WO2023138636A1 - Cd24疫苗 - Google Patents

Cd24疫苗 Download PDF

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Publication number
WO2023138636A1
WO2023138636A1 PCT/CN2023/073026 CN2023073026W WO2023138636A1 WO 2023138636 A1 WO2023138636 A1 WO 2023138636A1 CN 2023073026 W CN2023073026 W CN 2023073026W WO 2023138636 A1 WO2023138636 A1 WO 2023138636A1
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Prior art keywords
immunogenic
seq
spytag
spycatcher
hbcag
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PCT/CN2023/073026
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English (en)
French (fr)
Inventor
楼觉人
王文伟
胡海涛
王蓓
朱琳
张群
蔡蓓蓓
仝光杰
胡一幸
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上海至成生物科技有限公司
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Publication of WO2023138636A1 publication Critical patent/WO2023138636A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6075—Viral proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of biomedicine, in particular to a CD24 vaccine.
  • HSA heat-stable antigen
  • human CD24 is the homologous molecule of the heat-stable antigen in humans, located on chromosome 6q21.
  • CD24 molecule is a highly glycosylated sialic acid glycoprotein, which is anchored on the cell membrane surface by phosphatidylinositol (GPI) at the C-terminus.
  • GPI phosphatidylinositol
  • the amino acid numbers of mature mouse and human CD24 molecules are 27aa and 31aa respectively.
  • CD24 molecule has many potential glycosylation sites, and its apparent molecular weight is about 25-75KDa.
  • Mouse CD24 is mainly expressed in hematopoietic cell subsets, especially B lymphocytes, erythrocytes, neutrophils and thymocytes, while human CD24 is only expressed on the surface of B lymphocytes and is lost during plasma cell maturation.
  • CD24 has a variety of biological activities and is involved in cell proliferation and intercellular interactions. Its sialic acid sugar structure can interact with insulin-like lectin 10 (sialic-acid-binding Ig-like lectin 10, Siglec1-10) on innate immune cells, negatively regulate the immune response of the body, and inhibit the destructive inflammatory response caused by infection, sepsis, liver injury, and chronic graft-versus-host disease.
  • Ig-like lectin 10 insulin-like lectin 10
  • Siglec1-10 insulin-like lectin 10
  • CD24 is expressed in almost all tumor cells, including ovarian cancer, breast cancer, pancreatic cancer, kidney cancer, lung cancer, nasopharyngeal cancer, and liver cancer.
  • the expression level of CD24 is closely related to tumor invasion, metastasis, and prognosis. On the one hand, CD24 can promote tumor metastasis by strengthening the adhesion of tumor cells to the surrounding tissue matrix.
  • Vaccines usually consist of two parts: antigen (immunogen) and adjuvant. Whether the immune system of the body can respond to the vaccine depends on the characteristics of the antigen (immunogen) itself.
  • Effective vaccine antigens usually have the following characteristics: 1. Foreign body property; 2. Large molecular weight; 3. Complex composition and structure.
  • CD24 molecule is the body's own Peptides do not have the characteristics of foreign bodies. The mature CD24 molecule is only about 30 amino acids and has a small molecular weight. Therefore, a single CD24 molecule cannot be used as a vaccine antigen.
  • the inventors recombinantly expressed CD24 through genetic engineering technology and connected it to the surface of nanoparticle protein to construct a nanoparticle vaccine with CD24 as an antigen for immunotherapy of tumors.
  • the first aspect of the present invention provides an immunogenic complex comprising: (a) CD24 or an immunogenic fragment thereof and (b) a carrier protein.
  • the carrier protein is a carrier protein capable of forming nanoparticles.
  • the carrier protein is Ferritin protein, or, hepatitis B core antigen (HBcAg) or its variants.
  • (a) and (b) are covalently linked.
  • said covalent link is a peptide bond or an isopeptide bond formed by a SpyCatcher-SpyTag covalent link.
  • the CD24 is mouse CD24 or human CD24.
  • the CD24 has the sequence shown in SEQ ID NO: 1 or 2.
  • the immunogenic complex is a fusion polypeptide comprising: (a) CD24 or an immunogenic fragment thereof and (b) hepatitis B core antigen (HBcAg) or a variant thereof, wherein the HBcAg variant is (1) a variant having a C-terminal deletion of 34 or fewer amino acids of HBcAg, or (2) a variant of HBcAg having an insertion sequence after amino acid position 78 and a deletion of 34 or fewer amino acids at the C-terminus.
  • HBcAg hepatitis B core antigen
  • the insertion sequence is ((G) o S(G) p T) q , wherein o, p, and q are each independently an integer selected from 0, 1, 2, 3, 4, 5, and 6.
  • o, p, and q are each independently an integer selected from 0, 1, 2, 3, 4, 5, and 6.
  • q is 1, and o and p are each independently an integer selected from 0, 1, 2, 3. More preferably, q is 1 and o and p are 4.
  • the CD24 or its immunogenic fragment is located after the 78th amino acid of HBcAg or its variant or after the insertion sequence, and HBcAg or its variant is divided into two parts: A at the N-terminus and B at the C-terminus.
  • said CD24 or its immunogenic fragment The stretch is located after amino acid 78 of SEQ ID NO:6 or after amino acid 88 of SEQ ID NO:7.
  • the C-terminus of said CD24 or an immunogenic fragment thereof is connected to said part B via a linker.
  • the linker is ((G) mS ) n , wherein m and n are each independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; preferably the linker is GGGGGSGGGGS.
  • the immunogenic complex comprises covalently linked by SpyCatcher-SpyTag:
  • CD24 or an immunogenic fragment thereof linked to SpyCatcher (a) CD24 or an immunogenic fragment thereof linked to SpyCatcher, and (b) Ferritin protein linked to SpyTag, or
  • (b) or (b') self-assembles to form a nanoparticle carrier.
  • each of said linkages is independently a direct linkage or a linkage via a linker.
  • the linker is ((G) mS ) n , wherein m and n are each independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; the linker is preferably GGGGGSGGGGSGGGGS.
  • the CD24 is mouse CD24 or human CD24.
  • the CD24 has the sequence shown in SEQ ID NO: 1 or 2.
  • the SpyCatcher is shown in SEQ ID NO: 5.
  • the CD24 is mouse CD24 or human CD24.
  • the CD24 has the sequence shown in SEQ ID NO: 1 or 2.
  • the invention also provides a nucleic acid construct having a nucleic acid molecule according to any one of the embodiments herein.
  • the nucleic acid construct is a cloning vector or an expression vector.
  • the host cells include, but are not limited to, Escherichia coli, Pichia pastoris, Hansenula, Saccharomyces cerevisiae, insect cells and mammalian cells, such as E. coli BL21(DE3), Rosseta or JM109 strains.
  • the second aspect of the present invention also provides an immunogenic composition comprising the immunogenic complex described in any embodiment herein and an adjuvant.
  • the adjuvant is aluminum hydroxide.
  • the present invention also provides a pharmaceutical composition, which comprises: the immunogenic complex or immunogenic composition described in any embodiment herein, and pharmaceutically acceptable excipients.
  • the invention also provides a kit comprising the immunogenic or pharmaceutical composition of any embodiment herein, and a container for administering them.
  • the medicament is used to prevent, alleviate or treat CD24-related diseases or disorders.
  • the CD24-associated disease is cancer, preferably ovarian cancer, breast cancer, pancreatic cancer, kidney cancer, lung cancer, nasopharyngeal cancer or liver cancer.
  • the CD24-associated disorder is tumor invasion, metastasis, or immune escape.
  • Figure 1 SpyTag-Ferritin purification SDS-page.
  • M pre-stained protein maker
  • lane1 broken bacteria
  • lane2 supernatant of broken bacteria
  • lane3 supernatant after heating
  • lane4 Q-FF elution.
  • FIG. 2 Spycatcher-mCD24 purification SDS-page.
  • M prestained protein maker
  • lane1 bacterial supernatant
  • lane2 Q-FF elution
  • lane3 phenyl-Hisub flow-through
  • lane4 Q-HP elution.
  • Figure 3 mCD24-Ferritin purification SDS-page.
  • M pre-stained protein maker
  • lane1 supernatant of broken bacteria
  • lane2 flow-through of DEAE FF
  • lane3 flow-through of Poros-50HQ.
  • Figure 4 HBcAg-mCD24 purification SDS-page.
  • M pre-stained protein maker
  • lane1 whole bacteria
  • lane2 supernatant of bacteria
  • lane3 Poros-50HQ elution
  • lane4 Chromdex-200 exclusion peak.
  • Figure 5 Assembly SDS-page of SpyTag-Ferritin and Spycatcher-mCD24.
  • M prestained protein maker
  • lane1 SpyTag-Ferritin
  • lane2 Spycatcher-mCD24
  • lane3 0.5:1
  • lane4 1:1
  • lane5 1:0.5
  • lane6 1:0.25.
  • Figure 6 Purified SDS-page after assembly of SpyTag-Ferritin and Spycatcher-mCD24.
  • M prestained protein maker
  • lane1 ligation product concentrated by ultrafiltration
  • lane2 Chromdex-200 exclusion peak.
  • CD24 cannot be used as an immunogen for tumor vaccines. This is because: the CD24 molecule is the body's own polypeptide and does not have the characteristics of foreign bodies; the mature CD24 molecule is only about 30 amino acids and has a small molecular weight. Therefore, a single CD24 molecule cannot be used as a vaccine antigen. It is also verified in the examples of the present invention that CD24 molecules alone cannot induce antibody production.
  • CD24 has outstanding immunogenicity after being coupled (eg, covalently linked) with a carrier protein, and a tumor vaccine can be prepared, which can effectively prevent and treat CD24-related diseases represented by breast cancer.
  • CD24 described herein may be from any species, preferably CD24 (SEQ ID NO: 1 or 2) of mouse or human origin.
  • CD24 herein also includes functional variants.
  • a variant of a polypeptide has, for example, at least 80%, 85%, 90%, 95%, 97% or 99% identity at the amino acid level compared to its wild-type counterpart (eg, SEQ ID NO: 1 or 2).
  • functionally active variants of CD24 comprise naturally occurring functionally active variants, such as allelic variants and species variants as well as non-naturally occurring functionally active variants which can be produced, for example, by mutagenesis techniques or by direct synthetic methods.
  • the CD24 functional variant differs from the peptide shown in SEQ ID NO: 1 or 2 by about, eg, 1, 2, 3, 4 or 5 amino acid residues, and still retains antigenic CD24 biological activity.
  • the site of variation can occur at any position in the peptide, as long as the biological activity is substantially similar to the peptide shown in SEQ ID NO: 1 or 2.
  • Examples of groups of amino acids with side chains of similar chemical nature include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; cystine and methionine.
  • Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.
  • CD24 herein also includes immunogenic fragments thereof. Knowing that CD24 found herein can be used as an immunogen to prepare tumor vaccines, those skilled in the art can Ex situ identification of fragments of CD24 that are immunogenic.
  • the first aspect of the present invention provides an immunogenic complex comprising: (a) CD24 or an immunogenic fragment thereof and (b) a carrier protein.
  • the preferred covalent connection between component a and component b may be a peptide bond or an isopeptide bond formed by covalent connection of SpyCatcher-SpyTag.
  • the immunogenic complex can be a polypeptide capable of fusion expression or a complex covalently linked by SpyCatcher-SpyTag.
  • the present invention also relates to a method for preparing the immunogenic complex, comprising: fusion expressing the fusion polypeptide comprising CD24 or its immunogenic fragment and a carrier protein, or providing (for example expressing separately) (a) and (b) components respectively and then forming an immunogenic complex through chemical cross-linking or covalent linkage (such as isopeptide bonds) between the components.
  • the inventors selected carrier proteins capable of forming nanoparticles.
  • the carrier protein is Ferritin protein, or hepatitis B core antigen (HBcAg) or its variants.
  • the invention provides an immunogenic complex in the form of a fusion polypeptide comprising: (a) CD24 or an immunogenic fragment thereof and (b) hepatitis B core antigen (HBcAg) or a variant thereof.
  • Hepatitis B core antigen (HBcAg) is a 183 amino acid polypeptide. Both HBcAg and its truncated variants can be used as the carrier protein of CD24 nanoparticle vaccine.
  • HBsAg suitable for use in the present invention may be derived from any organism.
  • HBsAg suitable for use in the present invention can be derived from any organism as long as it can form virus-like particles and be used as an immunogenic carrier.
  • the present invention also relates to the preparation method of the immunogenic complex, comprising: expressing the fusion polypeptide comprising CD24 or its immunogenic fragment and HBcAg or its variant.
  • the fusion polypeptide herein can undergo sequence changes, and the resulting mutant has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity with the fusion polypeptide (such as SEQ ID NO: 9) and retains the biological activity (such as immunogenicity) of the fusion polypeptide.
  • Sequence identity between two aligned sequences can be calculated using, for example, NCBI's BLASTp.
  • the present invention provides immunogenic complexes in which the carrier protein is Ferritin protein.
  • the immunogenic complex comprises covalently linked by SpyCatcher-SpyTag: (a) CD24 or immunogenic fragment thereof linked to SpyCatcher, and (b) Ferritin protein linked to SpyTag, or (a') CD24 or immunogenic fragment thereof linked to SpyTag, and (b') Ferritin protein linked to SpyCatcher.
  • Ferritin protein is from the ferritin of thermophilic archaea (Pyrococcus furiosus), which is an octahedron composed of 24 subunits, preferably has the sequence shown in SEQ ID NO: 3 or a functional variant with at least 80% sequence identity thereto.
  • Ferritin proteins can self-assemble to form nanoparticle carriers. Furthermore, compositions comprising (a) and (b) or (a') and (b') above without SpyCatcher-SpyTag covalent linkage are also within the scope of the present invention.
  • the present invention also relates to a method for preparing the immunogenic complex, comprising: expressing the polypeptides in (a) and (b) or (a') and (b'), co-incubating after purification, and self-assembling to obtain the immunogenic complex.
  • the SpyCatcher is located at the N-terminus of CD24 or Ferritin protein
  • the SpyTag is located at the N-terminus of CD24 or Ferritin protein.
  • the SpyTag contains the amino acid sequence shown in SEQ ID NO: 4 or a functional variant having at least 80% sequence identity thereto
  • the SpyCatcher contains the amino acid sequence shown in SEQ ID NO: 5 or a functional variant having at least 80% sequence identity thereto.
  • the link between components in a polypeptide can be direct or via joints.
  • the amino acids of the linker are nonsense polypeptides that do not have additional functions (such as protein positioning, enzyme cleavage sites, etc.) other than connection.
  • the linker is ((G) m S) n , wherein m and n are each independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • the (a) component is SpyCatcher-mCD24, which contains the amino acid sequence shown in SEQ ID NO: 13 or a functional variant having at least 80% sequence identity thereto;
  • component is SpyTag-Ferritin, which contains the amino acid sequence shown in SEQ ID NO: 11 or a functional variant having at least 80% sequence identity thereto.
  • Each polypeptide (such as SpyCatcher-CD24, SpyCatcher-carrier protein, SpyTag-CD24, SpyTag-carrier protein) that is connected with SpyCatcher or SpyTag herein can change in sequence, and the obtained mutant has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity and retains the biological activity of the polypeptide (such as with the corresponding SpyCatcher or SpyTag Activity of covalent linkage, self-assembly activity of carrier protein, immunogenicity after covalent linkage of SpyCatcher and SpyTag, etc.).
  • Sequence identity between two aligned sequences can be calculated using, for example, NCBI's BLASTp.
  • the method for the preparation of said immunogenic complex comprises the step of expressing the corresponding polypeptide.
  • expression can be achieved by incubating a host cell comprising an expression vector with a coding sequence for the polypeptide to be expressed. Therefore, the present invention also relates to the coding sequence of the polypeptide described herein and the nucleic acid construct containing the coding sequence, which may be a cloning vector, an integrating vector or an expression vector.
  • the expression vector also contains other components required for polypeptide expression, such as promoters, terminators and the like. Host cells, expression vectors and components are all well known in the art. Exemplary expression vectors include pet24a.
  • the host cells described herein can be animal, plant or microbial cells, including but not limited to Escherichia coli, Pichia pastoris, Hansenula, Saccharomyces cerevisiae, insect cells and mammalian cells, such as Escherichia coli BL21(DE3), Rosseta or JM109 strains.
  • Adjuvants suitable for use in the vaccines of the present invention include those that enhance anti-CD24 or immunogenic fragments thereof adjuvants that enhance the cell-mediated response against CD24 or immunogenic fragments thereof.
  • Such adjuvants are well known in the art and are commercially available.
  • the adjuvant is selected from one or more of Sigma Adiuvant System, AddaVax, squalene, muramyl dipeptide, MF59, AS03, monophosphatidyl lipid A, flagellin, CPG, and small molecules of aluminum or calcium salts.
  • the preferred adjuvant is aluminum hydroxide.
  • Examples of pharmaceutically acceptable excipient ingredients include binders (syrup, gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone, etc.), fillers (lactose, sucrose, starch, calcium phosphate, sorbitol, glycine, etc.), lubricants (magnesium stearate, talc, polyethylene glycol, etc.), disintegrants (starch, microcrystalline cellulose, etc.), wetting agents (lauryl sulfate, etc.) Sodium (sodium lauryl sulphate, etc.), suspending agent (sorbitol, syrup, methylcellulose, glucose syrup, gelatin, hydrogenated edible fat, etc.), emulsifier (lecithin, sorbitan monooleate, gum arabic, etc.), non-aqueous vehicle (almond oil, fractionated coconut oil or glycerin, propylene glycol, ethanol, etc.
  • binders lactos
  • Hydrophobic esters, etc. preservatives (methylparaben or propylparaben, Sorbic acid, etc.), fragrances (synthetic flavors, natural flavors, etc.), sweeteners (sucrose, stevia, xylitol, etc.), pH regulators (sodium bicarbonate, potassium carbonate, etc.), powders (pigments, dyes, resins, etc.), thickeners (gum arabic, methylcellulose, etc.), antioxidants (vitamin C, vitamin E, etc.), etc.
  • compositions are preferably produced under GMP conditions.
  • compositions of the present invention may be prepared, packaged or sold in bulk as a single unit dose or as a plurality of single unit doses.
  • a "unit dose” refers to an individual quantity of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of said active ingredient is usually equal to the dose of active ingredient administered to the subject, or a convenient fraction of such a dose, for example one-half such a dose.
  • parenteral administration includes, but is not limited to, administering a pharmaceutical composition by injection of the composition, applying the composition through a surgical incision, applying the composition through tissue penetration into a non-surgical wound, and the like.
  • parenteral administration includes, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, Intraventricular, intraurethral, intracranial, intrasynovial injection or infusion; and renal dialysis perfusion techniques.
  • Typical immunizations are vaccinations by the intramuscular route, but the present invention also contemplates oral, subcutaneous (SC), nasal (IN), intravenous (IV), intraperitoneal (IP) or intradermal (ID) administration.
  • compositions suitable for parenteral administration will typically contain an active ingredient acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Formulations for injection can be prepared, packaged, or sold in unit dosage form, eg, in ampoules with a preservative, or in multi-dose containers. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more other ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • an active ingredient acceptable carrier such as sterile water or sterile isotonic saline.
  • Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration.
  • Formulations for injection can be prepared, packaged, or sold in unit dosage form, eg, in ampoules with
  • the immunogenic or pharmaceutical compositions described above are administered in a manner compatible with the dosage formulation, and in amounts such as a therapeutically effective amount and an immunogenically effective amount.
  • a “pharmaceutically effective dose” or “therapeutically effective dose” is the dose required to treat or prevent or alleviate one or more CD24-associated diseases or symptoms in a subject.
  • Such pharmaceutically effective doses will depend on the particular compound administered, the severity of the symptoms, the susceptibility of the subject to side effects, the type of disease, the composition employed, the route of administration, the type of mammal being treated, physiological characteristics of the particular mammal such as health and physiological condition, concomitant drug use, the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, and other factors known to those skilled in the art.
  • the invention also relates to a kit comprising an immunogenic or pharmaceutical composition as described herein, and a container for administering said immunogenic or pharmaceutical composition.
  • the container is preferably a medical syringe.
  • the present invention further provides methods for reducing or eliminating the possibility of a subject suffering from a CD24-related disease or disorder or treating a subject for a CD24-related disease or disorder, comprising administering to said subject an effective amount of an immunogenic complex, immunogenic composition or pharmaceutical composition according to the present invention.
  • a CD24-associated disease or disorder is, for example, a disease or disorder in which inhibition of CD24 activity or inhibition of CD24 interaction with a receptor is beneficial.
  • CD24-related diseases include but not limited to cancer, such as ovarian cancer, breast cancer, pancreatic cancer, kidney cancer, lung cancer, nasopharyngeal cancer or liver cancer; CD24-related disorders include but not limited to tumor invasion, metastasis or immune escape.
  • the CD24 immunogenic complex, immunogenic composition or pharmaceutical composition herein can be administered together with another drug, and the two can be administered sequentially in a certain order or simultaneously.
  • the subject is especially a mammal, preferably a primate, more preferably a human.
  • Subjects include healthy subjects, patients with CD24-related diseases or disorders, and recovered subjects.
  • Protein sequence SETTTGTSSNSSQSTSNSGLAPNPTNATTKV (SEQ ID NO: 2)
  • HBcAg1-149a a modified nucleotide sequence:
  • SpyTag-Ferritin, Spycatcher-mCD24a, mCD24-Ferritin and HBcAg-mCD24 and DNA sequences were all cloned into pet24a(+) expression vector through Nde1 and EcoR1 restriction sites.
  • the bacteria solution was centrifuged at 5000rpm for 30 minutes to collect the bacteria, resuspended the bacteria in the destructive buffer (20mM Tris, 50mM Nacl, PH7.0-8.0), crushed the bacteria in a high-pressure homogenizer at 800bar, centrifuged at 12000rpm for 20 minutes, and collected the supernatant; heated the supernatant at 75°C for 15 minutes, centrifuged at 12000rpm for 20 minutes, and collected the supernatant; centrifuged after heating The supernatant was purified by Q-FF anion exchange chromatography. After loading and equilibrating the column, gradient elution was carried out with elution buffer (20mM Tris, 500mM Nacl, pH7.0-8.0). The target protein was present in the eluent ( Figure 1).
  • the bacteria liquid was centrifuged at 5000rpm for 30 minutes to collect the bacteria, resuspended in the bacteria-breaking buffer (20mM Tris, 50mM Nacl, PH7.0-8.0), crushed the bacteria in a high-pressure homogenizer at 800 bar, centrifuged at 12000rpm for 20 minutes, and collected the supernatant of the bacteria; 0-8.0 solution gradient elution, the target protein can be bound to the Q-FF chromatography column and eluted between 0.1-0.3M Nacl; add ammonium sulfate to the eluate containing the target protein in the Q-FF column chromatography to a final concentration of 0.5M, perform Phenyl-Hisub hydrophobic chromatography, and elute with 20mM Tris, 50mM Nacl, PH7.0-8.0 buffer.
  • the bacteria-breaking buffer 20mM Tris, 50mM Nacl, PH7.0-8.0
  • Miscellaneous proteins can be removed after low-salt elution by hanging on the column; Phenyl-Hisub flow-through is used for Q-HP anion exchange chromatography, 20mM Tris, 500mM Nacl, pH7.0-8.0 solution gradient elution, the target protein exists in the eluent ( Figure 2).
  • the induced bacterial liquid was centrifuged at 5000rpm for 30 minutes to collect the bacterial cells, and the bacterial cell was resuspended in the bacterial destruction buffer (50mM Tris, 50mM Nacl, PH7.0-8.0).
  • the elution buffer (elution buffer 50mmTris, 1000mmNacl, 0.03% Tween80, PH7.0-8.0) adopts 0%-50% linear elution, and the target protein basically does not hang on the column, and mainly exists in the flow-through; 00mmNacl, 0.03% Tween80, PH7.0-8.0) phase elution, the target protein basically does not hang on the column, mainly exists in the flow-through ( Figure 3).
  • the bacterial solution was centrifuged at 5000rpm for 30 minutes to collect the bacterial cells, and the bacterial cells were resuspended in the bacterial destruction buffer (50mM Tris, 50mM Nacl, PH7.0-8.0).
  • elution buffer (elution buffer 50mmTris, 1500mmNacl, 0.03% Tween80, PH7.0-8.0) adopts 15% and 30% two-stage elution, all has target protein in 15% and 30% elution peak, and wherein the purity of target protein in 30% elution peak is higher;
  • the second step adopts Chromdex-200 size exclusion chromatography, and loading buffer is P In BS, 30% of the eluted components of Poros-50HQ anion exchange chromatography were loaded as samples, and the exclusion peaks were collected (Fig. 4).
  • mCD24-Ferritin 100 ⁇ L of purified samples of mCD24-Ferritin, SpyTag-Ferritin, SpymCD24-Ferritin and HBcAg-mCD24 were taken for transmission electron microscope observation.
  • mCD24-Ferritin, SpyTag-Ferritin, SpymCD24-Ferritin and HBcAg-mCD24 were all in the form of nanoparticles (Fig. 7-Fig. 10).
  • Purified SpymCD24-Ferritin, mCD24-Ferritin, HBcAg-mCD24 and mCD24 polypeptides were mixed with 200 ⁇ g/mL aluminum hydroxide adjuvant in equal volume, adsorbed overnight at 4°C, 8 mice in each group, each mouse was immunized with 0.5 mL intraperitoneally, and immunized three times. The interval between two immunizations was 4 weeks, and blood was collected from the tail vein 4 weeks after each immunization. , Antibody titers were detected by ELISA (Table 2).
  • mice After three times of immunization with SpymCD24-Ferritin, mCD24-Ferritin, HBcAg-mCD24 and mCD24, each mouse was subcutaneously inoculated with 1.0 ⁇ 106 4T1 mouse breast cancer cells, and the mice were sacrificed 7 days after inoculation, and the tumor tissues were removed and weighed. The results showed that, compared with the mCD24 control group, the tumor weight of the mice in the SpymCD24-Ferritin and HBcAg-mCD24 groups was significantly smaller than that of the mCD24 control group, indicating that the antibodies against mCD24 produced by the mice after immunization could inhibit the growth of tumor cells (Table 3 and Figure 11).

Abstract

提供一种免疫原性复合物CD24疫苗，其包含：(a)CD24或其免疫原性片段以及(b)载体蛋白。该CD24疫苗具有突出的免疫原性，可以预防和治疗CD24相关疾病。

Description

CD24疫苗 技术领域
本发明涉及生物医药领域，具体涉及CD24疫苗。
背景技术
20世纪70年代末，一种具有有机溶剂可溶性和热稳定的脂样结构的小鼠膜糖蛋白被发现，称为热稳定抗原(heat-stable antigen，HSA)，即小鼠CD24，人CD24是热稳定抗原在人类的同源分子，位于染色体6q21。CD24分子是一种高度糖基化的唾液酸糖蛋白，通过C端的磷脂酰肌醇(glycosylphoshphatidylinositol，GPI)锚固在细胞膜表面，成熟的小鼠和人CD24分子的氨基酸数目分别为27aa和31aa，CD24分子具有许多潜在的糖基化位点，其表观分子量约为25-75KDa。小鼠CD24主要在造血细胞亚群中表达，特别是B淋巴细胞、红细胞、中性粒细胞和胸腺细胞，人的CD24仅表达于B淋巴细胞表面，并在浆细胞成熟过程中丢失。
CD24具有多种生物学活性，参与细胞增殖和细胞间的相互作用，其唾液酸糖结构可与固有免疫细胞上的类胰岛素样凝集素10(sialic-acid-binding Ig-like lectin 10，Siglec1-10)相互作用，可对机体的免疫反应进行负调控，能够抑制感染、败血症、肝损伤和慢性移植物抗宿主疾病等导致的破坏性炎症反应。此外，CD24在几乎所有的肿瘤细胞中表达，包括卵巢癌、乳腺癌、胰腺癌、肾癌、肺癌、鼻咽癌和肝癌等，CD24的表达水平和肿瘤的侵袭、转移和预后密切相关，一方面，CD24可通过加强肿瘤细胞与周围组织基质的黏附而促进肿瘤转移，另一方面，CD24与Siglec1-10结合，阻止巨噬细胞对肿瘤细胞的吞噬作用，介导肿瘤的免疫逃逸。
但是，本领域尚无以CD24为抗原的肿瘤疫苗或免疫治疗产品。疫苗通常由抗原(免疫原)和佐剂两部分组成，机体免疫系统能否对疫苗起反应取决于抗原(免疫原)本身的特性，有效的疫苗抗原(免疫原)通常具有以下几个特征：1、异物性；2、分子量大；3、组成和结构复杂。CD24分子为机体自身多 肽，不具有异物性的特征，成熟的CD24分子仅约30个氨基酸，分子量小，因此单独的CD24分子无法做为疫苗抗原使用。
发明内容
发明人通过基因工程技术对CD24进行重组表达并将其连接到纳米颗粒蛋白表面，构建以CD24为抗原的纳米颗粒疫苗，用于肿瘤的免疫治疗。
本发明第一方面提供一种免疫原性复合物，其包含：(a)CD24或其免疫原性片段以及(b)载体蛋白。
在一个或多个实施方案中，所述载体蛋白是能形成纳米颗粒的载体蛋白。优选地，所述载体蛋白是Ferritin蛋白，或，乙肝核心抗原(HBcAg)或其变体。
在一个或多个实施方案中，(a)和(b)共价连接。优选地，所述共价连接是肽键或通过SpyCatcher-SpyTag共价连接形成的异肽键。
在一个或多个实施方案中，所述CD24是小鼠CD24或人CD24。
在一个或多个实施方案中，所述CD24具有SEQ ID NO：1或2所示的序列。
在第一方面的一些实施方案中，所述免疫原性复合物是融合多肽，包含：(a)CD24或其免疫原性片段以及(b)乙肝核心抗原(HBcAg)或其变体，其中，所述HBcAg变体是(1)HBcAg的C末端缺失34或更少个氨基酸的变体，或(2)HBcAg在第78位氨基酸之后具有插入序列的并在C末端缺失34或更少个氨基酸的变体。
在一个或多个实施方案中，所述插入序列是((G)oS(G)pT)q，其中o、p和q各自独立是选自0、1、2、3、4、5和6的整数。优选地，q是1，o和p各自独立是选自0、1、2、3的整数。更优选地，q是1，o和p是4。
优选地，所述HBcAg变体具有SEQ ID NO：6或7所示序列。
在一个或多个实施方案中，所述CD24或其免疫原性片段位于HBcAg或其变体的第78位氨基酸之后或所述插入序列之后，将HBcAg或其变体分成位于N端的A和位于C端的B两个部分。优选地，所述CD24或其免疫原性片 段位于SEQ ID NO：6的第78位氨基酸之后或SEQ ID NO：7的第88位氨基酸之后。
在一个或多个实施方案中，所述CD24或其免疫原性片段的C端与所述B部分通过接头连接。在一些实施方案中，接头是((G)mS)n，其中m和n各自独立是选自0、1、2、3、4、5、6、7、8、9和10的整数；接头优选是GGGGGSGGGGS。
在一个或多个实施方案中，所述免疫原性复合物的序列如SEQ ID NO：9所示。
在第一方面的另一些实施方案中，所述免疫原性复合物包含通过SpyCatcher-SpyTag共价连接的：
(a)与SpyCatcher连接的CD24或其免疫原性片段，和(b)与SpyTag连接的Ferritin蛋白，或者
(a’)与SpyTag连接的CD24或其免疫原性片段，和(b’)与SpyCatcher连接的Ferritin蛋白。
在一个或多个实施方案中，(b)或(b’)自组装形成纳米颗粒载体。
在一个或多个实施方案中，所述各连接各自独立是直接连接或通过接头连接。在一些实施方案中，接头是((G)mS)n，其中m和n各自独立是选自0、1、2、3、4、5、6、7、8、9和10的整数；接头优选是GGGGGSGGGGSGGGGS。
在一个或多个实施方案中，所述Ferritin蛋白来自人、马、牛蛙或细菌。优选地，所述Ferritin蛋白具有SEQ ID NO：3所示的序列。
在一个或多个实施方案中，所述SpyTag如SEQ ID NO：4所示。
在一个或多个实施方案中，所述SpyCatcher如SEQ ID NO：5所示。
在一个或多个实施方案中，所述CD24是小鼠CD24或人CD24。优选地，所述CD24具有SEQ ID NO：1或2所示的序列。
在一个或多个实施方案中，(a)如SEQ ID NO：13所示；(b)如SEQ ID NO：11所示。
本发明另一方面还提供一种组合物，包含(1)与SpyCatcher连接的CD24或其免疫原性片段，和与SpyTag连接的Ferritin蛋白，和/或(2)与SpyTag 连接的CD24或其免疫原性片段，和与SpyCatcher连接的Ferritin蛋白。所述各连接各自独立是直接连接或通过接头连接。
在一些实施方案中，接头是((G)mS)n，其中m和n各自独立是选自0、1、2、3、4、5、6、7、8、9和10的整数；接头优选是GGGGGSGGGGSGGGGS。
在一个或多个实施方案中，所述Ferritin蛋白来自人、马、牛蛙或细菌。优选地，所述Ferritin蛋白具有SEQ ID NO：3所示的序列。
在一个或多个实施方案中，所述SpyTag如SEQ ID NO：4所示。
在一个或多个实施方案中，所述SpyCatcher如SEQ ID NO：5所示。
在一个或多个实施方案中，所述CD24是小鼠CD24或人CD24。优选地，所述CD24具有SEQ ID NO：1或2所示的序列。
在一个或多个实施方案中，所述组合物包含具有SEQ ID NO：11和13所示序列的多肽。
本发明另一方面还提供核酸分子，其具有编码选自以下任一项的多肽的序列：
(1)本文第一方面中所述的包含由肽键连接的(a)CD24或其免疫原性片段以及(b)载体蛋白，所述载体蛋白优选乙肝核心抗原(HBcAg)或其变体的融合多肽，
(2)本文第一方面中所述的与SpyCatcher连接的CD24或其免疫原性片段，和/或与SpyTag连接的载体蛋白，优选本文第一方面中所述的与SpyTag连接的Ferritin蛋白，
(3)本文第一方面中所述的与SpyTag连接的CD24或其免疫原性片段，和/或与SpyCatcher连接的载体蛋白，优选本文第一方面中所述的与SpyCatcher连接的Ferritin蛋白，
(4)(1)-(3)的互补序列。
本发明还提供核酸构建物，其具有本文任一实施方案所述的核酸分子。所述核酸构建物是克隆载体或表达载体。
本发明还提供宿主细胞，其包含本文所述的核酸构建物。
在一个或多个实施方案中，所述宿主细胞包括但不限于大肠杆菌、毕赤酵母、汉逊酵母、酿酒酵母、昆虫细胞及哺乳动物细胞，例如大肠杆菌BL21(DE3)、Rosseta或JM109菌株。
本发明第二方面还提供一种免疫原性组合物，其包含本文任一实施方案所述的免疫原性复合物和佐剂。
在一个或多个实施方案中，所述免疫原性组合物是纳米颗粒疫苗。
在一个或多个实施方案中，所述佐剂为氢氧化铝。
本发明还提供一种药物组合物，其包含：本文任一实施方案所述的免疫原性复合物或免疫原性组合物，和药学上可接受的辅料。
本发明还提供一种试剂盒，其包含本文任一实施方案所述的免疫原性组合物或药物组合物，以及用于施用它们的容器。
本发明还提供本文任一实施方案所述的免疫原性复合物或免疫原性组合物在制备药物中的用途。
在一个或多个实施方案中，其中所述药物用于预防、减轻或者治疗CD24相关疾病或病症。
在一个或多个实施方案中，所述CD24相关疾病或病症是受益于CD24被抑制的疾病或病症。
在一个或多个实施方案中，所述CD24相关疾病是癌症，优选卵巢癌、乳腺癌、胰腺癌、肾癌、肺癌、鼻咽癌或肝癌。
在一个或多个实施方案中，所述CD24相关病症是肿瘤侵袭、转移或免疫逃逸。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将 对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1：SpyTag-Ferritin纯化SDS-page。M：预染蛋白maker；lane1：破菌全菌；lane2：破菌上清；lane3：加热后上清；lane4：Q-FF洗脱。
图2：Spycatcher-mCD24纯化SDS-page。M：预染蛋白maker；lane1：破菌上清；lane2：Q-FF洗脱；lane3：phenyl-Hisub流穿；lane4：Q-HP洗脱。
图3：mCD24-Ferritin纯化SDS-page。M：预染蛋白maker；lane1：破菌上清；lane2：DEAE FF流穿；lane3：Poros-50HQ流穿。
图4：HBcAg-mCD24纯化SDS-page。M：预染蛋白maker；lane1：破菌全菌；lane2：破菌上清；lane3：Poros-50HQ洗脱；lane4：Chromdex-200排阻峰。
图5：SpyTag-Ferritin和Spycatcher-mCD24的组装SDS-page。M：预染蛋白maker；lane1：SpyTag-Ferritin；lane2：Spycatcher-mCD24；lane3：0.5∶1；lane4：1∶1；lane5：1∶0.5；lane6：1∶0.25。
图6：SpyTag-Ferritin和Spycatcher-mCD24的组装后的纯化SDS-page。M：预染蛋白maker；lane1：连接产物超滤浓缩；lane2：Chromdex-200排阻峰。
图7：mCD24-Ferritin电镜观察
图8：SpyTag-Ferritin电镜观察
图9：SpymCD24-Ferritin电镜观察
图10：HBcAg-mCD24电镜观察
图11：小鼠肿瘤大小
具体实施方式
除非另有定义，否则与本发明关联使用的科学及技术术语应具有本领域普通技术人员通常所了解的含义。通常，本文所述的与细胞及组织培养、分子生物学、免疫学、微生物学、遗传学、蛋白质与核酸化学、杂交、医学及药物化学关联使用的命名及其技术是本领域公知和常用的。本文中所引用的所有出版物、专利及专利申请(无论上文或下文)均以全文引用的方式并入本文中。
本领域的共识是，CD24无法作为免疫原用于肿瘤疫苗，这是因为：CD24分子为机体自身多肽，不具有异物性的特征；成熟的CD24分子仅约30个氨基酸，分子量小，因此单独的CD24分子无法做为疫苗抗原使用。本发明实施例中也验证了单独的CD24分子无法诱导抗体生成。
发明人经过长期深入的研究，发现在与载体蛋白偶联(例如共价连接)后，CD24具有突出的免疫原性，可以制备得到肿瘤疫苗，该疫苗可以有效预防和治疗以乳腺癌为代表的CD24相关疾病。
本文所述的CD24可以来自任何物种，优选小鼠或人来源的CD24(SEQ ID NO：1或2)。本文的CD24还包括功能变体。多肽的变体与其野生型相应物(例如SEQ ID NO：1或2)相比在氨基酸水平具有例如至少80％、85％、90％、95％、97％或者99％相同性。
本文中，CD24的功能活性变体包含天然发生的功能活性变体，如等位基因变体和物种变体以及可通过例如诱变技术或者通过直接合成方法产生的非天然发生的功能活性变体。优选CD24功能变体与SEQ ID NO：1或2所示肽具有大约如1、2、3、4或5个氨基酸残基不同，且仍保留抗原性CD24生物学活性。变异的位点可以发生在所述肽中的任何位置，只要所述生物学活性与SEQ ID NO：1或2所示肽基本上相似即可。
可以在本发明的肽之一中进行的氨基酸取代的类型是保守氨基酸取代。“保守氨基酸取代”是其中氨基酸残基由具有相似化学性质(如电荷或疏水性)的侧链R基团的另一氨基酸残基取代。通常，保守氨基酸取代基本上不改变蛋白质的功能性质。具有相似化学性质侧链的氨基酸组的实例包括：1)脂肪族侧链：甘氨酸、丙氨酸、缬氨酸、亮氨酸和异亮氨酸；2)脂肪族-羟基侧链：丝氨酸和苏氨酸；3)含有酰胺的侧链：天冬酰胺和谷氨酰胺；4)芳族侧链：苯丙氨酸、酪氨酸和色氨酸；5)碱性侧链：赖氨酸、精氨酸和组氨酸；6)酸性侧链：天冬氨酸和谷氨酸；及7)含硫侧链：半胱氨酸和甲硫氨酸。优选的保守氨基酸取代组是：缬氨酸-亮氨酸-异亮氨酸，苯丙氨酸-酪氨酸，赖氨酸-精氨酸，丙氨酸-缬氨酸，谷氨酸-天冬氨酸，以及天冬酰胺-谷氨酰胺。
本文的CD24还包含其免疫原性片段。在知晓了本文所发现的CD24可以作为免疫原制备肿瘤疫苗的基础上，本领域技术人员可以根据CD24的序列容 易地确定具有免疫原性的CD24的片段。
本发明第一方面提供一种免疫原性复合物，其包含：(a)CD24或其免疫原性片段以及(b)载体蛋白。a组分和b组分之间优选共价连接，可以是肽键或通过SpyCatcher-SpyTag共价连接形成的异肽键。因此，免疫原性复合物可以是能融合表达的多肽或者是通过SpyCatcher-SpyTag共价连接的复合物。
本发明还涉及所述免疫原性复合物的制备方法，包括：融合表达所述包含CD24或其免疫原性片段以及载体蛋白的融合多肽，或者，分别提供(例如分别表达)(a)和(b)组分后通过组分间的化学交联或共价连接(例如异肽键)形成免疫原性复合物。
作为示例性的实施方案，发明人选用能形成纳米颗粒的载体蛋白。如本发明实施例中，载体蛋白是Ferritin蛋白，或乙肝核心抗原(HBcAg)或其变体。
因此，在一些实施方案中，本发明提供一种融合多肽形式的免疫原性复合物，包含：(a)CD24或其免疫原性片段以及(b)乙肝核心抗原(HBcAg)或其变体。乙型肝炎核心抗原(HBcAg)是183个氨基酸的多肽。HBcAg及其截短变体均可作为CD24纳米颗粒疫苗的载体蛋白。
可用于本发明中的特别感兴趣的HBcAg变体是其中一或多个天然存在的半胱氨酸残基已经被缺失或取代的那些变体。此外，本发明也可以使用加工形式的HBcAg，其缺少乙型肝炎核心抗原前体蛋白的N末端前导序列，特别是当HBcAg是在未发生加工的条件下产生的情况中(例如在细菌系统中表达)。
本发明的其它HBcAg变体包括：i)与野生型HBcAg氨基酸序列之一或者其亚部分具有至少80％、85％、90％、95％、97％或99％相同性的多肽序列，使用常规的已知计算机程序确定；ii)C-末端截短突变体，包括其中在C末端缺失34或更少(例如1、5、10、15、20、25、30)个氨基酸的突变体；ii)N-末端截短突变体，包括其中从N末端已经除去1、2、5、7、9、10、12、14、15或17个氨基酸的突变体；iii)在第78位氨基酸之后具有插入序列的并在C末端缺失34或更少(例如1、5、10、15、20、25、30)个氨基酸的HBcAg变体。适用于本发明中的HBsAg可衍生自任何生物体。适用于本发明中的HBsAg可衍生自任何生物体，只要其能形成病毒样颗粒并用作免疫原性载体即可。
本发明还涉及所述免疫原性复合物的制备方法，包括：表达所述包含CD24或其免疫原性片段以及HBcAg或其变体的融合多肽。
在一些实施方案中，所述HBcAg变体具有SEQ ID NO：6或7所示序列或与其具有至少80％序列相同性的功能变体；所述CD24或其免疫原性片段位于SEQ ID NO：6或其功能变体的第78位氨基酸之后或SEQ ID NO：7或其功能变体的第88位氨基酸之后。在优选实施方案中，所述免疫原性复合物的序列如SEQ ID NO：9所示或与其具有至少80％序列相同性的功能变体。
本文的融合多肽可以发生序列变化，得到的突变体与该融合多肽(例如SEQ ID NO：9)具有至少80％，优选至少85％，优选至少90％，优选至少95％，优选至少97％的序列相同性并保留该融合多肽的生物学活性(如免疫原性)。可采用例如NCBI的BLASTp计算两条比对的序列之间的序列相同性。
在另一些实施方案中，本发明提供载体蛋白为Ferritin蛋白的免疫原性复合物。所述免疫原性复合物包含通过SpyCatcher-SpyTag共价连接的：(a)与SpyCatcher连接的CD24或其免疫原性片段，和(b)与SpyTag连接的Ferritin蛋白，或者(a’)与SpyTag连接的CD24或其免疫原性片段，和(b’)与SpyCatcher连接的Ferritin蛋白。Ferritin蛋白来自嗜热古细菌(Pyrococcus furiosus)的铁蛋白，其是由24个亚基组成的八面体，优选具有SEQ ID NO：3所示的序列或与其具有至少80％序列相同性的功能变体。Ferritin蛋白可以自组装形成纳米颗粒载体。此外，包含未发生SpyCatcher-SpyTag共价连接的上述(a)和(b)或(a’)和(b’)的组合物也在本发明的范围内。本发明还涉及所述免疫原性复合物的制备方法，包括：表达(a)和(b)或(a’)和(b’)中的多肽，纯化后共孵育，自组装得到所述免疫原性复合物。
在一些实施方案中，所述SpyCatcher位于CD24或Ferritin蛋白的N端，所述SpyTag位于CD24或Ferritin蛋白的的N端。示例性地，所述SpyTag含有SEQ ID NO：4所示的氨基酸序列或与其具有至少80％序列相同性的功能变体，所述SpyCatcher含有SEQ ID NO：5所示的氨基酸序列或与其具有至少80％序列相同性的功能变体。
本文中，一条多肽中各组分(例如CD24和载体蛋白、SpyTag和CD24、SpyCatcher和CD24、SpyTag和载体蛋白、SpyCatcher和载体蛋白)之间的连 接可以是直接连接或通过接头连接。所述接头的氨基酸是不具有除连接以外的额外功能(例如蛋白定位、酶切位点等)的无意义多肽。是否使用接头以及接头序列可由本领域技术人员根据需要选择。在优选实施方案中，接头是((G)mS)n，其中m和n各自独立是选自0、1、2、3、4、5、6、7、8、9和10的整数。
在一些实施方式中，(a)组分为SpyCatcher-mCD24，其含有SEQ ID NO：13所示的氨基酸序列或与其具有至少80％序列相同性的功能变体；(b)组分为SpyTag-Ferritin，其含有SEQ ID NO：11所示的氨基酸序列或与其具有至少80％序列相同性的功能变体。
本文的连接有SpyCatcher或SpyTag的各多肽(例如SpyCatcher-CD24、SpyCatcher-载体蛋白、SpyTag-CD24、SpyTag-载体蛋白)可以发生序列变化，得到的突变体与相应多肽具有至少80％，优选至少85％，优选至少90％，优选至少95％，优选至少97％的序列相同性并保留该多肽的生物学活性(如与对应的SpyCatcher或SpyTag共价连接的活性、载体蛋白的自组装活性、发生SpyCatcher和SpyTag共价连接后的免疫原性等)。可采用例如NCBI的BLASTp计算两条比对的序列之间的序列相同性。
如上所述，所述免疫原性复合物的制备方法包括表达相应多肽的步骤。本文中，“表达”可通过孵育宿主细胞实现，该宿主细胞包含具有待表达多肽的编码序列的表达载体。因此，本发明还涉及本文所述多肽的编码序列和含有该编码序列的核酸构建物，所述核酸构建物可以是克隆载体、整合载体或表达载体。所述表达载体还含有多肽表达所需的其他组件，例如启动子、终止子等。宿主细胞、表达载体和组件均是本领域所熟知。示例性的表达载体包括pet24a。
本文所述宿主细胞可以是动物、植物或微生物细胞，包括但不限于大肠杆菌、毕赤酵母、汉逊酵母、酿酒酵母、昆虫细胞及哺乳动物细胞，例如大肠杆菌BL21(DE3)、Rosseta或JM109菌株。
本发明还涉及免疫原性组合物，其是包含如上所述的免疫原性复合物和佐剂的纳米颗粒疫苗。此外，本发明还提供药物组合物，其包含：本文所述的免疫原性复合物或免疫原性组合物，以及药学上可接受的辅料。
适用于本发明疫苗的佐剂包括可增强针对CD24或其免疫原性片段的抗 体反应的佐剂，以及可增强细胞介导的针对CD24或其免疫原性片段的反应的佐剂。这些佐剂均是本领域所熟知并可通过商业渠道获得的。在一些实施方式中，所述佐剂选自Sigma Adiuvant Systerm、AddaVax、角鲨烯、胞壁酰二肽、MF59、AS03、单磷脂酰脂质A，鞭毛蛋白、CPG、以及铝或钙盐的小分子中的一种或多种。其中，优选的佐剂为氢氧化铝。
药学上可接受的辅料成分的示例包括结合剂(糖浆、阿拉伯树胶、明胶、山梨醇、黄芪胶(tragacanth)、聚乙烯吡咯烷酮等)、填充剂(乳糖、蔗糖、淀粉、磷酸钙、山梨糖醇、甘氨酸等)、润滑剂(硬脂酸镁、滑石、聚乙二醇等)、崩解剂(淀粉、微晶纤维素(microcrystalline cellulose)等)、湿润剂(十二烷基硫酸钠(sodium lauryl sulphate)等)、悬浮剂(山梨糖醇、糖浆、甲基纤维素、葡萄糖浆(glucose syrup)、明胶、氢化食用脂肪等)、乳化剂(卵磷脂、山梨醇单油酸酯、阿拉伯树胶等)、非水性载体(杏仁油、分馏椰子油或甘油、丙二醇、乙醇等疏水酯等)、防腐剂(对羟基苯甲酸甲酯或对羟基苯甲酸丙酯、山梨酸等)、芳香剂(合成香料、天然香料等)、甜味剂(蔗糖、甜叶菊、木糖醇等)、pH调节剂(碳酸氢钠、碳酸钾等)、粉剂(色素、染料、树脂等)、增稠剂(阿拉伯树胶、甲基纤维素等)、抗氧化剂(维生素C、维生素E等)等等。
本发明的免疫原性组合物、药物组合物和制备其的方法为本领域技术人员显而易见。这种组合物和制备其的方法可见于例如Remington’s Pharmaceutical Sciences，19th Edition(Mack Publishing Company，1995)。药物组合物优选在GMP条件下生产。
本发明的药物组合物可以作为单一单位剂量或者多个单一单位剂量形式大批量制备、包装或者销售。如本文所用，“单位剂量”是指包含预定量活性成分的药物组合物的个别量。所述活性成分的量通常等于施用给对象的活性成分的剂量或者这种剂量的方便分数，例如这种剂量的一半。
本发明的疫苗和药物组合物典型地被配制用于肠胃外施用。如本文所用，肠胃外施用包括但不限于通过注射所述组合物施用药物组合物、通过手术切口应用所述组合物、通过组织穿透非手术创口应用所述组合物等。特别地，肠胃外施用包括但不限于皮下、腹膜内、肌内、胸骨内、静脉内、动脉内、鞘内、 心室内、尿道内、颅内、滑膜内注射或者输注；以及肾透析灌注技术。典型的免疫接种是通过肌内途径的疫苗接种，但本发明还考虑了口腔、皮下(SC)、鼻腔(IN)、静脉内(IV)、腹膜内(IP)或真皮内(ID)施用实现。
适于肠胃外施用的药物组合物的配制典型通常包含活性成分可接受的载体，如无菌水或无菌等渗盐水。这种配制物可以适于推注施用或者连续施用的形式制备、包装或者销售。注射用配制物可以单位剂量形式如在含有防腐剂的安瓿或在多剂量容器中制备、包装或销售。肠胃外施用的配制物包括但不限于悬浮液、溶液、在油或水性载体中的乳状液、糊剂等。这种配制物可进一步包含一或多种其它成分，包括但不限于悬浮剂、稳定剂或者分散剂。在肠胃外施用的配制物的一个实施方案中，活性成分以干燥形式提供(即粉末或颗粒)，在肠胃外施用之前用合适的载体(如无菌无热原水)重建之后肠胃外施用所述重建的组合物。肠胃外施用的配制物可以配制成立即、缓释、持续、脉冲、控制、靶向以及程序化释放的释放形式。示例性的本发明药物组合物是无菌水溶液，pH范围在大约5.0-6.5，包含约0.1mg/mL至大约20mg/mL本发明的免疫原性组合物、约1mmol至约100mmol的组氨酸缓冲液，约0.01mg/mL至约10mg/mL聚山梨酯80、约100mmol至约400mmol海藻糖，以及约0.01mmol至约1.0mmol的EDTA二钠二水合物。
上述免疫原性组合物或药物组合物是以与剂量配方相容的方式，以及诸如治疗有效量和免疫原性有效量的用量被施用的。“药物有效剂量”或者“治疗有效剂量”是治疗或预防或减轻对象的一或多种CD24相关疾病或症状需要的剂量。所述药物有效剂量依赖于施用的特定化合物、症状的严重性、对象对于副作用的易感性、疾病的类型、使用的组合物、施用途径、治疗的哺乳动物类型、特定哺乳动物的生理特性如健康和生理状况、同时服药情况、个体免疫系统合成抗体的能力、希望的保护作用程度以及本领域技术人员已知的其它因素。出于预防目的，选择的每个剂量中肽的量是在典型的疫苗接种个体中诱导免疫保护性应答且无明显不利副作用的量。在最初的免疫接种之后，所述对象可以接受一或多次足够间隔时间的加强免疫。所述有效量优选经皮下或口鼻或肌内施用(一次或多次)。
应理解对于任何特定患者的特定剂量水平根据各种因素确定，包括应用的 特定化合物的活性、年龄、体重、一般健康状况、性别、饮食、施用时间、施用途径及排泄率，药物组合及进行治疗的特定疾病的严重性。例如，本发明的CD24免疫原性组合物或者药物组合物可以以如下剂量施用给对象：约0.1μg至约5mg，如约0.1μg至约5μg，约5μg至约10μg，约10μg至约25μg，约25μg至约50μg，约50μg至约100μg，约100μg至约500μg，约500μg至约1mg，约1mg至约2mg，任选地加强剂量在例如1周、2周、3周、4周、2个月、3个月、6个月和/或1年之后。
本发明还涉及试剂盒，其包含本文所述的免疫原性组合物或药物组合物，以及用于施用所述免疫原性组合物或药物组合物的容器。容器优选为医用注射器。
本发明再一方面还涉及如上所述的免疫原性复合物或免疫原性组合物在制备用于预防、减轻或者治疗CD24相关疾病或病症的药物中的用途。
本发明进一步提供了降低或消除受试者患CD24相关疾病或病症的可能性或治疗受试者的CD24相关疾病或病症的方法，包括给所述受试者施用有效量的根据本发明的免疫原性复合物、免疫原性组合物或药物组合物。
另一方面，本发明提供了在对象体内诱导针对自身CD24免疫应答的方法，包括给所述对象施用治疗或免疫学有效量的本发明的免疫原性复合物、免疫原性组合物或药物组合物。
在本发明的用途或方法的一些方面中，CD24相关疾病或病症是例如其中CD24活性被抑制或者CD24与受体相互作用被抑制是有益的疾病或病症。CD24相关疾病包括但不限于癌症，例如卵巢癌、乳腺癌、胰腺癌、肾癌、肺癌、鼻咽癌或肝癌；CD24相关病症包括但不限于肿瘤侵袭、转移或免疫逃逸。
在一些实施方案中，本文的CD24免疫原性复合物、免疫原性组合物或者药物组合物可以与另一药物一起施用，两者可以以一定顺序相继施用或者同时施用。
在本发明的应用或方法中，受试者特别是哺乳动物，优选为灵长类动物，更优选为人。受试者包括健康对象、CD24相关疾病或病症患者、康复对象等。
除非另有指示，否则本发明的方法及技术一般根据本领域公知的和常规的方法来进行且如本说明书中所引用及论述的各种一般及更特定的参考文献中 所述来进行。例如参见Sambrook J.及Russell D.Molecular Cloning：A Laboratory Manual，第3版(2000)。根据制造商的说明书进行酶促反应及纯化技术，通常如本领域或如本文所述完成。
实施例
1、序列及表达载体构建
1.1、小鼠CD24序列(mCD24)
蛋白质序列：NQTSVAPFPGNQNISASPNPTNATTRG(SEQ ID NO：1)
1.2、人CD24序列(hCD24)
蛋白质序列：SETTTGTSSNSSQSTSNSGLAPNPTNATTKV(SEQ ID NO：2)
1.3、Ferritin序列
蛋白质序列：
1.4、SpyTag序列
蛋白质序列：AHIVMVDAYKPTK(SEQ IDNO：4)
1.5、Spycatcher序列
蛋白质序列：
1.6、HBcAg序列
HBcAg 1-149aa蛋白序列：
HBcAg 1-149aa改造蛋白序列：
HBcAg1-149aa改造核苷酸序列：

1.7、HBcAg-mCD24序列
蛋白质序列：
核苷酸序列：
1.8、SpyTag-Ferritin序列
蛋白质序列：
核苷酸序列：
1.9、Spycatcher-mCD24序列
蛋白质序列：
核苷酸序列：
1.10、mCD24-Ferritin序列
蛋白质序列：
核苷酸序列：
SpyTag-Ferritin、Spycatcher-mCD24a、mCD24-Ferritin和HBcAg-mCD24和DNA序列均通过Nde1和EcoR1酶切位点克隆至pet24a(+)表达载体上。
2、纳米颗粒抗原的表达和纯化
2.1、诱导表达
取SpyTag-Ferritin--pet24a、Spycatcher-mCD24-pet24a、mCD24-Ferritin-pet24a和HBcAg-mCD24-pet24a质粒各1μL，分别转化大肠杆菌BL21(DE3)感受态细胞，吸取200μL菌液涂布在含卡那霉素的LB平板上，置37℃培养箱中培养过夜。于平板上挑取单克隆菌落，接种于含5mL LK培养基的试管中培养至菌体OD600＝0.6-0.8，取1mL菌液接种到含1.5L LK培养基的三角瓶中，37℃培养至菌体OD600＝1.0左右，加入终浓度为1mM IPTG 37℃诱导表达6h。
2.2、分离纯化
2.2.1、SpyTag-Ferritin分离纯化
诱导后的菌液5000rpm离心30分钟收菌体，破菌缓冲液(20mM Tris，50mM Nacl，PH7.0-8.0)重悬菌体，高压均质匀浆机800bar破碎菌体，12000rpm离心20分钟，收集破菌上清；将破菌上清于75℃加热15分钟，12000rpm离心20分钟，收集上清液；加热后离心上清液进行Q-FF阴离子交换层析纯化，上样、平衡洗柱后，以洗脱缓冲液(20mM Tris，500mM Nacl，PH7.0-8.0)进行梯度洗脱，目的蛋白存在于洗脱液中(图1)。
2.2.2、Spycatcher-mCD24分离纯化
诱导后的菌液5000rpm离心30分钟收菌体，破菌缓冲液(20mM Tris，50mM Nacl，PH7.0-8.0)重悬菌体，高压均质匀浆机800bar破碎菌体，12000rpm离心20分钟，收集破菌上清；破菌上清进行Q-FF阴离子交换层析，以20mM Tris，500mM Nacl，PH7.0-8.0溶液梯度洗脱，目的蛋白可结合到Q-FF层析柱，并在0.1-0.3M Nacl之间被洗脱；将Q-FF柱层析含有目的蛋白的洗脱液，加硫酸铵至终浓度为0.5M，进行Phenyl-Hisub疏水层析，以20mM Tris，50mM Nacl，PH7.0-8.0缓冲液洗脱，目的蛋白未挂柱，存在于流穿液中，大部分杂蛋白挂柱，低盐洗脱后可去除；取Phenyl-Hisub流穿液进行Q-HP阴离子交换层析，20mM Tris，500mM Nacl，PH7.0-8.0溶液梯度洗脱，目的蛋白存在于洗脱液中(图2)。
2.2.3、mCD24-Ferritin分离纯化
诱导后的菌液5000rpm离心30分钟收集菌体，破菌缓冲液(50mM Tris，50mM Nacl，PH7.0-8.0)重悬菌体，高压均质匀浆机800bar破碎菌体，12000rpm离心20分钟，收集破菌上清；破菌上清采用DEAE FF阴离子交换层析(上样缓冲液50mmTris，50mmNacl，PH7.0-8.0)，洗脱缓冲液(洗脱缓冲液50mmTris，1000mmNacl，0.03％Tween80，PH7.0-8.0)采用0％-50％线性洗脱，目的蛋白基本不挂柱，主要存在于流穿中；第二步采用Poros-50HQ阴离子交换层析，DEAE FF阴离子交换层析的流穿上样，洗脱缓冲液(洗脱缓冲液50mmTris，1000mmNacl，0.03％Tween80，PH7.0-8.0)阶段洗脱，目的蛋白基本不挂柱，主要存在于流穿中(图3)。
2.2.4、HBcAg-mCD24分离纯化
诱导后的菌液5000rpm离心30分钟收集菌体，破菌缓冲液(50mM Tris，50mM Nacl，PH7.0-8.0)重悬菌体，高压均质匀浆机800bar 3遍破碎菌体，12000rpm离心20分钟，收集破菌上清；破菌上清采用Poros-50HQ阴离子交换层析(上样缓冲液50mmTris，50mmNacl，PH7.0-8.0)，洗脱缓冲液(洗脱缓冲液50mmTris，1500mmNacl，0.03％Tween80，PH7.0-8.0)采用15％和30％两阶段洗脱，15％和30％洗脱峰中均有目的蛋白，其中30％的洗脱峰中目的蛋白的纯度较高；第二步采用Chromdex-200体积排阻层析，上样缓冲液为PBS，以Poros-50HQ阴离子交换层析30％的洗脱组分上样，收集排阻峰(图4)。
2.2.5、SpyTag-Ferritin和Spycatcher-mCD24的组装
2.2.5.1、SpyTag-Ferritin和Spycatcher-mCD24组装条件优化
将SpyTag-Ferritin和Spycatcher-mCD24的蛋白浓度分别调整至0.5mg/ml左右。按照下表中所示比例将样品混合均匀，4℃连接过夜，SDS-PAGE电泳分析可见SpyTag-Ferritin和Spycatcher-mCD24比例在1∶0.25-0.5之间的连接效率较高，SpyTag-Ferritin和Spycatcher-mCD24连接后的蛋白命名为SpymCD24-Ferritin(表1和图5)。
表1 SpyTag-Ferritin和Spycatcher-mCD24的组装
2.2.5.2、SpyTag-Ferritin和Spycatcher-mCD24的组装后的纯化
按照SpyTag-Ferritin和Spycatcher-mCD24的蛋白浓度分别调整至0.5mg/ml左右。按照1∶0.25的体积比4℃连接过夜，连接产物经超滤浓缩后，采用Chromdex-200体积排阻层析纯化，收集排阻峰(图6)。
3、电镜观察
将mCD24-Ferritin、SpyTag-Ferritin、SpymCD24-Ferritin和HBcAg-mCD24纯化后的样品取100μL进行透射电镜观察。mCD24-Ferritin、SpyTag-Ferritin、SpymCD24-Ferritin和HBcAg-mCD24均呈纳米颗粒形态(图7-图10)。
4、动物实验
4.1、小鼠免疫
纯化后的SpymCD24-Ferritin、mCD24-Ferritin、HBcAg-mCD24和mCD24多肽(免疫剂量相当于1.4×1015个mCD24分子)分别与200μg/mL氢氧化铝佐剂等体积混合均匀，4℃吸附过夜，每组8只小鼠，每只小鼠腹腔免疫0.5mL，共免疫三次，两次免疫间隔4周，每次免疫后4周尾静脉取血，用ELISA方法检测抗体滴度(表2)。
表2，ELISA抗体滴度
4.2、小鼠肿瘤模型
经SpymCD24-Ferritin、mCD24-Ferritin、HBcAg-mCD24和mCD24免疫三次后，每只小鼠皮下接种1.0×106个4T1小鼠乳腺癌细胞，接种后第7天处死小鼠，摘取肿瘤组织并称重。结果显示，与mCD24对照组相比，SpymCD24-Ferritin和HBcAg-mCD24组的小鼠肿瘤重量明显小于mCD24对照组，表明小鼠免疫后产生的针对mCD24的抗体可抑制肿瘤细胞的生长(表3和图11)。
表3，肿瘤重量

Claims (10)

  1. 一种免疫原性复合物，其包含：(a)CD24或其免疫原性片段以及(b)载体蛋白，
    优选地，所述载体蛋白是能形成纳米颗粒的载体蛋白，和/或
    优选地，(a)和(b)共价连接；更优选地，所述共价连接是肽键或通过SpyCatcher-SpyTag共价连接形成的异肽键，和/或
    优选地，所述CD24是小鼠CD24或人CD24。
  2. 如权利要求1所述的免疫原性复合物，其特征在于，所述免疫原性复合物是融合多肽，包含：(a)CD24或其免疫原性片段以及(b)乙肝核心抗原(HBcAg)或其变体，其中，所述HBcAg变体是(1)HBcAg的C末端缺失34或更少个氨基酸的变体，或(2)HBcAg在第78位氨基酸之后具有插入序列的并在C末端缺失34或更少个氨基酸的变体，
    优选地，所述插入序列是((G)oS(G)pT)q，其中o、p和q各自独立是选自0、1、2、3、4、5和6的整数；更优选地，q是1，o和p各自独立是选自0、1、2、3的整数，
    优选地，所述CD24或其免疫原性片段位于HBcAg或其变体的第78位氨基酸之后或所述插入序列之后，将HBcAg或其变体分成位于N端的A和位于C端的B两个部分，
    优选地，所述CD24或其免疫原性片段的C端与所述B部分通过接头连接；更优选地，接头是((G)mS)n，其中m和n各自独立是选自0、1、2、3、4、5、6、7、8、9和10的整数。
  3. 如权利要求2所述的免疫原性复合物，其特征在于，
    所述HBcAg变体具有SEQ ID NO：6或7所示序列，所述CD24或其免疫原性片段位于SEQ ID NO：6的第78位氨基酸之后或SEQ ID NO：7的第88位氨基酸之后，
    优选地，所述免疫原性复合物的序列如SEQ ID NO：9所示。
  4. 如权利要求1所述的免疫原性复合物，其特征在于，所述免疫原性复合物包含通过SpyCatcher-SpyTag共价连接的：
    (a)与SpyCatcher连接的CD24或其免疫原性片段，和(b)与SpyTag连接的Ferritin蛋白，或者
    (a’)与SpyTag连接的CD24或其免疫原性片段，和(b’)与SpyCatcher连接的Ferritin蛋白，
    优选地，所述各连接各自独立是直接连接或通过接头连接；更优选地，接头是((G)mS)n，其中m和n各自独立是选自0、1、2、3、4、5、6、7、8、9和10的整数，
    所述Ferritin蛋白来自人、马、牛蛙或细菌。
  5. 如权利要求4所述的免疫原性复合物，其特征在于，
    所述Ferritin蛋白具有SEQ ID NO：3所示的序列，
    所述SpyTag具有SEQ ID NO：4所示的序列，
    所述SpyCatcher具有SEQ ID NO：5所示的序列，
    (a)组分如SEQ ID NO：13所示；(b)组分如SEQ ID NO：11所示。
  6. 一种组合物，包含(1)与SpyCatcher连接的CD24或其免疫原性片段，和与SpyTag连接的Ferritin蛋白，和/或(2)与SpyTag连接的CD24或其免疫原性片段，和与SpyCatcher连接的Ferritin蛋白，
    优选地，所述各连接各自独立是直接连接或通过接头连接；更优选地，所述接头是((G)mS)n，其中m和n各自独立是选自0、1、2、3、4、5、6、7、8、9和10的整数，
    优选地，所述Ferritin蛋白来自人、马、牛蛙或细菌；更优选地，所述Ferritin蛋白具有SEQ ID NO：3所示的序列，
    优选地，所述SpyTag具有SEQ ID NO：4所示的序列，
    优选地，所述SpyCatcher具有SEQ ID NO：5所示的序列，
    优选地，所述CD24是小鼠CD24或人CD24；更优选地，所述CD24具 有SEQ ID NO：1或2所示的序列，
    更优选地，所述组合物包含具有SEQ ID NO：11和13所示序列的多肽。
  7. 一种免疫原性组合物，其包含权利要求1-5中任一项所述的免疫原性复合物和佐剂，
    优选地，所述佐剂是氢氧化铝。
  8. 一种药物组合物，其包含：权利要求1-5中任一项所述的免疫原性复合物或权利要求7所述的免疫原性组合物，和药学上可接受的辅料。
  9. 一种试剂盒，其包含：权利要求7所述的免疫原性组合物或权利要求8所述的药物组合物，以及用于施用它们的容器。
  10. 权利要求1-5中任一项所述的免疫原性复合物或权利要求7所述的免疫原性组合物在制备药物中的用途，
    优选地，所述药物用于预防、减轻或者治疗CD24相关疾病或病症，
    更优选地，所述CD24相关疾病或病症是受益于CD24被抑制的疾病或病症。
PCT/CN2023/073026 2022-01-20 2023-01-19 Cd24疫苗 WO2023138636A1 (zh)

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