Figure 1I). The efficiency of nanoparticle coating with the stem cell membrane was 80% or higher.

[0215] Example 4 Characterization of iPM Nanovaccine Protein

[0216] To investigate the protein profile of the iPM nanovaccine, SDS-PAGE was performed and the resulting gel was stained with Coomassie Brilliant Blue (Solarbio, Beijing, China). All samples were dispersed in loading buffer (Invitrogen, Carlsbad, CA, USA) and equal amounts of protein (10 μg) were loaded on NUPAGE Novex 10% separating gels (Invitrogen) in MOPS running buffer (Invitrogen). Western blot analysis was also performed to identify the presence of specific protein markers SSEA-1 (SSEA-1 antibody, Novus, Catalog No. NB100-1831SS), Na+-K+ ATPase (Bufalin, Abcam; Catalog No. ab145636), and E-cadherin (E-cadherin antibody, Cell Signaling Technology; Catalog No. 3195T) on the nanoparticles. These proteins indicate that the vaccine preparation method can effectively retain key proteins on the stem cell membrane surface (Figure 1J, Figure 1K).

[0217] Example 5 Detection of the in vivo effect of iPM vaccine

[0218] method

[0219] To investigate the ability of iPM nanovaccines to accumulate in lymph nodes (LNs), the fluorescent dye DiD was used to label iPSC membrane antigen nanovaccines (iPM nanovax), liposome nanoadjuvants (Lipo-adjuvant), and iPSC membrane antigens only (iPM vesciles), which were then subcutaneously injected into C57BL6N mice. Lymph nodes were isolated and dispersed into single-cell suspensions 3, 12, and 24 hours later. CD11c expression was analyzed by flow cytometry. + 、CD11c high -MHC-II inter 、CD11c inter -MHC-II high Then, 24 hours after the mice were immunized with nanoparticles, the maturation of lymph node dendritic cells (DCs) was detected by labeling with FITC-CD11c, PECy7-CD80, and APC-CD86.

[0220] In addition, to evaluate whether iPM nanovaccines can effectively activate T cells. Lymph nodes were harvested 72 hours after nanovaccine injection in C57BL6N mice and analyzed by flow cytometry for CD69 (FITC; Biolegend; clone: ​​H1.2F3; Cat. No. 104505), CD4, CD8, and CD3. For memory cells, mice received four rounds of vaccination, delivered weekly. Two months later, splenocytes were harvested and analyzed by staining for CD44, CD8, CD4, and CD3.

[0221] result

[0222] In addition to the iPM vesicle group, a large amount of DiD was present in the lymph nodes in the iPM nanovaccine group (Figure 2A) and co-localized with DC (Figure 2B). Similarly, DID fluorescence at different time points after iPM nanovaccination was analyzed by flow cytometry. The results showed that the fluorescence signal of DID in migrating DCs (mDCs, Figure 2C) gradually increased over time, indicating that some iPM nanovaccines were actively carried to the draining lymph nodes by mDCs. In addition, the iPM nanovaccines passively dehydrated into the inguinal lymph nodes within 12 hours, and then DiD+ resident DCs (rDCs) increased in the lymph nodes (Figure 2D). These results suggest that iPM nanovaccines may be located at antigen-presenting cells (APCs) in the lymph nodes, which is an ideal site for vaccination accumulation to trigger potential T cell activation. Then, lymph node DC maturation can be promoted 24 hours after vaccination. The results showed that after in vivo vaccination with iPM nanovaccines, the expression of stimulatory markers and MHC-II on DCs, such as CD11c + 、CD80 + 、CD86 + ( Figure 2E-2F ) Furthermore, a significant increase in serum IL-1β concentration was observed after iPM nanovaccination, indicating the induction of a systemic innate immune response ( Figure 2G ).

[0223] Next, we explored the ability of nanovaccines based on iPSC cell membrane antigens to induce adaptive immunity in vivo. As described above, C57BL6N mice were subcutaneously injected with iPSC cell membrane nanovaccines (iPM nanovax), iPS cell membrane vesicles (iPM vesciles) and lipid adjuvant (Lipo-adjuvant), and LNs were collected 4 days later. Then, T cell activation in the lymph nodes was evaluated by flow cytometry. The iPSC cell membrane antigens in the iPM nanovaccine group were able to universally and effectively stimulate CD4 T cells in LNs. 4+ and CD 8+Activation of T cells (Figure 2H-Figure 2I). In addition, in the iPM nanovaccine group, activated Tfh cells and B cells in the germinal centers of lymph nodes also increased significantly (Figure 2J-Figure 2K). The above results indicate that iPS cell membrane antigens triggered these adaptive immune responses through the form of iPM nanovaccine. Importantly, the CD4+ T cells in the blood of mice two months after vaccination with iPM nanovaccine were significantly increased. 4+ The increase in memory T cells indicated that the iPM nanovaccine triggered a strong long-term memory immune response (Figure 2L). These results indicate that the iPM nanovaccine can effectively trigger cellular and humoral immunity against iPS cell membrane antigens.

[0224] Example 6 Anti-tumor effect of iPM vaccine in vivo

[0225] method

[0226] For each mouse, 100 μg of iPM nanovaccine, adjuvant control group, and solvent group were suspended in 100 μL of water and loaded into an insulin syringe. The mice were randomly divided into three groups, with 5 mice in each group. Immunization was then performed by subcutaneous injection in the flank of the mice, and the injection site was changed every week. The mice were monitored for vaccine safety every week by measuring body weight and performing gross examinations. Four weeks after vaccination, all mice were inoculated with cancer cells. For preventive treatment of C57BL6N mice, 5×10 4 MC38 cells.

[0227] At the end of the MC38 tumor prevention experiment, spleens were isolated from mice that had received PBS, iPM-nanovax, or adjuvant. 5 Splenocytes were co-cultured with iPS cell membrane antigen (10 μg / well), MC38 cell lysate (10 μg / well), or MC38 neoantigen (1 μg / well) in a specific culture medium for 20 hours (Catalog, 6015012; Dakewei). IFN-γ secretion was then measured by enzyme-linked immunosorbent assay (ELISPOT) according to the manufacturer's instructions (Cat. No. 2210005; Dakewei).

[0228] result

[0229] To understand the potential preventive benefits of iPS cell membrane antigen nanovaccine (iPM nanovax) in the setting of tumor progression, we immunized C57BL6N mice for 4 weeks and then subcutaneously inoculated MC38 cells in each mouse (Figure 3A). The results showed that iPM nanovax can effectively delay disease progression, and the changes in tumor growth are shown in Figures 3B-3C. Stimulation with iPM nanovax significantly increased the cytotoxic GranzymB in MC38 tumor tissue. + and IFN-γ+ CD8 + T cells and cytotoxic IFN-γ + CD4 + T cells (Figure 3D-3E). In addition, CD8 + and CD4 + T cell infiltration also increased (Figure 3F-3G). In addition, two months after iPM nanovaccine vaccination, memory CD4 + T cells and memory CD8 in dLN and spleen + T cells were still highly detectable (Figure 3H-J). Overall, these results indicate that co-delivery of iPSC membrane antigens with adjuvants accurately triggered anti-tumor CD8 + and CD4 + T cell responses play an important role in inhibiting tumor growth and enhancing the intensity and persistence of anti-tumor immunity. The cytotoxic effects of effector T cells and helper T cells are also mediated by CD8 + T depletion and CD4 + T depletion was confirmed (Figure 3K). + T and CD4 + Depletion of T cells abolished the antitumor effect of iPM nanovaccine.

[0230] To test whether the iPS cell membrane antigen nanovaccine provides immunity against cancer cells, experiments were performed to evaluate the tumor specificity of cytotoxic T cells. MC38 cells were injected into C57BL6N mice that were vaccinated four times within a month. Two weeks later, spleen T cells were isolated from the mice and restimulated with iPS cell membrane antigens and MC38 cell lysates. Specifically, C57BL / 6J mice were immunized once a week for 4 weeks and then vaccinated with 5×10 4 Two weeks later, the mice were euthanized, and the spleens were harvested and isolated into single cells. iPSC membrane protein (10 μg / well) and MC38 tumor lysate (10 μg / well) were mixed with spleen cells (1×10) from mice immunized with PBS, Lipo adjuvant, and iPM nanovaccine, respectively. 6 The cells were incubated for 20 h, and then IFN-γ secretion was detected by ELISPOT.

[0231] The results showed that increased IFN-γ secretion was observed in the spleen cells of mice treated with iPS cell membrane nanovaccine, indicating that iPS cell membrane antigens and MC38 cell lysates can re-stimulate T cells (Figures 3L-3M). In particular, the iPS cell membrane antigen nanovaccine can induce MC38 neoantigen-specific anti-tumor immunity, which means that the T cell response induced by the iPS cell membrane antigen nanovaccine can spread to non-vaccine neoantigen epitopes.

[0232] Example 7 In vivo anti-tumor effect of iPS-glucan vaccine

[0233] To verify that adjuvant particles of different properties help the immune system recognize iPS cell membranes, we designed another vaccine system, using alum aluminum hydroxide to adsorb iPSC cell membranes and yeast-derived mannan gulcan as an adjuvant. According to the method of Example 2, nano vaccine particles iPS-gulcan were prepared together with iPSC cell membranes. The particles were then injected into mice and lymph node enrichment was observed (Figure 4A). It was found that the vaccine can be effectively enriched in the lymph nodes and can induce a higher antibody response against iPSC cells (Figure 4B). After subcutaneously immunizing mice four times, the mice were injected with MC38 cells again. It can be seen that the mice in the iPS-gulcan group had a better effect of inhibiting tumor progression than the gulcan group (Figure 4C). This shows that the purpose of the present invention can also be achieved using adjuvants other than TLR agonists.

Claims

An immune composition comprising a cell membrane derived from pluripotent stem cells and an adjuvant.

2. The immunogenic composition of claim 1, wherein the adjuvant comprises a first adjuvant that is inserted into the lipid bilayer of the cell membrane.

3. The immune composition according to any one of claims 1-2, wherein the adjuvant comprises a second adjuvant encapsulated inside the cell membrane.

4. The immune composition according to any one of claims 1 to 3, wherein the first adjuvant and the second adjuvant are each independently selected from the group consisting of agents capable of enhancing antigen presentation, agents capable of stimulating and / or strengthening an immune response, and combinations thereof; optionally, the first adjuvant and the second adjuvant are each independently selected from the group consisting of aluminum salts, water-oil emulsions, manganese salts, liposomes, cytokines, antibodies, polypeptides, DNA, chitosan, polysaccharide derivatives, Poly(I:C), RNA, virus-like particles, flagellin, Freund's adjuvant, MF59, emulsions, AS03, viral particles, small molecules, TLR1 / 2, TLR4, TLR5, TLR7 / 8, TLR9, monophosphoryl lipid A (MPLA) or its analogs, DDA, muramyl dipeptide, threonyl-muramyl dipeptide, OM-174, cholera toxin, and inorganic salt nanoparticles that can elicit an immune response.

5. The immunological composition according to any one of claims 1 to 4, wherein the size of the first adjuvant and / or the second adjuvant is 200 nm to 1000 nm.

6. The immune composition according to any one of claims 1 to 5, wherein the first adjuvant is a TLR receptor agonist MPLA or an analog thereof; optionally, the second adjuvant is a TLR receptor agonist CpG and / or R848.

7. The immune composition according to any one of claims 1 to 6, wherein the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

8. The immune composition according to any one of claims 1 to 7, wherein the surface of the cell membrane comprises a tumor antigen.

9. The immune composition according to any one of claims 1 to 8, wherein the surface of the cell membrane is capable of expressing proteins having one or more functions selected from the group consisting of nutrient transport, cell proliferation, and cell division.

10. The immune composition according to any one of claims 1 to 9, wherein the tumor antigen on the surface of the cell membrane is not artificially introduced.

11. The immunocomposition according to any one of claims 1 to 10, wherein the mass ratio between the cell membrane and the second adjuvant is 1:20 to 20:

1.

12. The immunological composition according to any one of claims 1 to 11, comprising particles comprising the cell membrane and the second adjuvant.

13. The immunogenic composition according to any one of claims 1 to 12, wherein the hydrated particle size of the particles is between 80 nm and 200 nm.

14. The immunological composition according to any one of claims 1 to 13, wherein the surface potential of the particles is between +50 mV and -50 mV.

15. The immunological composition according to any one of claims 1 to 14, which does not comprise the cytoplasm and / or nucleus of the pluripotent stem cell.

16. A method for preparing the immune composition according to any one of claims 1 to 15.

17. A vaccine comprising the immunogenic composition of any one of claims 1 to 15.

18. A pharmaceutical composition comprising the immune composition according to any one of claims 1 to 15, the vaccine according to claim 17, and a pharmaceutically acceptable carrier.

19. Use of the immunogenic composition according to any one of claims 1 to 15, the vaccine according to claim 17 and / or the pharmaceutical composition according to claim 18 in the preparation of a medicament for preventing or treating a disease and / or condition.

20. A method for activating immune cells, comprising administering the immune composition of any one of claims 1 to 15, the vaccine of claim 17 and / or the pharmaceutical composition of claim 18.

21. A method for enhancing an immune response of an organism, comprising administering the immune composition of any one of claims 1 to 15, the vaccine of claim 17 and / or the pharmaceutical composition of claim 18.

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