Novel vaccine delivery system

Abstract

The invention relates to the technical field of biological pharmacy, in particular to a novel vaccine delivery system. According to the vaccine delivery system, lymph node co-delivery of the antigen and the adjuvant can be achieved, the immunity promoting effect of the adjuvant is effectively enhanced, the specific immune response of the antigen is remarkably improved, and the vaccine delivery system is superior to a clinically common oil type composite adjuvant system. Particularly, the vaccine delivery system shows a remarkable anti-tumor effect in tumor prevention and treatment, and a new thought is provided for development and application of a novel vaccine system.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and in particular relates to a novel vaccine delivery system. Background Art

[0002] In anti-tumor treatment, tumor vaccines, as one of the three pillars of tumor immunotherapy, have been widely studied in the past 50 years. Tumor vaccines work by strengthening existing or inducing new T lymphocyte-mediated tumor antigen-specific responses. [1,2] Since the target antigen is mainly related to tumor cells, tumor vaccines can avoid off-target effects and have high safety. [3] ; Moreover, tumor vaccines produce active immune effects, which can induce systemic anti-tumor effects and memory protection effects through the activation of the immune system [3] , has become a promising therapy in the field of oncology, especially peptide tumor vaccines with good biocompatibility [4] However, the clinical benefits of tumor vaccines are currently limited. [5,6] Insufficient activation of tumor-specific immune response is the main factor, and the level of immune activation is restricted by the cross-presentation of tumor antigens. [7] In the tumor vaccine system, adjuvant efficiency and delivery method are key factors affecting cross-presentation [8,9] .

[0003] First of all, adjuvants play an important role in the efficacy of vaccines, determining the type and intensity of immune responses caused by vaccines. Among them, CpG oligodeoxynucleotide (CpG ODN) has been widely concerned and used as a highly effective TLR9 agonist, which can not only enhance humoral immune responses, but also greatly enhance cellular immune responses, the latter of which cannot be achieved by aluminum adjuvants. The receptors of CpG are located on the intracellular endosome membrane, so CpG adjuvants need to enter antigen-presenting cells to work. How to efficiently deliver them into cells is a key issue in the application of such adjuvants in vaccines.

[0004] The delivery method of tumor vaccines can change the distribution and pharmacokinetics of vaccine components and thus affect cross-presentation. In tumor vaccine programs with CpG as an adjuvant, a good vaccine carrier is not only important for delivering CpG into cells, but also for the delivery of antigens. First, the cytoplasmic delivery of exogenous antigens in antigen-presenting cells is an important prerequisite for cross-presentation.

[10] Secondly, co-delivery of antigens and CpG adjuvants through vaccine carriers is another important way to promote cross-presentation. Currently, there are many methods that can be used to promote vaccine delivery. Among them, nanocarriers, which are widely studied, can effectively encapsulate antigens and adjuvants to achieve co-delivery and can enhance cross-presentation to varying degrees. However, most of the nanoparticles will be degraded by lysosomes after entering the cell.

[11] , resulting in low efficiency of antigen cytoplasmic delivery, and the production cost, safety and biocompatibility of nanoparticles are also issues to be solved [12,13] . Summary of the invention

[0005] It has been reported that cell-penetrating peptides (CPPs) have high efficiency and biocompatibility in transmembrane delivery and are potential vaccine delivery vectors. Among them, the most widely used is the cationic peptide TAT from HIV virus. However, although TAT can enhance antigen cross-presentation

[14] However, the endocytic vesicle escape efficiency is also low, and most of the delivered antigens cannot be effectively released into the cytoplasm. [10,15,16] The efficiency of cytoplasmic delivery needs to be further enhanced to promote cross-presentation. The feasibility and specific efficiency of co-delivery of antigens and adjuvants based on cell-penetrating peptides remain to be studied.

[0006] This research team has developed an intracellular delivery system called eTAT in the early stage, which has good cytoplasmic delivery ability. eTAT is described in detail in Chinese patent application CN202011240557.4, international patent application WO2021135647A1, and Yu, S., Yang, H., Li, T. et al. Nat Commun 12, 5131 (2021). The inventors of this application unexpectedly found that eTAT has great potential in promoting antigen cross-presentation and studied its immunopotentiating effect as a vaccine delivery carrier. Thus, the present invention provides a new vaccine delivery system, which can significantly improve lymph node co-delivery and cross-presentation, showing a significant immunopotentiating effect, and ultimately inducing a potent antigen-specific immune response, with significant tumor prevention and treatment effects.

[0007] Compound

[0008] In a first aspect, the present invention provides a composite comprising:

[0009] (i) a protein component comprising a carrier peptide and a cargo peptide; wherein the carrier peptide comprises a multimerization domain sequence, a cell-penetrating peptide, a pH-sensitive fusion peptide and a protease recognition sequence; and the cargo peptide comprises an antigenic peptide;

[0010] (ii) a nucleic acid component comprising a CpG adjuvant.

[0011] I. Connection method

[0012] In certain embodiments, the protein component and the nucleic acid component are conjugated via electrostatic interactions.

[0013] In certain embodiments, the protein component comprises a carrier peptide and a cargo peptide from the N-terminus to the C-terminus. In certain embodiments, the protein component comprises a cargo peptide and a carrier peptide from the N-terminus to the C-terminus.

[0014] In certain embodiments, the cargo peptide and the antigenic peptide in the protein component can be fused or covalently linked or non-covalently linked.

[0015] In some embodiments, the carrier peptide is linked to the cargo peptide by fusion.

[0016] In certain embodiments, the cargo peptide in the protein component is optionally fused to the N-terminal or C-terminal of the carrier peptide through a joint. In certain embodiments, the cargo peptide in the protein component is optionally fused to the C-terminal of the carrier peptide through a joint. In certain embodiments, the joint is preferably a peptide joint, such as a flexible peptide joint, such as a peptide joint comprising one or more glycine and / or one or more serines.

[0017] In other embodiments, the carrier peptide is covalently linked to the cargo peptide.

[0018] In certain embodiments, the carrier peptide in the protein component is optionally connected to a first member of a protein-protein binding pair via a linker to form a first protein, and the cargo peptide is optionally connected to a second member of the protein-protein binding pair via a linker to form a second protein; wherein the first member and the second member are bound by a covalent bond. In this article, "protein-protein binding pair" has the meaning generally understood by those skilled in the art, which comprises two proteins (e.g., a first member (e.g., a first polypeptide) and a second homologous member (e.g., a second polypeptide)), which interact to form a covalent bond (e.g., an isopeptide bond) under conditions that achieve or promote the formation of a covalent bond (e.g., an isopeptide bond), wherein the term "homologous" refers to components that act together, i.e., react together to form a covalent bond (e.g., an isopeptide bond).

[0019] In certain embodiments, the first member of the protein-protein binding pair is optionally linked to the C-terminus of the carrier peptide via a linker. In certain embodiments, the second member of the protein-protein binding pair is optionally linked to the N-terminus of the cargo peptide via a linker.

[0020] In certain embodiments, the first member of the protein-protein binding pair is optionally linked to the N-terminus of the carrier peptide via a linker. In certain embodiments, the second member of the protein-protein binding pair is optionally linked to the C-terminus of the cargo peptide via a linker.

[0021] In certain embodiments, the first member of the protein-protein binding pair is optionally linked to the C-terminus of the carrier peptide via a linker. In certain embodiments, the second member of the protein-protein binding pair is optionally linked to the C-terminus of the cargo peptide via a linker.

[0022] In certain embodiments, the first member of the protein-protein binding pair is optionally linked to the N-terminus of the carrier peptide via a linker. In certain embodiments, the second member of the protein-protein binding pair is optionally linked to the N-terminus of the cargo peptide via a linker.

[0023] In certain embodiments, the covalent bond is an isopeptide bond.

[0024] In certain embodiments, the protein-protein binding pair is selected from: (i) SpyTag:SpyCatcher pair, (ii) SpyTag:KTag pair, (iii) Isopeptag:pilin-C pair, (iv) SnoopTag or SnoopTagJr:SnoopCatcher pair, (v) SpyTag002:SpyCatcher002 pair, (vi) RrgATag, RrgATag2 or DogTag:RrgACatcher pair, (vii) IsopepTag-N:Pilin-N pair, and (viii) PsCsTag:PsCsCatcher pair.

[0025] In certain embodiments, the protein-protein binding pair is a SpyTag:SpyCatcher pair. For example, the SpyTag comprises the sequence shown in SEQ ID NO:9; for example, the SpyCatcher comprises the sequence shown in SEQ ID NO:10.

[0026] In certain embodiments, the linker is preferably a peptide linker, such as a flexible peptide linker, such as a peptide linker comprising one or more glycine and / or one or more serine.

[0027] In certain embodiments, the protein component is formed by linking a first protein and a second protein via an isopeptide bond, wherein:

[0028] The first protein comprises the carrier peptide and a first member of the SpyTag:SpyCatcher pair, wherein the first member is optionally connected to the carrier peptide via a first linker (eg, connected to the C-terminus of the carrier peptide);

[0029] The second protein comprises the cargo peptide and a second member of the SpyTag:SpyCatcher pair, wherein the second member is optionally linked to the cargo peptide via a second linker (eg, linked to the N-terminus of the cargo peptide).

[0030] In certain embodiments, the first protein comprises the carrier peptide and SpyCatcher, and the second protein comprises the cargo peptide and SpyTag.

[0031] In other embodiments, the carrier peptide is linked to the cargo peptide by non-covalent means. As used herein, the term "non-covalent binding" refers to specific binding, such as between an antibody and its antigen, between a ligand and its receptor, or between an enzyme and its substrate, for example, exemplified by the interaction between streptavidin binding protein and streptavidin or between an antibody and its antigen.

[0032] In certain embodiments, the cargo peptide in the protein component is optionally linked to a first member of a specific binding pair via a linker to form a first protein, and the carrier peptide is optionally linked to a second member of the specific binding pair via a linker to form a second protein; wherein the first member and the second member are bound by a non-covalent bond mediated by specific binding.

[0033] In certain embodiments, the first member of the specific binding pair is optionally linked to the C-terminus of the carrier peptide via a linker. In certain embodiments, the second member of the specific binding pair is optionally linked to the N-terminus of the cargo peptide via a linker.

[0034] In certain embodiments, the first member of the specific binding pair is optionally linked to the N-terminus of the carrier peptide via a linker. In certain embodiments, the second member of the specific binding pair is optionally linked to the C-terminus of the cargo peptide via a linker.

[0035] In certain embodiments, the first member of the specific binding pair is optionally linked to the C-terminus of the carrier peptide via a linker. In certain embodiments, the second member of the specific binding pair is optionally linked to the C-terminus of the cargo peptide via a linker.

[0036] In certain embodiments, the first member of the specific binding pair is optionally linked to the N-terminus of the carrier peptide via a linker. In certain embodiments, the second member of the specific binding pair is optionally linked to the N-terminus of the cargo peptide via a linker.

[0037] II. Multimerization Domain

[0038] As used herein, the term "multimerization domain" refers to any polypeptide or protein that is capable of multimerizing (i.e., forming a biomolecular complex) several copies of a fusion polypeptide comprising the multimerization domain. In certain embodiments, the multimerization domain is a homologous multimerization domain that aggregates the same fusion polypeptide and forms a multimer.

[0039] In certain embodiments, the multimerization domain can be a dimerization domain, a trimerization domain, a tetramerization domain, or essentially any higher order multimerization domain so long as the domain is capable of facilitating interactions between at least two domains (and the polypeptides of which they form a part).

[0040] In certain embodiments, the multimerization domain is selected from the group consisting of: leucine zipper, NOE (SEQ ID NO: 38), GCN4-P1 (SEQ ID NO: 39), Delta (SEQ ID NO: 40), and any combination thereof.

[0041] In certain embodiments, the multimerization domain is selected from a leucine zipper.

[0042] In certain embodiments, the multimerization domain sequence comprises the sequence set forth in SEQ ID NO:36 or 37. In certain embodiments, the multimerization domain sequence comprises the sequence set forth in SEQ ID NO:36.

[0043] III. pH-sensitive peptides

[0044] In this article, the term "pH-sensitive fusogenic peptide" is used interchangeably with "pH-sensitive peptide", which refers to a class of polypeptides that can undergo conformational changes under acidic conditions (e.g., pH <6.5) to promote fusion with the endocytic vesicle membrane. When the pH-sensitive peptide is endocytosed by the cell, during the maturation and acidification of the endocytic vesicle, once the pH value drops to the critical point, such polypeptides undergo conformational changes and bind to the lipid bilayer of the vesicle membrane, thereby violently disturbing the integrity of the phospholipid bilayer membrane, forming small holes thereon or causing the membrane to lyse, thereby releasing the transported biomacromolecules into the cytoplasm. Such polypeptides are well known in the art and are described in, for example, Varkouhi, Amir K., et al. Journal of Controlled Release 151.3 (2011): 220-228; Erazo-Oliveras A, Muthukrishnan N, Baker R, et al. Pharmaceuticals, 2012, 5(11): 1177-1209, all of which are incorporated herein by reference.

[0045] The pH-sensitive peptide that can be used as the carrier peptide of the present invention can be selected from the following proteins or polypeptides, or derived from the following proteins or polypeptides:

[0046] Viral protein sources: HA2 (influenza virus) and its mutants KALA, GALA; penton base (adenovirus or rhinovirus), gp41 (HIV), L2 (papillomavirus), envelope protein (West Nile virus);

[0047] Bacterial protein sources: Listeriolysin O (LLO), Pneumococcal pneumolysin (PLO), Streptococcal streptolysin O (SLO), Diphtheria toxin, Pseudomonas aeruginosa exotoxin A, Shiga toxin, Cholera toxin;

[0048] Plant protein sources: Ricin, Saporin, Gelonin toxin;

[0049] Human / animal protein sources: human calcitonin, fibroblast growth factor receptor, melittin;

[0050] Artificially synthesized peptides: (R-Ahx-R)(4)AhxB, Penetratin (pAntp), EB1, Bovine prion protein (bPrPp), Sweet Arrow Peptide (SAP), Poly (L-histidine), Proline-rich peptide (Proline-rich).

[0051] In certain embodiments, the pH sensitive fusion peptide is selected from influenza virus HA2 (SEQ ID NO: 47) or a mutant thereof, melittin (SEQ ID NO: 50), and any combination thereof. In certain embodiments, the mutant of influenza virus HA2 is selected from INF7 (SEQ ID NO: 46), KALA (SEQ ID NO: 48) or GALA (SEQ ID NO: 49).

[0052] In certain embodiments, the pH-sensitive fusion peptide comprises INF7. In certain embodiments, the pH-sensitive fusion peptide comprises or consists of the following sequence: SEQ ID NO:46.

[0053] IV. Cell-penetrating peptides

[0054] As used herein, the term "cell penetrating peptide (CPP)" is also referred to as "cell penetrating peptide", "protein translocation domain (PTD), "Trojan horse peptides" or "transduction peptide", etc., and refers to a polypeptide that can promote the cellular uptake of various molecules (e.g., various macromolecules including proteins or nucleic acids). Such polypeptides are well known in the art and are described in, for example, Stewart KM, et al. Org Biomol Chem. 2008 Jul 7; 6 (13): 2242-55 and Chinese patent application CN101490081A (all of which are incorporated herein by reference); or can be obtained by methods known in the art, such as the method described in detail in U.S. patent application US2008 / 0234183, all of which are incorporated herein by reference.

[0055] The CPPs that can be used for the fusion protein of the present invention include, but are not limited to, cationic: Penetratin, HIV-TAT-47-57, HIV-1 Rev 34-50, FHV coat-35-49, oligoarginines (R9-R12), CCMV Gag-7-25, S413-PV, VP22, BP16, DPV3, DPV6, FAH coat protein, protamine 1 (Protamine 1), human cJun, Engrailed-2, Islet-1, HoxA-13, TP10, etc.; amphipathic type: transport peptide (transportan), Transportan10, Pep-1, MPGα, MPGβ, CADY, Pepfect6, Pepfect14, Pepfect15, NickFect, Hel, sC18, pVEC, ARF (1-22), YTA2, PAR1 (Palmitoyl-SFLLRN), F2Pal10 (Palmitoyl-SFLLRN), BPrPp (1-30), hLF peptide (19-40), Buforin 2, Crotamine, Azurin p18, hCT peptide (18-32), S413-PVrev, etc.; hydrophobic type: Kaposi's sarcoma fibroblast growth factor, derived from Caiiman The signal peptide of Ig light chain of crocodylus, integrin β3 fragment, Grb2-SH2 domain, HIV-1gp41 (1-23), HBV translocation motif, sperm-egg fusion protein (89-111), human calcitonin (9-32), Pep-7, C105Y, K-FGF, etc.

[0056] In addition, the CPP used in the fusion protein of the present invention can also be selected from a polypeptide sequence having about 60, 70, 80, 90, 95, 99% or 100% sequence identity with any of the polypeptide sequences described above, as long as the polypeptide sequence still retains its biological activity, i.e., promotes cellular uptake of the molecule.

[0057] In certain embodiments, the cell-penetrating peptide is selected from Penetratin (SEQ ID NO: 51), Tat-derived peptide, Rev (34-50) (SEQ ID NO: 53), VP22 (SEQ ID NO: 54), transportan (SEQ ID NO: 55), Pep-1 (SEQ ID NO: 56), Pep-7 (SEQ ID NO: 57), and any combination thereof. In certain embodiments, the Tat-derived peptide is selected from Tat (48-60) (SEQ ID NO: 1) or Tat (47-57) (SEQ ID NO: 52).

[0058] In certain embodiments, the cell-penetrating peptide comprises a Tat-derived peptide, such as Tat(48-60). In certain embodiments, the cell-penetrating peptide comprises or consists of the following sequence: SEQ ID NO:1.

[0059] V. Protease recognition sequence

[0060] In certain embodiments, the protease recognition sequence comprises a furin recognition sequence and / or a cathepsin L recognition sequence.

[0061] In certain embodiments, the furin recognition sequence comprises or consists of the following sequence: RX 1 -X 2 -R ↓ (SEQ ID NO:41), wherein X 1 is any amino acid, X 2 is K or R, ↓ indicates the cleavage site.

[0062] In certain embodiments, the furin recognition sequence comprises or consists of the following sequence: RRX 1 -X 2 -R ↓ (SEQ ID NO:42).

[0063] In certain embodiments, X 1 Selected from alanine (A), arginine (R), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), histidine (H), isoleucine (I), glycine (G), aspartic acid (N), leucine (L), lysine (K), methionine (M), phenylalanine (F), coline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and valine (V).

[0064] In certain embodiments, the furin recognition sequence comprises or consists of the following sequence: RRHKR ↓ (SEQ ID NO:43).

[0065] In certain embodiments, the furin recognition sequence comprises or consists of the following sequence: QSVASSRRHKR ↓ FAGV (SEQ ID NO:44).

[0066] In certain embodiments, the cathepsin L recognition sequence comprises or consists of the following sequence: NNTHDLVGDVRLAGV (SEQ ID NO: 45).

[0067] In certain embodiments, the protease recognition sequence comprises a furin recognition sequence and a cathepsin L recognition sequence. In certain embodiments, the protease recognition sequence is a single-chain polypeptide comprising a furin recognition sequence and a cathepsin L recognition sequence from N-terminus to C-terminus, or comprising a cathepsin L recognition sequence and a furin recognition sequence from N-terminus to C-terminus.

[0068] In certain embodiments, the protease recognition sequence comprises RRHKR (SEQ ID NO: 43) and NNTHDLVGDVRLAGV (SEQ ID NO: 45). In certain embodiments, the protease recognition sequence comprises SEQ ID NO: 44 and SEQ ID NO: 45.

[0069] VI. Connection Order

[0070] In some embodiments, the carrier peptide comprises, from the N-terminus to the C-terminus: the cell-penetrating peptide, the pH-sensitive fusion peptide, the protease recognition sequence, and the multimerization domain sequence is located at the N-terminus or the C-terminus of the carrier peptide, or between any two adjacent domains. For example, the multimerization domain sequence is located at the N-terminus of the carrier peptide, or at the C-terminus of the carrier peptide.

[0071] In other embodiments, the carrier peptide comprises, from the N-terminus to the C-terminus: the pH-sensitive fusion peptide, the cell-penetrating peptide, the protease recognition sequence, and the multimerization domain sequence is located at the N-terminus or the C-terminus of the carrier peptide, or between any two adjacent domains. For example, the multimerization domain sequence is located at the N-terminus of the carrier peptide, or at the C-terminus of the carrier peptide.

[0072] In certain embodiments, the protease recognition sequence comprises the furin recognition sequence and the cathepsin L recognition sequence from the N-terminus to the C-terminus. In certain embodiments, the protease recognition sequence comprises the cathepsin L recognition sequence and the furin recognition sequence from the N-terminus to the C-terminus.

[0073] In certain exemplary embodiments, the carrier peptide comprises, from N-terminus to C-terminus: the multimerization domain sequence, a cell-penetrating peptide, a pH-sensitive fusion peptide, and a protease recognition sequence.

[0074] In certain exemplary embodiments, the carrier peptide comprises, from N-terminus to C-terminus: the multimerization domain sequence, the pH-sensitive fusion peptide, the cell-penetrating peptide, and the protease recognition sequence.

[0075] In certain embodiments, any adjacent domains contained in the carrier peptide are optionally connected by a peptide linker. In certain embodiments, the peptide linker is (G m S) n , wherein m is selected from an integer of 1-4 (e.g., 1, 2, 3 or 4), and n is selected from an integer of 1-3 (e.g., 1, 2 or 3). In certain embodiments, the peptide linker is SEQ ID NO: 58. In certain embodiments, the peptide linkers between any adjacent domains may be the same or different.

[0076] In certain exemplary embodiments, the carrier peptide comprises the sequence shown in SEQ ID NO:3 or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and the variant has substantially the same activity as SEQ ID NO:3 (e.g., cytoplasmic delivery ability and the ability to form a complex with a CpG adjuvant through electrostatic interaction).

[0077] In certain exemplary embodiments, the present invention provides a complex formed by a protein component and a nucleic acid component through electrostatic interaction, wherein:

[0078] (i) the protein component comprises a carrier peptide and a cargo peptide; wherein the carrier peptide comprises the sequence shown in SEQ ID NO: 3 or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and the variant has substantially the same activity as SEQ ID NO: 3 (e.g., cytoplasmic delivery ability and the ability to form a complex with a CpG adjuvant through electrostatic interaction); and the cargo peptide comprises an antigenic peptide;

[0079] (ii) The nucleic acid component comprises a CpG adjuvant.

[0080] VII. Antigenic Peptides

[0081] In some embodiments, the antigenic peptide comprised by the cargo peptide is derived from a tumor antigen, or is a tumor antigen protein or an epitope peptide thereof.

[0082] In certain embodiments, the tumor antigen is selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), or a tumor neoantigen.

[0083] In certain embodiments, the tumor antigen is a solid tumor antigen, such as a melanoma antigen, a colorectal cancer antigen, a breast cancer antigen, a prostate cancer antigen, a bladder cancer antigen, a lung cancer antigen, an ovarian cancer antigen, a brain cancer antigen, a gastric cancer antigen, a renal cell carcinoma antigen, a pancreatic cancer antigen, a liver cancer antigen, an esophageal cancer antigen, or a head and neck cancer antigen. In certain embodiments, the tumor antigen is a melanoma antigen. In certain embodiments, the tumor antigen is a colorectal cancer antigen.

[0084] In certain embodiments, the tumor antigen is a blood tumor antigen, such as a lymphoma antigen, a myeloma (such as multiple myeloma or plasma cell myeloma) antigen, an acute lymphoblastic leukemia antigen, a chronic myeloid leukemia antigen, or an acute myeloid leukemia antigen.

[0085] In certain embodiments, the tumor antigen is selected from RepS1 (e.g., SEQ ID NO: 26), Adpgk (e.g., SEQ ID NO: 27), HER2, GP100, TRP1, TRP2, M27, M30, WT1, CEA, HVP16 E7, Kras, P53, LCMVgp33, LCMV NP369, InfluenzaNP, HIV gag, MuLV gp70, MCMV IE1, VSV NP, HSV-1gB, HBchelper peptide, etc.

[0086] In certain exemplary embodiments, the tumor antigen is selected from GP33 (eg, SEQ ID NO: 7), E7 38～57 (e.g., SEQ ID NO: 18), CEA 567～584 (e.g., SEQ ID NO: 19), Trp1 (e.g., SEQ ID NO: 23), GP100 (e.g., SEQ ID NO: 24), M27 (e.g., SEQ ID NO: 25), RepS1 (e.g., SEQ ID NO: 26), Adpgk (e.g., SEQ ID NO: 27).

[0087] In certain exemplary embodiments, the tumor antigen is selected from melanoma antigens, such as Trp1 (SEQ ID NO: 23), GP100 (SEQ ID NO: 24), M27 (SEQ ID NO: 25).

[0088] In certain exemplary embodiments, the tumor antigen is selected from colorectal cancer antigens, such as RepS1 (SEQ ID NO: 26), Adpgk (SEQ ID NO: 27).

[0089] In certain exemplary embodiments, the tumor antigen is selected from human tumor antigens, such as E7 38～57 (SEQ ID NO: 18), CEA 567～584 (SEQ ID NO: 19).

[0090] In other embodiments, the antigenic peptide contained in the cargo peptide is derived from a pathogen antigen, or is a pathogen antigen protein or an epitope peptide thereof.

[0091] In certain embodiments, the pathogen is selected from the group consisting of bacteria, bacterial infection, fungus, virus, or parasite.

[0092] In certain embodiments, the cargo peptide described in any of the above embodiments optionally further comprises an additional functional peptide connected to the antigenic peptide. In certain embodiments, the additional functional peptide is a lytic tag, such as protein phosphatase 1B (Ppm1b).

[0093] VIII. Multimers

[0094] In certain embodiments, the protein component is a fusion protein monomer comprising the carrier peptide and the cargo peptide.

[0095] In certain embodiments, the protein component is a multimer formed by fusion protein monomers comprising the carrier peptide and the cargo peptide.

[0096] In certain embodiments, the polymer is a dimer, trimer or tetramer. In certain embodiments, the polymer is a dimer. In certain embodiments, the polymer is a trimer.

[0097] In certain embodiments, the multimer is a homomultimer. In certain embodiments, the multimer is a homodimer. In certain embodiments, the multimer is a homotrimer.

[0098] IX. Nucleic Acid Components

[0099] The complex of the present invention comprises a CpG adjuvant. CpG adjuvant has a meaning known to those skilled in the art, and refers to a DNA fragment containing an unmethylated cytosine-guanine dinucleotide (CpG) motif. It can activate B cells, NK cells, DC dendritic cells and other cells, induce the release of cytokines such as IFN-α, thereby inducing a strong Th1 type response and cellular immunity. CpG adjuvants are well known to those skilled in the art, and include, but are not limited to, CpG 2395 (SEQ ID NO: 17), CpG 1826 (SEQ ID NO: 22), CpG 7909 (SEQ ID NO: 59), CpG 1018 (SEQ ID NO: 60), CpG 1585 (SEQ ID NO: 61), CpG 1668 (SEQ ID NO: 62), CpG 2007 (SEQ ID NO: 63), CpG 2336 (SEQ ID NO: 64), CpG 2088 (SEQ ID NO: 65), CpG M362 (SEQ ID NO: 66), and the like.

[0100] In certain exemplary embodiments, the CpG adjuvant is a B-class or C-class CpG.

[0101] In certain exemplary embodiments, the CpG adjuvant comprises the sequence shown in SEQ ID NO:17 or 22.

[0102] In certain embodiments, in the complex, the protein component and the nucleic acid component are mixed in a molar ratio of about 1:1 to 10:1 (calculated based on monomers, not polymers). In certain embodiments, the molar ratio of the protein component to the nucleic acid component is about 1:1 to 8:1, for example, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1. In certain embodiments, the molar ratio of the protein component to the nucleic acid component is about 2:1 to 8:1, for example, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1.

[0103] Reagent test kit

[0104] In a second aspect, the present invention provides a kit comprising the protein component and the nucleic acid component of the complex of the first aspect contained in separate containers.

[0105] In some embodiments, the cargo peptide in the protein component is connected to the carrier peptide in a fusion manner, that is, the protein component is a fusion protein formed by the cargo peptide and the carrier peptide. In such embodiments, the kit comprises: a first container containing the protein component and a second container containing the nucleic acid component.

[0106] In other embodiments, the cargo peptide in the protein component is covalently linked to the carrier peptide: the cargo peptide in the protein component is optionally linked to the first member of the protein-protein binding pair through a linker to form a first protein, and the carrier peptide is optionally linked to the second member of the protein-protein binding pair through a linker to form a second protein; wherein the first member and the second member are bound by a covalent bond. In such embodiments, the kit comprises: a first container containing the first protein in the protein component, a second container containing the second protein in the protein component, and a third container containing the nucleic acid component.

[0107] In other embodiments, the cargo peptide in the protein component is non-covalently linked to the carrier peptide: the cargo peptide in the protein component is optionally linked to the first member of the specific binding pair through a linker to form a first protein, and the carrier peptide is optionally linked to the second member of the specific binding pair through a linker to form a second protein; wherein the first member and the second member are bound by a non-covalent bond. In such embodiments, the kit comprises: a first container containing the first protein in the protein component, a second container containing the second protein in the protein component, and a third container containing the nucleic acid component.

[0108] In a third aspect, the present invention provides a kit comprising:

[0109] (i) a nucleic acid molecule or a vector comprising a nucleotide sequence encoding a protein component of the complex described in the first aspect; and

[0110] (ii) CpG adjuvant.

[0111] In some embodiments, the cargo peptide in the protein component is connected to the carrier peptide in a fusion manner, that is, the protein component is a fusion protein formed by the cargo peptide and the carrier peptide. In such embodiments, the nucleic acid molecule or vector described in (i) comprises a nucleotide sequence encoding the protein component.

[0112] In certain embodiments, the kit comprises: a first container comprising (i) the nucleic acid molecule or vector, and a second container comprising a CpG adjuvant.

[0113] In other embodiments, the cargo peptide in the protein component is covalently linked to the carrier peptide: the cargo peptide in the protein component is optionally connected to the first member of the protein-protein binding pair through a linker to form a first protein, and the carrier peptide is optionally connected to the second member of the protein-protein binding pair through a linker to form a second protein; wherein the first member and the second member are bound by a covalent bond. In such embodiments, the nucleic acid molecule or vector described in (i) comprises: a first nucleotide sequence encoding the carrier peptide and the first member of the protein-protein binding pair connected thereto, and a second nucleotide sequence encoding the antigenic peptide and the second member of the protein-protein binding pair connected thereto. In certain embodiments, the first nucleotide sequence and the second nucleotide sequence are present on the same or different nucleic acid molecules or vectors. The protein-protein binding pair is as defined in the first aspect.

[0114] In certain embodiments, the kit comprises one or more vectors (e.g., expression vectors) comprising the first nucleotide sequence and the second nucleotide sequence. The first nucleotide sequence and the second nucleotide sequence optionally comprise a regulatory element (e.g., a promoter) operably connected thereto.

[0115] In certain embodiments, the kit comprises one or more vectors (e.g., expression vectors) comprising the first nucleotide sequence and the second nucleotide sequence. The first nucleotide sequence and the second nucleotide sequence optionally comprise a regulatory element (e.g., a promoter) operably connected thereto.

[0116] In certain embodiments, the kit comprises: a first container comprising a vector comprising the first nucleotide sequence, a second container comprising a vector comprising the second nucleotide sequence, and a third container comprising a CpG adjuvant.

[0117] In other embodiments, the cargo peptide in the protein component is non-covalently connected to the carrier peptide: the cargo peptide in the protein component is optionally connected to the first member of the specific binding pair through a joint to form a first protein, and the carrier peptide is optionally connected to the second member of the specific binding pair through a joint to form a second protein; wherein the first member and the second member are bound by a non-covalent bond. In such embodiments, the nucleic acid molecule or vector described in (i) comprises: a first nucleotide sequence encoding the carrier peptide and the first member of the specific binding pair connected thereto, and a second nucleotide sequence encoding the antigenic peptide and the second member of the specific binding pair connected thereto. In certain embodiments, the first nucleotide sequence and the second nucleotide sequence are present on the same or different nucleic acid molecules or vectors. The specific binding pair is as defined in the first aspect.

[0118] In certain embodiments, the kit comprises one or more vectors (e.g., expression vectors) comprising the first nucleotide sequence and the second nucleotide sequence. The first nucleotide sequence and the second nucleotide sequence optionally comprise a regulatory element (e.g., a promoter) operably connected thereto.

[0119] In certain embodiments, the kit comprises: a first container comprising a vector comprising the first nucleotide sequence, a second container comprising a vector comprising the second nucleotide sequence, and a third container comprising a CpG adjuvant.

[0120] Composite preparation

[0121] In a fourth aspect, the present invention provides a method for preparing the composite of the first aspect, comprising:

[0122] (1) providing a protein component and a nucleic acid component in the complex;

[0123] (2) The protein component and nucleic acid component are mixed and optionally incubated to form a complex.

[0124] In certain embodiments, prior to mixing, the protein component and / or nucleic acid component is purified. The purification of protein or nucleic acid can be carried out by conventional purification procedures well known in the art, and the selection of appropriate methods and substrates and buffers used therein is well known in the art and is therefore not described in detail herein.

[0125] In certain embodiments, the protein component and the nucleic acid component are mixed in a molar ratio of about 1:1 to 10:1 (calculated based on monomers, not polymers). In certain embodiments, the molar ratio of the protein component and the nucleic acid component is about 1:1 to 8:1, for example, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1. In certain embodiments, the molar ratio of the protein component and the nucleic acid component is about 2:1 to 8:1, for example, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1.

[0126] Nucleic acid components

[0127] The nucleic acid component in the complex of the present invention can be prepared by various methods known in the art, for example, by genetic engineering methods, or by chemical synthesis methods (such as solid phase synthesis). The nucleic acid component of the present invention is not limited by the way it is produced. In certain embodiments, the nucleic acid component is preferably produced by solid phase synthesis, for example, using an oligonucleotide synthesizer.

[0128] Protein components

[0129] The protein components in the complex of the present invention can be prepared by various methods known in the art, for example, by genetic engineering methods (recombinant technology) or by chemical synthesis methods (such as Fmoc solid phase method). The protein components of the present invention are not limited by the way they are produced.

[0130] In some embodiments, the cargo peptide in the protein component is connected to the carrier peptide in a fusion manner, that is, the protein component is a fusion protein formed by the cargo peptide and the carrier peptide. In such embodiments, step (1) includes: introducing a nucleic acid molecule or a vector encoding a nucleotide sequence of the protein component into a host cell, culturing the host cell under conditions that allow protein expression, and recovering (e.g., and purifying) the protein component from the cultured host cell culture, wherein the protein component is optionally present in a polymeric form.

[0131] In other embodiments, the cargo peptide in the protein component is covalently linked to the carrier peptide via a protein-protein binding pair (e.g., an isopeptide bond). In such embodiments, step (1) comprises: (a) providing a first protein formed by the carrier peptide and a first member of the protein-protein binding pair linked thereto, and a second protein formed by the antigenic peptide and a second member of the protein-protein binding pair linked thereto; (b) mixing the first protein with the second protein and optionally incubating them to form an isopeptide bond, thereby providing a protein component.

[0132] In certain embodiments, the first protein is provided by the following steps: introducing a first nucleotide sequence encoding the first protein or a nucleic acid molecule or vector comprising the first nucleotide sequence into a host cell, culturing the host cell under conditions allowing protein expression, and recovering (e.g., and purifying) the first protein from the cultured host cell culture, wherein the first protein is optionally present in a multimeric form.

[0133] In certain embodiments, the second protein is provided by the following steps: introducing a second nucleotide sequence encoding the second protein or a nucleic acid molecule or vector comprising the second nucleotide sequence into a host cell, culturing the host cell under conditions allowing protein expression, and recovering (e.g., and purifying) the second protein from the cultured host cell culture.

[0134] It is understood by those skilled in the art that the nucleotide sequences encoding the first protein and the second protein may be present in the same nucleic acid molecule or vector, or in different nucleic acid molecules or vectors. Therefore, the first protein and the second protein may be obtained simultaneously, separately or sequentially in any order.

[0135] In other embodiments, the cargo peptide in the protein component is non-covalently linked to the carrier peptide via a specific binding pair. In such embodiments, step (1) comprises: (a) providing a first protein formed by the carrier peptide and a first member of the specific binding pair linked thereto, and a second protein formed by the antigenic peptide and a second member of the specific binding pair linked thereto; (b) mixing the first protein with the second protein and optionally incubating to form a non-covalent link mediated by the specific binding of the first member and the second member, thereby providing a protein component.

[0136] In certain embodiments, the first protein is provided by the following steps: introducing a first nucleotide sequence encoding the first protein or a nucleic acid molecule or vector comprising the first nucleotide sequence into a host cell, culturing the host cell under conditions allowing protein expression, and recovering (e.g., and purifying) the first protein from the cultured host cell culture, wherein the first protein is optionally present in a multimeric form.

[0137] In certain embodiments, the second protein is provided by the following steps: introducing a second nucleotide sequence encoding the second protein or a nucleic acid molecule or vector comprising the second nucleotide sequence into a host cell, culturing the host cell under conditions allowing protein expression, and recovering (e.g., and purifying) the second protein from the cultured host cell culture.

[0138] It is understood by those skilled in the art that the nucleotide sequences encoding the first protein and the second protein may be present in the same nucleic acid molecule or vector, or in different nucleic acid molecules or vectors. Therefore, the first protein and the second protein may be obtained simultaneously, separately or sequentially in any order.

[0139] Pharmaceutical composition

[0140] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the complex described in the first aspect.

[0141] In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0142] In certain embodiments, the pharmaceutical composition is a vaccine.

[0143] In certain embodiments, in the complex, the protein component and the nucleic acid component are mixed in a molar ratio of about 1:1 to 10:1 (calculated based on monomers, not polymers). In certain embodiments, the molar ratio of the protein component to the nucleic acid component is about 1:1 to 8:1, for example, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1. In certain embodiments, the molar ratio of the protein component to the nucleic acid component is about 2:1 to 8:1, for example, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1.

[0144] The complex or pharmaceutical composition of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The complex or pharmaceutical composition of the present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such an injection can be a sterile injection solution. In addition, the sterile injection solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0145] The complex or pharmaceutical composition of the present invention can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic, inguinal, intravesical, topical (e.g., powders, ointments or drops), or nasal routes. However, for many therapeutic uses, the preferred route / mode of administration is parenteral administration (e.g., intravenous or bolus, subcutaneous, intraperitoneal, intramuscular). The skilled person will appreciate that the route and / or mode of administration will vary depending on the intended purpose. In certain exemplary embodiments, the complex or pharmaceutical composition of the present invention is administered by subcutaneous injection.

[0146] The complex or pharmaceutical composition of the present invention should be administered in an effective dose. The appropriate amount can be determined based on the specific disease or condition to be treated or prevented, the severity, the age of the subject, and other personal attributes of the specific subject (e.g., the general state of health of the subject and the robustness of the subject's immune system). The determination of the effective dose is also guided by animal model studies, followed by human clinical trials, and guided by an administration regimen that significantly reduces the occurrence or severity of the target disease symptoms or conditions in the subject.

[0147] Uses and methods

[0148] In a sixth aspect, the present invention provides a method for inducing an immune response in a subject (e.g., a human), comprising administering an effective amount of a complex or pharmaceutical composition of the present invention to a subject in need thereof. Also provided is the use of the complex or pharmaceutical composition of the present invention in the preparation of a preparation (e.g., a drug) for inducing an immune response in a subject (e.g., a human).

[0149] In certain embodiments, the complexes or pharmaceutical compositions of the invention are used to induce an antigen-specific immune response in a subject (eg, a human).

[0150] In certain embodiments, the antigenic peptide contained in the complex of the present invention is derived from a tumor antigen, for example, selected from a tumor antigen protein or an epitope peptide thereof. In such embodiments, the complex or pharmaceutical composition of the present invention is used to induce an anti-tumor immune response in a subject (eg, a human).

[0151] In certain embodiments, the antigenic peptide contained in the complex of the present invention is derived from a pathogen antigen, for example, selected from a pathogen antigen protein or an epitope peptide thereof. In such embodiments, the complex or pharmaceutical composition of the present invention is used to induce an immune response against the pathogen in a subject (e.g., a human).

[0152] In certain embodiments, the immune response is a cell-mediated response or a humoral response. In certain embodiments, the immune response comprises an enhancement of B cell proliferation, CD4+T cell proliferation, CD8+T cell proliferation, or a combination thereof.

[0153] In certain embodiments, the immune response is a T cell-mediated immune response.

[0154] In certain embodiments, the immune response comprises enhancement of T cell proliferation, IL-2 production, IFN-γ production, or a combination thereof.

[0155] In a seventh aspect, the present invention provides a method for preventing and / or treating a tumor in a subject (e.g., a human), comprising administering an effective amount of the complex or pharmaceutical composition of the present invention to a subject in need thereof. Also provided is the use of the complex or pharmaceutical composition of the present invention in the preparation of a preparation (e.g., a drug) for preventing and / or treating a tumor in a subject (e.g., a human).

[0156] In certain embodiments, the tumor is a solid tumor, such as melanoma, colorectal cancer, breast cancer, prostate cancer, bladder cancer, lung cancer, ovarian cancer, brain cancer, gastric cancer, renal cell carcinoma, pancreatic cancer, liver cancer, esophageal cancer, head and neck cancer. The complex of the present invention comprises a tumor antigen corresponding to the above tumor.

[0157] In certain embodiments, the tumor is a blood tumor, such as lymphoma, myeloma (such as multiple myeloma or plasma cell myeloma), acute lymphocytic leukemia, chronic myeloid leukemia or acute myeloid leukemia. The complex of the present invention comprises a tumor antigen corresponding to the above tumor.

[0158] In certain embodiments, the tumor is a metastatic tumor, such as a metastatic solid tumor, such as a metastatic melanoma.

[0159] In certain embodiments, the subject is a non-tumor-affected subject or a tumor-affected subject.

[0160] In certain embodiments, the complex or pharmaceutical composition is administered alone or in combination with another therapeutic agent (anti-tumor agent).

[0161] In certain embodiments, the complex or pharmaceutical composition is administered in combination with an immune checkpoint inhibitor (eg, PD-L1 antibody, PD-1 antibody, CTLA-4 antibody).

[0162] In certain embodiments, the complexes or pharmaceutical compositions of the invention can be administered topically, intratumorally, intranasally, intraperitoneally, intracerebrospinal, subcutaneously, intraarticularly, intrasynovially, intrathecally, orally, topically, dermally, intravenously, or by inhalation to, for example, readily accessible tumors such as melanoma, head and neck cancer, breast cancer, colon cancer, ovarian cancer, bladder cancer, and lymphoma; or systemically for other cancers such as lung cancer, liver cancer, pancreatic cancer, prostate cancer, brain cancer, kidney cancer, blood cancer, and metastatic cancer, etc.

[0163] For preventive applications, the complex or pharmaceutical composition of the invention is provided before any symptoms occur, for example, when the patient is not suffering from a tumor. Preventive administration is used to prevent or improve the expected severity, duration or extent of any subsequent related disease symptoms. In some embodiments, the subject to be treated is a subject who does not suffer from a tumor.

[0164] For therapeutic applications, the complexes or pharmaceutical compositions of the invention are administered at or after the onset of symptoms of a disease, for example, after the onset of symptoms of a tumor or after the diagnosis of a tumor. In some embodiments, the subject to be treated is a subject with a tumor.

[0165] In an eighth aspect, the present invention provides a method for preventing and / or treating infection in a subject (e.g., a human), comprising administering an effective amount of a complex or pharmaceutical composition of the present invention to a subject in need thereof. Also provided is the use of a complex or pharmaceutical composition of the present invention in the preparation of a preparation (e.g., a drug) for preventing and / or treating infection in a subject (e.g., a human).

[0166] In certain embodiments, the infection is selected from a viral infection, a bacterial infection, a fungal infection, or a parasitic infection.

[0167] In certain embodiments, the subject is a subject that does not have an infection or a subject that has an infection.

[0168] For prophylactic applications, the complex or pharmaceutical composition of the invention is provided before any symptoms occur, for example, before infection. Prophylactic administration is used to prevent or ameliorate any subsequent infection, to reduce the expected severity, duration or extent of infection and / or related disease symptoms after exposure or suspected exposure to a virus or after actual onset of infection. In some embodiments, the subject to be treated may be a subject at risk of developing an infection, for example due to exposure or potential exposure to a pathogen.

[0169] For therapeutic applications, the complexes or pharmaceutical compositions of the invention are administered at or after the onset of symptoms of a disease or infection, for example, after symptoms of infection develop or after an infection is diagnosed. In some embodiments, the subject to be treated may also be an already infected subject.

[0170] In the ninth aspect, the present invention provides a method for targeted delivery of an active agent to a lymphoid organ in a subject, the method comprising: administering to the subject an effective amount of a complex or pharmaceutical composition of the present invention, wherein the active agent comprises a cargo peptide in the complex and a CpG adjuvant. Also provided is the use of the complex or pharmaceutical composition of the present invention in preparing a preparation for targeted delivery of an active agent to a lymphoid organ in a subject.

[0171] In certain embodiments, the lymphoid organ is a secondary lymphoid organ. In certain embodiments, the lymphoid organ is a lymph node.

[0172] In certain embodiments, the methods are used to induce an immune response, prevent and / or treat a tumor, or prevent and / or treat an infection in a subject (eg, a human).

[0173] In certain embodiments, the complex of the present invention has improved lymph node delivery efficiency, for example, at least 2 times, at least 5 times, or at least 10 times compared to a complex not comprising the carrier peptide provided by the present invention (e.g., a complex with TAT as a carrier peptide).

[0174] Definition of terms

[0175] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0176] As used herein, the term "antigen" is a substance that induces an immune response. "Tumor-specific antigen (TSA)" refers to an antigen that is specific to tumor cells and is not present on other cells in the body. "Tumor-associated antigen (TAA)" is not specific to tumor cells and may be present at very low levels on normal cells, but is highly expressed when tumor cells proliferate. "Tumor neoantigens" are neoantigens that are present in tumor cells or tissues of a subject but not in the corresponding normal cells or tissues of the subject. Exemplary methods for identifying neoantigen sequences are described in Kreiter et al. (2015) Nature 520 (7549): 692-696, Ott et al. (2017) Nature 547: 217-221, and Sahin et al. (2017) Nature 547: 222-226, the entire contents of which are incorporated herein by reference.

[0177] As used herein, the term "CpG adjuvant" refers to a DNA fragment containing a non-methylated cytosine-guanine dinucleotide (CpG) motif. "CpG motif" refers to a 5'C nucleotide connected to a 3'G nucleotide by a phosphodiester internucleotide bond or a phosphodiester derivative internucleotide bond. In some embodiments, the CpG motif comprises a phosphodiester internucleotide bond. In some embodiments, the CpG motif comprises a phosphodiester derivative internucleotide bond. CpG adjuvants typically include Class A CpG ODN, Class B CpG ODN, and Class C CpG ODN.

[0178] Class A CpG ODN refers to: a CpG motif comprising an oligodeoxynucleotide, wherein the oligodeoxynucleotide comprises one or more poly-G sequences at 5', 3' or both ends; an internal palindromic sequence comprising the CpG motif; or one or more phosphodiester derivatives connected to the deoxynucleotide. In an embodiment, the Class A CpG ODN comprises a poly-G sequence at 5', 3' or both ends; an internal palindromic sequence comprising the CpG motif; and one or more phosphodiester derivatives connected to the deoxynucleotide. In an embodiment, the phosphodiester derivative is a thiophosphate. Examples of Class A CpG ODNs include ODN D19, ODN 1585, ODN 2216, and ODN 2336.

[0179] Class B CpG ODN refers to: a CpG motif comprising an oligodeoxynucleotide, wherein the oligodeoxynucleotide comprises one or more 6-mer motifs comprising the CpG motif; a phosphodiester derivative connecting all deoxynucleotides. In an embodiment, a Class B CpG ODN comprises one or more copies of a 6-mer motif comprising a CpG motif and a phosphodiester derivative connecting all deoxynucleotides. In an embodiment, the phosphodiester derivative is a thiophosphate. In an embodiment, a Class B CpG ODN comprises one copy, two copies, three copies or four copies of a 6-mer motif containing a CpG motif. Examples of Class B CpG ODNs include ODN 1668, ODN 1826, ODN 2006 and ODN 2007.

[0180] Class C CpG ODN refers to an oligodeoxynucleotide comprising a palindromic sequence comprising a CpG motif and a phosphodiester derivative (phosphorothioate) linking all deoxynucleotides. Examples of Class C CpG ODN include ODN 2395 and ODN M362.

[0181] As used herein, the term "epitope peptide" refers to a peptide segment on an antigen that can serve as an epitope. In some cases, a single epitope peptide can be specifically recognized / bound by an antibody against the epitope.

[0182] As used herein, the term "protein-protein binding pair" comprises two proteins (e.g., a first member (e.g., a first polypeptide) and a second homologous member (e.g., a second polypeptide)) that interact to form a covalent isopeptide bond under conditions that achieve or promote isopeptide bond formation, wherein the term "homologous" refers to components that work together, i.e., react together to form an isopeptide bond. Thus, two proteins that effectively react together to form an isopeptide bond under conditions that achieve or promote isopeptide bond formation may also be referred to as a "complementary" peptide linker pair. Specific binding pairs capable of interacting to form covalent isopeptide bonds are reviewed in Veggiani G, Zakeri B, Howarth M. Superglue from bacteria: unbreakable bridges for protein nanotechnology. Trends Biotechnol. 2014; 32(10): 506-512. Non-limiting examples include SpyTag: SpyCatcher, SpyTag002: SpyCatcher002, SpyTag: KTag, isopeptag: pilinC, SnoopTag: SnoopCatcher, etc.

[0183] The term "isopeptide bond" refers to an amide bond between a carboxyl or carboxamide group and an amino group, at least one of the groups not being derived from the protein backbone, or in another view, not being part of the protein backbone. An isopeptide bond may be formed within a single protein or may occur between two peptides or between a peptide and a protein. Thus, an isopeptide bond may be formed intramolecularly within a single protein, or intermolecularly, i.e., between two peptide / protein molecules, such as between two peptide linkers. Typically, an isopeptide bond may occur between a lysine residue and an asparagine, aspartic acid, glutamine or glutamic acid residue or a terminal carboxyl group of a protein or peptide chain, or may occur between the α-amino terminus of a protein or peptide chain and asparagine, aspartic acid, glutamine or glutamic acid.

[0184] The SpyTag:SpyCatcher system is described in U.S. Pat. No. 9,547,003 and Zakeri et al. Peptidetag forming a rapid covalent bond to a protein, through engineering abacterial adhesin. Proc Natl Acad Sci US A. 2012; 109(12): E690-E697 (which are incorporated herein by reference in their entireties), and is derived from the CnaB2 domain of the Streptococcus pyogenes fibronectin binding protein FbaB. Another specific binding pair derived from the CnaB2 domain is SpyTag:KTag, which forms an isopeptide bond in the presence of SpyLigase, see Fierer JO, Veggiani G, Howarth M. SpyLigase peptide-peptideligation polymerizes affibodies to enhance magnetic cancer cell capture. Proc Natl Acad Sci US A. 2014; 111 (13): E1176-E1181, which is incorporated herein by reference in its entirety. The SpyTag002: SpyCatcher002 system is described in Keeble AH et al. Evolving Accelerated Amidation by SpyTag / SpyCatcher to Analyze Membrane Dynamics. Angew Chem Int Ed Engl. 2017; 56 (52): 16521-16525., which is incorporated herein by reference in its entirety.

[0185] The SnoopTag: SnoopCatcher system is described in Veggiani G, Nakamura T, Brenner MD, et al. Programmable polyproteams built using twin peptide superglues. Proc Natl Acad Sci US A. 2016; 113(5): 1202-1207., which is incorporated herein by reference in its entirety. The D4Ig-like domain of RrgA (adhesion to Streptococcus pneumoniae) was isolated to form SnoopTag and SnoopCatcher.

[0186] The isopeptag:pilin-C specific binding pair is derived from the major pilin protein Spy0128 from Streptococcus pyogenes, see Zakeri B, Howarth M. Spontaneous intermolecular amide bond formation between side chains for irreversible peptide targeting. J Am Chem Soc. 2010; 132(13):4526-4527., which is incorporated herein by reference in its entirety.

[0187] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0188] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0189] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0190] The compilation of the twenty conventional amino acids involved in this article follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0191] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active ingredient, which is well known in the art and includes, but is not limited to, pH regulators, surfactants, ionic strength enhancers, agents that maintain osmotic pressure, agents that delay absorption, diluents, adjuvants, preservatives, stabilizers, and the like. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like. Adjuvants include, but are not limited to, aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvants (such as complete Freund's adjuvant), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like.

[0192] As used herein, the terms "cancer" or "tumor" are used interchangeably and refer to a cellular disease characterized by uncontrolled or dysregulated cell proliferation, reduced cell differentiation, inappropriate ability to invade surrounding tissues, and / or the ability to establish new growth at ectopic sites. The term "cancer" includes, but is not limited to, solid tumors and hematogenous tumors. The term "cancer" encompasses diseases of the skin, tissues, organs, bones, cartilage, blood, and blood vessels. The term "cancer" further encompasses primary and metastatic cancers. Non-limiting examples of solid tumors include: pancreatic cancer; bladder cancer; colorectal cancer; breast cancer; prostate cancer; kidney cancer; hepatocellular carcinoma; lung cancer; ovarian cancer; cervical cancer; gastric cancer; esophageal cancer; head and neck cancer; skin cancer, including, for example, malignant melanoma; neuroendocrine cancer; brain tumors, including, for example, gliomas, oligodendrogliomas, adult glioblastoma multiforme, and adult anaplastic astrocytomas; bone cancer; soft tissue sarcomas; and thyroid cancer. Non-limiting examples of hematological malignancies include: acute myeloid leukemia (AML); chronic myeloid leukemia (CML); acute lymphocytic leukemia (ALL); chronic lymphocytic leukemia (CLL); Hodgkin's disease (HD); non-Hodgkin's lymphoma (NHL); B-cell lymphoma; T-cell lymphoma; multiple myeloma (MM), etc.

[0193] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or disorder or symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to the expected survival (if not receiving treatment).

[0194] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human.

[0195] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., a tumor); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient who already has the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0196] Advantageous Effects of the Invention

[0197] The present invention provides a novel vaccine delivery system that can efficiently deliver antigens and adjuvants to lymph nodes, promote cross-presentation of antigens, and induce potent antigen-specific immune responses. It is superior to the oil-based composite adjuvant system commonly used in clinical practice, and the immune response stimulated also has a strong memory effect, which is of great significance for the prevention and treatment of diseases such as tumors and infections.

[0198] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0199] Figure 1 The cloning design of the model antigen and its control antigen based on eTAT fusion expression is shown.

[0200] Figure 2 The levels of specific T cell responses based on model antigens expressed by eTAT fusion are shown.

[0201] Figure 3 Interaction analysis of model antigens expressed based on eTAT fusion with CpG is shown.

[0202] Figure 4 The evaluation of the intracellular efficiency of a mixture of fluorescently labeled model antigen and CpG on DC cells is shown.

[0203] Figure 5 Shown is the fluorescence imaging and intensity calculation of lymph nodes after subcutaneous co-injection of fluorescently labeled pattern antigen and CpG into mice.

[0204] Figure 6 Shown are the fluorescence imaging and intensity calculation of major organs in mice after subcutaneous co-injection of fluorescently labeled model antigen and CpG.

[0205] Figure 7 The ratio of antigen- and CpG-double-positive cells in the lymph nodes of mice after subcutaneous co-injection of fluorescently labeled model antigen and CpG is shown.

[0206] Figure 8 Evaluation of the immune response levels based on the model antigen expressed by eTAT fusion with or without CpG adjuvant is shown.

[0207] Fig. 9 The design of a universal eTAT vaccine system is shown.

[0208] Fig.10 The evaluation of the ligation of eTAT-Catcher and its control protein to SpyTag-GP33 and the purification of the product is shown.

[0209] Fig.11 Interaction analysis of eTAT-Catcher and its ligation products with CpG is shown.

[0210] Fig.12 The figure shows the proportion of antigen- and CpG-double-positive cells in the lymph nodes of mice after subcutaneous injection of the universal eTAT vaccine system.

[0211] Fig.13 The figure shows the evaluation of the specific T cell response level after subcutaneous immunization of mice with the universal eTAT vaccine system based on the model antigen GP33.

[0212] Fig.14 The effect of the universal eTAT vaccine system on enhancing the immunogenicity of human-related tumor antigens was demonstrated.

[0213] Fig.15The CD8 + Evaluation of T cell responses.

[0214] Fig.16 The melanoma preventive effect of the universal eTAT vaccine system evaluated based on the model antigen GP33 was shown.

[0215] Fig.17 The melanoma therapeutic effect of the universal eTAT vaccine system evaluated based on the model antigen GP33 is shown.

[0216] Fig.18 The melanoma therapeutic effect of the universal eTAT vaccine system evaluated based on MC38 cell neoantigens was shown.

[0217] Fig.19 The results showed that the eTAT vaccine system based on tumor-associated antigens and neoantigens has therapeutic effects on subcutaneous malignant melanoma.

[0218] Fig. 20 The results showed that the eTAT vaccine system based on tumor-associated antigens and neoantigens has therapeutic effects on metastatic malignant melanoma.

[0219] Sequence information

[0220] The information of the partial sequences involved in the present invention is provided in Table 1 below.

[0221] Table 1: Description of sequences

[0222]

[0223]

[0224]

[0225]

[0226] Example

[0227] The invention is now described in the following non-limiting examples.

[0228] Those skilled in the art will appreciate that the embodiments describe the present invention by way of example and are not intended to limit the scope of protection claimed in the present application. The experimental methods in the embodiments are conventional methods unless otherwise specified. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0229] The sources of the main reagents involved in the following examples are as follows:

[0230] The materials required for cloning construction related reagents are as follows: DNA polymerase (TaKaRa, R040A), DNA recovery kit (TianGen, DP214-03), plasmid mini-extraction kit (TianGen, DP103-03), plasmid large-extraction kit (QIAGEN, 12663), 5 tubes of Gibson assembly premix (NEB, E2611L), DNA marker (ThmeroFisher, SM0331), agarose (Biowest, BW-R0100);

[0231] The materials required for large-scale protein expression are as follows: peptone (BiSIGMA-ALDRICH, T7293-1KG), yeast powder (OXOID, LP0021B), sodium chloride (Xilong Chemical, 10011012AR), IPTG (Inalco, 1758-1400);

[0232] The media required for protein purification are as follows: SP SEPHAROSE FAST FLOW (GE Healthcare, 17-0729-01), NI SEPHAROSE (GE Healthcare, 17-5268-02).

[0233] The reagents required for protein purification and storage are as follows: glycerol / propylene glycol / C 3 H 8 O 3 (SIGMA-ALDRICH, G5516), KCl (Xilong Chemical, 1002007), NaCl (Xilong Chemical, 1001012AR), Na 2 HPO 4 12H 2 O(Xilong Chemical, 1001067AR), KH 2 PO 4 (Xilong Chemical, 1002048AR500), imidazole (SIGMA-ALDRICH, V900153), Tris base (Seebio, 183995), glucose (Xilong Chemical, 1064008AR500), BCA protein concentration determination kit (TianGen, PA115-02);

[0234] Reagents required for cell culture: FBS (GIBCO, 10099-133), RPMI 1640 (Source Culture, L220KJ) DMEM (GIBCO, 11965092), trypsin (AMRESCO, 0458);

[0235] The relevant plasmids used in the experiment were synthesized by the companies (Sangon Biotech and Universal Biotech).

[0236] Flow cytometry reagents: The flow cytometry antibodies used in the experiment were purchased from Biolegend (USA), BD biosciences (USA) and MBL (Beijing) Company.

[0237] Other reagents: GM-CSF (Sino Biological, 51048-MNAH), IL-4 (Sino Biological, 51084-MNAE), CpG ODN-2395 (InvivoGen, tlrl-2395-1);

[0238] Cell lines and animals: B16-F10 (mouse melanoma cells) were purchased from ATCC. DC2.4 (mouse DC cell line) was preserved by our laboratory. MC38 (mouse colorectal cell line) was purchased from the Basic Medical Cell Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. B16-GP33 (mouse melanoma cells stably expressing LCMV GP33 epitope) was donated by the University of California, USA. C57BL / 6 mice were purchased from Shanghai Slake Laboratory Animal Co., Ltd.

[0239] Example 1: eTAT fusion expression can improve cross-presentation of antigens

[0240] In previous studies, an intracellular delivery system called eTAT has been developed, which has good cytoplasmic delivery ability. eTAT is described in detail in Chinese patent application CN202011240557.4, international patent application WO2021135647A1, and Yu, S., Yang, H., Li, T. et al. Efficient intracellular delivery of proteins by a multifunctional chimaeric peptide in vitro and in vivo. Nat Commun 12, 5131 (2021). eTAT contains a cell-penetrating peptide (TAT, SEQ ID NO: 1), a pH-sensitive peptide (INF7, SEQ ID NO: 46), a protease recognition sequence (cathepsin L recognition sequence N + furin recognition sequence Ne, SEQ ID NO: 45 + SEQ ID NO: 44), and a multimerization domain (leucine zipper, SEQ ID NO: 36) from N-terminus to C-terminus.

[0241] The inventors of the present application unexpectedly discovered that eTAT has great potential in promoting antigen cross-presentation and studied its immunopotentiating effect as a vaccine delivery vector.

[0242] 1.1 Construction of expression vectors for eTAT-pGP33 fusion protein and other control proteins

[0243] A recombinant protein expression vector containing a cell-penetrating peptide TAT (SEQ ID NO: 1) or eTAT (SEQ ID NO: 3), a lytic tag protein phosphatase 1B (Ppm1b) (SEQ ID NO: 5), and a model antigen epitope GP33 (SEQ ID NO: 7) derived from lymphocytic choriomeningitis virus (LCMV) was constructed. The structural schematic diagrams of each recombinant protein are shown in Figure 1 As shown, the amino acid sequences of each component from N-terminal to C-terminal are shown in Table 2. The construction method is as follows: the nucleic acid sequences of eTAT, Ppm1b and GP33 are obtained by PCR amplification, and the parts are connected by multiple rounds of PCR, and in the last round of PCR, the NdeI restriction site and its overlapping sequence upstream of the NdeI restriction site corresponding to pET-21b(+) are introduced at the 5' end of the fragment by upstream priming, and the HindIII restriction site and its overlapping sequence downstream of the HindIII restriction site corresponding to pET-21b(+) are introduced at the 3' end of the fragment by downstream priming. The pET-21b(+) plasmid is treated with double restriction digestion of NdeI and HindIII. The inserted fragment with overlapping sequences is connected to the restriction digestion vector pET-21b(+) by GIBSON assembly method.

[0244] Table 2: Components of the delivery system-antigen molecule complex

[0245]

[0246] 1.2 Expression and purification of eTAT-pGP33 fusion protein and other control proteins

[0247] The expression plasmid described in 1.1 was transformed into the expression strain E. coli BL21 (DE3); a single colony was selected from the transformed plate and inoculated into 5 ml LB liquid culture medium containing ampicillin resistance and cultured overnight, then 1 ml of the overnight culture was transferred to 500 ml LB liquid culture medium containing ampicillin resistance, and cultured at 37°C, 180 rpm until the OD 600 of the culture was about 0.6, then the inducer IPTG was added to a final concentration of 0.2 mM, and induced at 25°C for 8 hours; after the induction expression was completed, the cells were collected by centrifugation at 25°C, 7000g for 10 minutes; then the cells were resuspended in 10 ml protein purification equilibrium buffer (PBS, 5% glycerol) and ultrasonically disrupted. Then centrifuge and take the supernatant. There are two ways to purify. First, for eTAT-pGP33, load it onto the SP strong cation chromatography column of the protein purification system; then use the protein purification system to elute the target protein with protein elution buffer (PBS+0.2M NaCl~PBS+0.8MNaCl to remove impurities, and PBS+1.2M NaCl to collect the eluted product); for pGP33 and TAT-pGP33, use NI nickel column for purification, and elute the target protein with buffer containing 250mM imidazole. Use Detoxi-Gel pre-packed column to remove endotoxin. The protein concentration can be determined by spectrophotometer or BCA protein concentration determination kit. Each purified fusion protein is stored at -20℃ after aliquoting.

[0248] 1.3 Study on immune response of eTAT-pGP33 fusion protein and other control proteins

[0249] C57BL / 6 mice were subcutaneously immunized with 2 nmol of antigen protein (divided into pGP33, TAT-pGP33 and eTAT-pGP33). A week later, booster immunization was performed with the same dosage as the first immunization. On the 14th day after the first immunization, the mice were euthanized, the spleen was removed, and single cell suspension was prepared by grinding. After lysing the red blood cells with commercial red blood cell lysis buffer, the obtained cells were used to detect T cells that specifically secrete IFN-γ. 5*10 of the above obtained cells were taken 6 Resuspend the cells in 1 ml RPMI1640 + 10% FBS, add 5 μg / ml stimulatory peptide (GP33: KAVYNFATM), mix well and culture in a 37°C cell culture incubator for 2 hours, then add 1 μl of protein transport inhibitor BD GolgiPlug TM , and continue to culture for 4 hours after mixing. Collect the cells into EP tubes, centrifuge at 400g to collect the cell pellet, wash and resuspend in 100μl PBS + 2% FBS, add CD8 antibody and dead and live dye Aqua for staining, fix and permeabilize the cells after washing, wash again, and then stain with IFN-γ antibody for 20 minutes, and use for flow analysis after washing. The results are as follows Figure 2 As shown in the figure, the specific T cell content after immunization with TAT-pGP33 and eTAT-pGP33 was higher than that of the pGP33 group, indicating that the fusion expression of the cell-penetrating peptide and the antigen can indeed enhance the specific T cell response of the antigen. Among them, the T cell response level of eTAT-pGP33 was further improved compared with TAT-pGP33. The above results fully demonstrate that eTAT is superior to the traditional cell-penetrating peptide TAT and significantly improves the cross-presentation of antigens.

[0250] Example 2: Analysis of the interaction between eTAT fusion expressed antigen and CpG

[0251] On the basis of proving that eTAT is an effective antigen delivery carrier, the next step is to conduct a study on the coordination of adjuvants. In cancer vaccine applications, the choice of adjuvant is crucial for the anti-tumor effect of cancer vaccines. CpG is a TLR-9 agonist that shows good clinical effectiveness. The inventors of this application found that positively charged eTAT can be combined with negatively charged CpG, and when the antigen (Ag) is fused with eTAT and mixed with CpG, the co-delivery of antigen adjuvants can be achieved. The inventors then analyzed their interaction by agarose gel electrophoresis shift experiment.

[0252] Prepare a 3% agarose gel with 1 / 10,000 AG-CelRed nucleic acid gel dye, mix the fusion protein obtained in 1.2 above with CpG (CpG ODN 2395) at different molar ratios (calculated as monomers), centrifuge the mixed product, add it to the gel wells for electrophoresis. Figure 3 As shown in the figure, we found that even when the fusion antigen / CpG molar ratio was 8:1, the binding interaction between the control pGP33 and CpG was very weak; in contrast, when the fusion antigen / CpG molar ratio was 2:1 and above, almost 100% of CpG bound to eTAT-pGP33, and it was much stronger than TAT-pGP33. The above results show that the antigen fused with eTAT can achieve efficient binding with the CpG adjuvant through electrostatic interaction, and its binding efficiency is significantly better than that of the conventional transmembrane peptide TAT.

[0253] Example 3: Intracellular co-delivery of antigen and adjuvant based on eTAT fusion expression

[0254] The realization of cross-presentation requires the entry of antigens into cells, and the CpG adjuvant also needs to enter cells to work. The co-delivery of antigens and adjuvants into the same cell can maximize the activation of immune responses. Therefore, based on Example 1, we further analyzed the intracellular co-delivery of eTAT fusion expression and CpG adjuvant.

[0255] Label the fusion expressed antigen with Alexa FluorTM 647 fluorescein, CpG labeled TAMRA fluorescein, CpG and fluorescently labeled GP33-related antigens (pGP33, TAT-pGP33, eTAT-pGP33) were mixed in DC2.4 cells at a molar ratio of antigen:CpG = 2:1, and then added to the cells for 3 hours. The co-delivery of antigen and CpG was studied by flow cytometry. Figure 4 As shown in A, compared with the conventional formula of pGP33 mixed with CpG, the fusion expression of eTAT resulted in a 20-fold and 18-fold increase in the intracellular delivery efficiency of antigen (Ag) and CpG, and was significantly better than TAT fusion expression. Figure 4 As shown in B, almost 100% of the cells loaded with the eTAT system showed double-positive characteristics of Ag+CpG+, further illustrating that the fusion expression of eTAT and antigen can effectively promote the intracellular co-delivery of antigen and CpG adjuvant.

[0256] Example 4: eTAT promotes lymph node co-delivery of fusion protein and adjuvant

[0257] In the process of vaccine immune activation, lymph nodes are the main site of immune response. Whether it is humoral immunity or cellular immune response, lymph node delivery is a necessary condition. The delivery efficiency determines the strength of the immune response. Therefore, we further studied the delivery of Ag / CpG in vivo, especially the efficiency of lymph node delivery. The fused expressed antigen was labeled with Alexa Fluor TM 647 fluorescein, CpG-labeled TAMRA, then 2 nmol antigen and 1 nmol CpG were mixed for 30 minutes and injected subcutaneously into C57BL / 6 mice. After 24 hours, the inguinal lymph nodes and other major organs were harvested for fluorescence imaging. Figure 5 As shown in A and 5B, the TAT-pGP33 preparation mixed with CpG showed a weaker Ag / CpG signal in the lymph nodes similar to that of pGP33+CpG. In contrast, mice immunized with a mixture of eTAT-pGP33 and CpG showed significantly enhanced Ag and CpG fluorescence signals in the lymph nodes (dLNs). In addition, fluorescence imaging observations were also performed on other major organs besides lymph nodes, such as Figure 6As shown, it shows the antigen fluorescence signal 24 hours after injection. Figure A is the fluorescence imaging of each organ, and Figure B is the corresponding fluorescence intensity value. In the eTAT-pGP33+CpG group, the signal density of the lymph nodes was significantly higher than that of other organs, while the antigen signal in the other two control groups after immunization did not increase significantly in the lymph nodes. Further analysis by flow cytometry showed that the ratio of Ag+CpG+ double-positive antigen-presenting cells in dLNs (mainly DC cells and macrophages, DC cells were stained with CD11c fluorescent antibody for 20 minutes, and macrophages were stained with F4 / 80 fluorescent antibody) was more than 20 times higher than that in the pGP33+CpG group ( Figure 7 ). These results indicate that the fusion expression of Ag and eTAT enables the efficient co-delivery of Ag and CpG into dLNs.

[0258] Example 5: Study on the cellular immune response level of the eTAT vaccine system

[0259] After observing the efficient co-delivery of antigens and adjuvants to lymph nodes of the eTAT vaccine system, the cellular immune response level of the eTAT vaccine system was further evaluated. 2nmol of antigen (divided into pGP33, TAT-pGP33 and eTAT-pGP33) was subcutaneously immunized in C57BL / 6 mice with or without the addition of 1nmol of CpG. A booster immunization was performed one week later with the same dosage as the first immunization. On the 14th day after the initial immunization, the mice were euthanized, the spleen was removed, and a single cell suspension was prepared by grinding. After the red blood cells were lysed with a commercial red blood cell lysis solution, the obtained cells were used to analyze GP33-specific T cells. There are two analysis methods. The first is to use a tetramer that recognizes GP33-specific T cells for staining. Take 1*10 of the above-obtained cells. 6 The cells were resuspended in PBS + 2% FBS buffer, stained with tetramer for 30 minutes, and then further stained with CD8 antibody. Finally, the dead and live dye Aqua was added for staining. After washing, the cells were filtered with a 0.75 μm mesh for flow cytometry analysis. Another analysis method is to detect T cells that specifically secrete IFN-γ. 5*10 cells were obtained as above. 6 Resuspend the cells in 1 ml RPMI 1640 + 10% FBS, add 5 μg / ml stimulatory peptide (GP33: KAVYNFATM), mix well and culture in a 37°C cell culture incubator for 2 hours, then add 1 μl of protein transport inhibitor BD GolgiPlug TM, and continue to culture for 4 hours after mixing. Collect the cells into EP tubes, centrifuge at 400g to collect the cell pellet, wash and resuspend with 100μl PBS+2% FBS, add CD8 antibody and dead-live dye Aqua for staining, fix and permeabilize the cells after washing, wash again, and then stain with IFN-γ antibody for 20 minutes, and use for flow analysis after washing.

[0260] The results are as follows Figure 8 As shown, Figure 8 A is the quantitative analysis of GP33-specific CD8 T cells stained with tetramer. Figure 8 B is the quantitative analysis of CD8 T cells that specifically secrete IFN-γ. First, in the group without CpG, the specific T cell response after eTAT-pGP33 immunization was higher than that of pGP33 and TAT-pGP33. After further immunization with CpG, the CD8 + IFN-γ + The number of GP33-specific CD8+T cells stained with tetramers increased by nearly 300%, which was significantly higher than that of the other two groups (CpG+pGP33 group and CpG+TAT-pGP33 group) (≤70%), indicating that the adjuvant effect of CpG was significantly improved. The above results fully demonstrate that eTAT fusion expression can significantly improve the response level of the vaccine.

[0261] Example 6: Establishment of a universal eTAT vaccine system

[0262] Tumors can acquire a large number of cell mutations during transformation and development, thereby generating diverse tumor antigens. Although the fusion of antigens with eTAT can significantly improve the response efficiency of cancer vaccines, it is still a challenge to cater to the diversity and heterogeneity of tumor antigens with flexibility. To overcome this problem, we utilized a protein cross-linking system composed of SpyTag / SpyCatcher molecular pairs. Fig. 9 As shown, SpyCatcher (Catcher) and eTAT are fused and expressed to form eTAT-Catcher protein, and the epitope peptide labeled by SpyTag can be rapidly covalently bound to eTAT-Catcher through the formation of covalent bonds.

[0263] 6.1 Construction of expression vectors for eTAT-Catcher fusion protein and other control proteins

[0264] The amino acid sequences of each component of each recombinant protein from N-terminus to C-terminus are shown in Table 3. The method for constructing the recombinant protein expression vector is as follows: the nucleic acid sequences of eTAT and Catcher are obtained by PCR amplification, and the various parts are connected by multiple rounds of PCR, and in the last round of PCR, the NdeI restriction site and its overlapping sequence upstream of the NdeI restriction site corresponding to pET-21b(+) are introduced at the 5' end of the fragment by the upstream primer, and the HindⅢ restriction site and its overlapping sequence downstream of the HindⅢ restriction site corresponding to pET-21b(+) are introduced at the 3' end of the fragment by the downstream primer. The pET-21b(+) plasmid is treated with double restriction enzymes of NdeI and HindⅢ. The inserted fragment with overlapping sequences is connected to the restriction vector pET-21b(+) by GIBSON assembly.

[0265] Table 3: Components of the delivery system-antigen molecule complex

[0266]

[0267] 6.2 Expression and purification of eTAT-Catcher fusion protein and other control proteins

[0268] The expression plasmid described in 6.1 was transformed into the expression strain E. coli BL21 (DE3); a single colony was selected from the transformed plate and inoculated into 5 ml LB liquid culture medium containing ampicillin resistance and cultured overnight, then 1 ml of the overnight culture was transferred to 500 ml LB liquid culture medium containing ampicillin resistance, and cultured at 37°C, 180 rpm until the OD 600 of the culture was about 0.6, then the inducer IPTG was added to a final concentration of 0.2 mM, and induced at 25°C for 8 hours; after the induction expression was completed, the cells were collected by centrifugation at 25°C, 7000g for 10 minutes; then the cells were resuspended in 10 ml protein purification equilibrium buffer (PBS, 5% glycerol) and ultrasonically disrupted. Then centrifuge and take the supernatant. There are two ways to purify. First, for eTAT-Catcher, load it onto the SP strong cation chromatography column of the protein purification system; then use the protein purification system to elute the target protein with protein elution buffer (PBS+0.2M NaCl~PBS+0.8M NaCl to remove impurities, and PBS+1.2M NaCl to collect the eluted product); for Catcher and TAT-Catcher, use NI nickel column for purification, and elute the target protein with buffer containing 250mM imidazole. Use Detoxi-Gel pre-packed column to remove endotoxin. The protein concentration can be determined by spectrophotometer or BCA protein concentration determination kit. Each purified fusion protein is stored at -20℃ after aliquoting.

[0269] 6.3 Evaluation of the ligation efficiency of eTAT-Catcher and SpyTag-Ag and product purification

[0270] GP33 was selected as the model antigen, and its N-terminus was connected to SpyTag (SEQ ID NO: 9) to form SpyTag-Ag. Catcher, TAT-Catcher, eTAT-Catcher and excess SpyTag-GP33 were mixed, incubated at 4°C for 30 minutes, and purified using a 10kD pore size ultrafiltration centrifuge tube to remove excess SpyTag-GP33 polypeptide. The samples before and after purification were subjected to SDS-PAGE polyacrylamide gel electrophoresis. The results are as follows: Fig.10 As shown in the figure, the connection between the three Catcher-related proteins and SpyTag-GP33 is very thorough, and no Catcher-related proteins remain. After purification by ultrafiltration tube, the remaining SpyTag-GP33 polypeptide can be effectively removed, and the purified product obtained has a single band and high purity. The above results show that the connection reaction between eTAT-Catcher and SpyTag-Ag is fast and complete, which can replace the fusion expression method to achieve effective series connection between eTAT and antigen.

[0271] 6.4 Analysis of the interaction between eTAT-Catcher and its ligation products and CpG

[0272] A 3% agarose gel with 1 / 10,000 AG-CelRed nucleic acid gel dye was prepared, and the eTAT-Catcher fusion protein and its connection purification product with the SpyTag-GP33 polypeptide (eTAT-Catcher:SpyTag-GP33) were mixed with CpG at a protein:CpG molar ratio of 2:1 and 4:1. The mixed product was centrifuged and subjected to agarose gel electrophoresis. Fig.11 As shown, consistent with previous results, eTAT-Catcher or its ligation product eTAT-Catcher:SpyTag-GP33 and CpG can fully bind to each other when the molar ratio is 2:1 or above; however, Catcher and its ligation product Catcher:SpyTag-GP33 cannot effectively bind to CpG.

[0273] 6.5 Evaluation of the lymph node delivery efficiency of the universal vaccine system based on eTAT-Catcher

[0274] SpyTag-GP33 was labeled with fluorescein FAM, and Catcher: SpyTag-GP33 and eTAT-Catcher: SpyTag-GP33 fusion antigens with FAM were prepared by ligation reaction and subsequent ultrafiltration tube purification. 2 nmol of antigen plus 1 nmol of CpG-TAMRA were mixed and injected subcutaneously into C57BL / 6 mice. Lymph nodes were collected 24 hours later. Single cell suspensions were prepared for flow cytometry analysis, and DC cells and macrophages were labeled with CD11c and F4 / 80 antibodies. After washing, they were analyzed by flow cytometry. The results are shown in Fig.12 As shown, the number of cells double-positive for antigen and CpG adjuvant in the lymph nodes of the eTAT-Catcher:SpyTag-GP33+CpG group was significantly higher than that of the SpyTag-GP33+CpG and Catcher:SpyTag-GP33+CpG groups, indicating that the universal vaccine system based on SpyCatcher&SpyTag is consistent with the previous fusion expression and can achieve effective lymph node co-delivery of Ag and CpG.

[0275] 6.6 Evaluation of specific T cell response levels of the universal vaccine system based on eTAT-Catcher

[0276] like Fig.13 The group setting shown in the figure was that the immune preparation was prepared according to the dosage of 2nmol antigen + 1nmol CpG adjuvant. After 2 injections of C57BL / 6 mice were immunized 1 week apart, 100 μl of mouse orbital blood was collected using lithium heparin anticoagulant, and the cells were lysed using commercial red blood cell lysis buffer. The obtained cells were used to analyze T cells that specifically secreted IFN-γ and TNF-α after GP33 epitope stimulation. The process was as described in the method of Example 5 above. The results are shown in Fig.13As shown, the eTAT-based vaccine system (eTAT-Catcher: SpyTag-GP33 + CpG) induced the production of approximately 15% Ag-specific CD8 + T cells, which was 10 times higher than the conventional peptide formula SpyTag-GP33 + CpG, 8 times higher than Catcher: SpyTag-GP33 + CpG, and 4 times higher than TAT-Catcher: SpyTag-GP33 + CpG. However, when the naked GP33 peptide without the SpyTag tag was mixed with the eTAT-Catcher instead of being connected, the immunogenicity was significantly reduced, indicating that the connection with eTAT is indispensable. It is worth noting that the frequency of CTL induced by the eTAT-based vaccine is also significantly higher than that of the formulation based on one of the strongest adjuvant systems in clinical tumor vaccines, that is, the system of emulsifying Ag and CpG with the oil adjuvant Montanide (Mon). Even when the dose of epitope peptide is increased by 5 times (SpyTag-GP33 (500%) + Mon + CpG), it is significantly lower than the eTAT system. The above results fully demonstrate that the universal vaccine system based on eTAT is consistent with fusion expression and can significantly improve the T cell response level of the "protein + CpG" vaccine system.

[0277] In addition to the GP33 model antigen, human tumor antigens E738-57 (SEQ ID NO: 18) and CEA 567～584 (SEQ ID NO: 19) was used as the target antigen for vaccine system evaluation. These two antigens were synthesized in series with SpyTag to form SpyTag-E7 38～57 and SpyTag-CEA 567～584 , further mixed with eTAT-Catcher protein for connection, incubated at 4°C for 30 minutes, purified using a 10kD pore size ultrafiltration centrifuge tube to remove excess polypeptide antigens, and the purified samples were subjected to SDS-PAGE polyacrylamide gel electrophoresis. The results are as follows Fig.14 As shown in A, the purified product obtained has a single band and high purity. The groups included the traditional peptide vaccine system and the eTAT vaccine system. The immune preparation was prepared according to the dosage of 2nmol antigen + 1nmol CpG adjuvant. After immunizing C57BL / 6 mice twice with an interval of 1 week, heparin lithium anticoagulant was used to collect mouse orbital blood. After the cells were lysed with commercial red blood cell lysis buffer, the corresponding T cell epitope peptides were used for stimulation. The epitope peptides were E7 49～57 (SEQ ID NO: 20) and CEA 572～579 (SEQ ID NO: 21), the stimulation concentration was 5 μg / ml, and T cells that specifically secreted IFN-γ and TNF-α were detected, and the process was as described in the method in Example 5 above.

[0278] The results are as follows Fig.14 B and Fig.14 As shown in C, for E7 38～57 Antigen, conventional SpyTag-E7 38～57 +CpG peptide vaccine system has a low response, and cytokine-positive CD8 + The proportion of T is less than 1%. eTAT:SpyTag-E7 38～57 IFN-γ after immunization with CpG system + CD8 + The proportion of T cells is about 20%, and IFN-γ + TNF-α + The percentage of multifunctional CD8+T cells reached about 10%, which was significantly higher than that of the conventional peptide system (P<0.0001) ( Fig.14 B) In addition, for CEA 567～584 Antigen, IFN-γ in the eTAT system + CD8 + T and IFN-γ + TNF-α + CD8 + T cells also exceeded 20% and 10%, respectively, which is about 30 times higher than that of the peptide system ( Fig.14 C). The above results fully demonstrate that the vaccine system based on eTAT-Catcher has the effect of widely enhancing the immunogenicity of tumor antigens and can induce strong antigen-specific T cell responses.

[0279] Example 7: CD8 of the eTAT vaccine system based on CpG 1826 + T cell response assessment

[0280] In the above examples, the type of CpG adjuvant is ODN 2395, which belongs to CpG class. In order to study whether the eTAT vaccine system delivers nucleic acid adjuvants with a broad immunogenicity enhancement effect, the CD8 + T cell response. GP33 was used as the evaluation antigen. The groups included conventional peptide vaccine system (SpyTag-GP33+CpG1826), clinical double adjuvant vaccine system based on Montanide (Mon) emulsification (SpyTag-GP33+CpG1826+Mon), and eTAT vaccine system (eTAT-Catcher: SpyTag-GP33+CpG1826). The immune preparation was prepared according to the dosage of 2nmol antigen + 1nmol CpG adjuvant, and the immunization time was as follows. Fig.15As shown in A, after immunizing C57BL / 6 mice twice with an interval of 12 days, heparin lithium anticoagulant was used on the 19th day to collect mouse orbital blood, and the cells were lysed with commercial red blood cell lysis buffer, and then stimulated with GP33 epitope peptide to detect CD8 + T cells, the process is as described in the method in Example 5 above.

[0281] The results are as follows Fig.15 B and Fig.15 As shown in C, the CpG1826-based eTAT vaccine system can activate about 3% of GP33-specific IFN-γ + CD8 + T cells, the ratio is more than twice that of conventional vaccine system and double adjuvant vaccine system; in addition, the IFN-γ induced by the eTAT vaccine system + TNF-α + Pluripotent CD8 + The proportion of T cells was also significantly higher than that of the other two groups, about 4 times. The above results fully demonstrate that the eTAT vaccine system can be used to deliver other types of CpG, which can also significantly improve the immunogenicity of the vaccine and enhance the antigen-specific T cell response.

[0282] Example 8: Application of eTAT vaccine system in tumor prevention

[0283] To evaluate the immune memory effect of the vaccine, C57BL / 6 mice were subcutaneously inoculated with 5*10 5 B16-GP33 melanoma cells (B16-F10 cell line stably expressing GP33 epitope) were used for the specific time points. Fig.16 As shown in A, there were 8 mice in each group. The growth of tumors in mice was observed and the tumor size was recorded every 2 days. Fig.16 As shown in B and C, after 60 days of tumor bearing, only 1 of 8 mice immunized with the eTAT vaccine system (eTAT-Catcher: SpyTag-GP33+CpG) grew tumors, with a complete protection rate of more than 85%, while almost all mice in other groups had tumor growth, which fully demonstrated that the eTAT vaccine system has a highly effective tumor prevention effect after immunization. This is related to the strong immune response induced by the eTAT vaccine system.

[0284] Example 8: Application of eTAT vaccine system in tumor treatment

[0285] 8.1 Evaluation of tumor therapeutic effects based on tumor pattern antigens

[0286] On the basis of effective tumor prevention, the tumor treatment effect of the universal vaccine system was further evaluated, first based on the model antigen GP33. In order to block the immunosuppressive PD-1 / PD-L1 pathway, the vaccine was combined with anti-PD-1 therapy. The specific time is as follows Fig.17 As shown in A, each C57BL / 6 mouse was injected subcutaneously at the back with 5*10 5 Each group of mice received 3 injections of vaccine (2 nmol GP33-related antigen + 1 nmol CpG 2395 adjuvant, 4 days between each injection); PD1 antibody was injected intraperitoneally, 100 μg per mouse, for a total of 5 injections. Fig.17 As shown in BD, first of all, when the vaccine is used alone for treatment, the eTAT vaccine system (eTAT: SpyTag-GP33 + CpG) can significantly inhibit the growth of mouse tumors, and can effectively reduce the tumor volume in the middle of the treatment. However, the conventional peptide formula (SpyTag-GP33 + CpG) has little inhibitory effect on tumor growth. When eTAT: SpyTag-GP33 + CpG and anti-PD-1 antibodies are further combined for immunotherapy (eTAT: SpyTag-GP33 + CpG + PD-1), in the more aggressive model of melanoma, 25% of mouse tumors can be completely regressed, proving that the eTAT-based vaccine and PD-1 antibody treatment have good synergistic effects. On the contrary, the conventional peptide formula combined with PD-1 antibody (SpyTag-GP33 + CpG + PD-1) has little effect. The above results fully demonstrate that the eTAT system can significantly improve the immunogenicity of the model antigen vaccine and greatly enhance the tumor efficacy.

[0287] 8.2 Evaluation of the therapeutic effect of the neoantigen-based eTAT vaccine system on MC38 tumors

[0288] In addition to evaluating the model antigens, we also evaluated the tumor neoantigens with relatively weak immunogenicity. We selected two neoantigens RepS1 (SEQ ID NO: 26) and Adpgk (SEQ ID NO: 27) from the mouse colorectal cell line MC38 as immunogens. Six-week-old C57BL / 6 female mice were injected subcutaneously on the back with 5*10 5MC38 cells were added, and then the treatment was carried out, with 8 mice in each group. The vaccine included the traditional peptide vaccine Ags+CpG (SpyTag-RepS1+SpyTag-Adpgk+CpG), and eTAT:Ags+CpG (i.e., a mixture of eTAT:SpyTag-RepS1, eTAT:SpyTag-Adpgk and CpG) prepared based on the eTAT-Catcher universal system. Each shot of the vaccine included the equivalent of 2nmol Adpgk antigen, 2nmol RepS1 antigen and 2nmol CpG adjuvant, for a total of 3 shots. In order to study the effect of the vaccine combined with immune checkpoint inhibitors, the therapeutic effect of the combined PD1 antibody was also studied. The antibody was injected intraperitoneally, 100μg per mouse, for a total of 5 shots. The specific treatment time points are as follows: Fig.18 As shown in A. First, on the 23rd day of treatment, orbital blood was collected from mice using lithium heparin anticoagulant, and red blood cells were lysed to detect antigen-specific T cell responses. 5*10 6 Resuspend the cells in 1 mL RPMI 1640 + 10% FBS, add 5 μg / mL of stimulatory peptide RepS1 or Adpgk, mix well and culture in a 37°C cell culture incubator for 2 hours, then add 1 μL of protein transport inhibitor BD GolgiPlug TM , mix well and continue to culture for 4 hours. Collect cells into EP tubes, centrifuge at 400g to collect cell pellets, wash and resuspend with 100μl PBS + 2% FBS, add CD8 antibody and dead and live dye Aqua for staining, fix and permeabilize cells after washing, wash again, and then stain with IFN-γ antibody for 20 minutes, wash and use for flow cytometry analysis. Fig.18 As shown in B, it can be seen that after immunization in the eTAT:Ags+CpG group, the number of RepS1 and Adpgk-specific T cells stimulated were significantly higher than those in the Ags+CpG group.

[0289] Tumor treatment results Fig.18 As shown in CF, first, when the vaccine is used alone for treatment, the eTAT vaccine system (eTAT: Ags + CpG) can significantly inhibit the growth of mouse tumors, and the tumor of 1 mouse out of 8 mice was completely eliminated. In contrast, the conventional peptide vaccine Ags + CpG has a negligible inhibitory effect on tumor growth, and no mouse can be completely cured. After further combination with PD1 antibodies, the eTAT vaccine group was able to completely cure 50% of the mice and achieve complete elimination of the tumor. However, the combination of the peptide vaccine system with PD1 antibodies has limited therapeutic effects, and the inhibitory effect on the growth of mouse tumors is not significant enough.

[0290] For the cured mice in the eTAT:Ags+CpG+PD1 group, MC38 cells were used to bear tumors again on day 80, with the number of cells being 5*10 per mouse. 5 At the same time, blank mice of similar age were also loaded with tumors as controls, and the size of the tumors was recorded. Fig.18 As shown in G and H, the tumor volume of cured mice in the eTAT:Ags+CpG+PD1 group was significantly smaller than that in the control group, and 50% of the cured mice showed no tumor growth when they were re-bearing tumors, indicating that the anti-tumor immune response stimulated by the eTAT vaccine system has a strong memory effect.

[0291] 8.3 Evaluation of the therapeutic effect of the eTAT vaccine system based on tumor-associated antigens and neoantigens on malignant melanoma

[0292] In addition to the MC38 tumor model, we also evaluated the anti-tumor effect of the eTAT vaccine system in the more malignant B16-F10 mouse melanoma. Two tumor-associated antigens Trp1 (SEQID NO: 23) and GP100 (SEQ ID NO: 24) of the mouse melanoma cell line B16-F10, as well as a tumor neoantigen M27 (SEQ ID NO: 25) were selected as immunogens. These three antigens were synthesized in series with SpyTag to form SpyTag-Trp1, SpyTag-GP100 and SpyTag-M27, which were further mixed with eTAT-Catcher protein for connection. After incubation at 4°C for 30 minutes, they were purified using a 10kD pore size ultrafiltration centrifuge tube to remove excess polypeptide antigens. The purified samples were subjected to SDS-PAGE polyacrylamide gel electrophoresis. The results are shown in Figure 2. Fig.19 As shown in A, the purified product has a single band and high purity. The vaccine includes the traditional peptide vaccine Ags+CpG (SpyTag-Trp1+SpyTag-GP100+SpyTag-M27+CpG), and the eTAT:Ags+CpG (i.e., a mixture of eTAT-Catcher:SpyTag-Trp1, eTAT-Catcher:SpyTag-GP100, eTAT-Catcher:SpyTag-M27 and CpG 2395) prepared based on the eTAT-Catcher universal system. Each vaccine includes the equivalent of 2nmol Trp1 antigen, 2nmol GP100 antigen, 2nmol M27 antigen and 3nmol CpG adjuvant, and the molar ratio of total antigen to CpG follows 2:1.

[0293] First, the treatment of subcutaneous tumors was studied. 6-8 week old C57BL / 6 female mice were injected subcutaneously on the back with 5*10 5B16-F10 cells were used for treatment, and 6 mice were treated in each group. Fig.19 As shown in B, the vaccine was subcutaneously injected on the 5th, 9th and 13th day after tumor loading, for a total of 3 injections, and the tumor volume was monitored every two days. First, on the 17th day of treatment, orbital blood was collected from mice using lithium heparin anticoagulant, and red blood cells were lysed to detect antigen-specific T cell responses. 5*10 6 Resuspend the cells in 1 mL RPMI 1640 + 10% FBS and plate them in a 12-well plate. Add 5 μg / mL of stimulatory peptides Trp1, GP100 or M27 to each well, mix well and culture in a 37°C cell culture incubator for 2 hours. Then add 1 μL of the protein transport inhibitor BD GolgiPlug TM , mix well and continue to culture for 4 hours. Collect cells into EP tubes, centrifuge at 400g to collect cell pellets, wash and resuspend with 100μL PBS + 2% FBS, add CD8 antibody and dead-live dye Aqua for staining, fix and permeabilize cells after washing, wash again, and then stain with IFN-γ antibody for 20 minutes, wash and use for flow cytometry analysis. Fig.19 As shown in C, it can be seen that after immunization with the eTAT:Ags+CpG group, activated Trp1, GP100 and M27-specific CD8 + The number of T cells in the group was significantly higher than that in the Ags+CpG group, and the total positive cell ratio was about 4 times that of the peptide vaccine group, indicating that the eTAT vaccine system significantly improved the immunogenicity of tumor-associated antigens and neoantigens, and effectively enhanced their CD8 + T cell response. Tumor treatment results such as Fig.19 As shown in DE, the conventional peptide vaccine system (Ags+CpG) had almost no tumor inhibitory effect, while the eTAT vaccine system (eTAT:Ags+CpG) could significantly inhibit the growth of mouse tumors, with tumors less than 100 mm in the first 20 days. 3 , effectively prolonging the survival of mice, demonstrating a powerful therapeutic effect on malignant melanoma.

[0294] Malignant melanoma is highly invasive and prone to tumor metastasis in clinical practice. How to treat metastatic melanoma is a major difficulty. Therefore, in addition to the treatment of subcutaneous melanoma, the above-mentioned eTAT vaccine system is also studied for the treatment of metastatic melanoma to explore its application potential. Fig. 20 As shown in A, 5*10 5B16-F10 cells were injected into mice through the tail vein. On the 3rd, 7th and 11th days, the mice were treated by subcutaneous injection using the conventional peptide vaccine system (Ags+CpG) and eTAT vaccine system (eTAT:Ags+CpG) containing the above three antigens. The mice were euthanized on the 20th day and the melanoma metastasis in their lungs and backs was observed. The treatment results are shown in Figure 2. Fig. 20 As shown in B, mice treated with the conventional peptide vaccine system had obvious metastatic melanoma in the lungs and back, which was not significantly different from the PBS control group. However, mice treated with the eTAT vaccine system had no obvious melanoma in the lungs and back, indicating that it can effectively inhibit the development of metastatic melanoma.

[0295] Based on the above results, it is shown that the eTAT vaccine system based on tumor-associated antigens and neoantigens can effectively treat subcutaneous solid malignant melanoma and hematogenous metastatic melanoma, showing good application potential.

[0296] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

[0297] References

[0298] 1.Finn,OJ,The dawn of vaccines for cancer prevention.Nat RevImmunol,2018.18(3):p.183-194.

[0299] 2. Falzone, L., S. Salomone, and M. Libra, Evolution of CancerPharmacological Treatments at the Turn of the Third Millennium. FrontPharmacol, 2018.9: p.1300.

[0300] 3. Shemesh, CS, et al., Personalized Cancer Vaccines: ClinicalLandscape, Challenges, and Opportunities. Mol Ther, 2021.29(2):p.555-570.

[0301] 4.Mehta,N.K.,et al.,Pharmacokinetic tuning of protein-antigen fusionsenhances the immunogenicity of T-cell vaccines.Nat Biomed Eng,2020.4(6):p.636-648.

[0302] 5.Rahma,O.E.,et al.,Is the"3+3"dose-escalation phase I clinical trialdesign suitable for therapeutic cancer vaccine development?A recommendationfor alternative design.Clin Cancer Res,2014.20(18):p.4758-67.

[0303] 6.Tan,A.C.,A.Goubier,and H.E.Kohrt,A quantitative analysis oftherapeutic cancer vaccines in phase 2 or phase 3 trial.J Immunother Cancer,2015.3:p.48.

[0304] 7.Colbert,J.D.,F.M.Cruz,and K.L.Rock,Cross-presentation of exogenousantigens on MHC I molecules.Curr Opin Immunol,2020.64:p.1-8.

[0305] 8.van der Burg,S.H.,Correlates of immune and clinical activity ofnovel cancer vaccines.Semin Immunol,2018.39:p.119-136.

[0306] 9.Tran,T.,et al.,Therapeutic cancer vaccine:building the future fromlessons of the past.Semin Immunopathol,2019.41(1):p.69-85.

[0307] 10.Tacken,P.J.,et al.,Dendritic-cell immunotherapy:from ex vivoloading to in vivo targeting.Nat Rev Immunol,2007.7(10):p.790-802.

[0308] 11.Mosquera,J.,I.García,and L.M.Liz-Marzán,Cellular Uptake ofNanoparticles versus Small Molecules:A Matter of Size.Acc Chem Res,2018.51(9):p.2305-2313.

[0309] 12.Mi,Y.,et al.,Emerging Nano- / Microapproaches for CancerImmunotherapy.Adv Sci(Weinh),2019.6(6):p.1801847.

[0310] 13.Kuai,R.,et al.,Designer vaccine nanodiscs for personalized cancerimmunotherapy.Nat Mater,2017.16(4):p.489-496.

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[0313] 16.Erazo-Oliveras,A.,et al.,Improving the endosomal escape of cell-penetrating peptides and their cargos:strategies andchallenges.Pharmaceuticals(Basel),2012.5(11):p.1177-1209。

Claims

1. A compound comprising: (i) a protein component comprising a carrier peptide and a cargo peptide; wherein, The carrier peptide comprises a multimerization domain sequence, a cell-penetrating peptide, a pH-sensitive fusion peptide, and a protease recognition sequence; the cargo peptide comprises an antigenic peptide; (ii) a nucleic acid component comprising a CpG adjuvant.

2. The complex according to claim 1, wherein The protein component and the nucleic acid component are conjugated via electrostatic interaction.

3. The complex according to claim 1 or 2, wherein The cargo peptide in the protein component is optionally fused to the N-terminus or C-terminus, such as the C-terminus, of the carrier peptide via a linker.

4. The complex according to claim 1 or 2, wherein The carrier peptide in the protein component is optionally connected to a first member of a protein-protein binding pair through a linker to form a first protein, and the cargo peptide is optionally connected to a second member of the protein-protein binding pair through a linker to form a second protein; wherein the first member and the second member are bound by a covalent bond; Preferably, the covalent bond is an isopeptide bond; Preferably, the protein-protein binding pair is selected from: (i) SpyTag:SpyCatcher pair, (ii) SpyTag:KTag pair, (iii) Isopeptag:pilin-C pair, (iv) SnoopTag or SnoopTagJr:SnoopCatcher pair, (v) SpyTag002:SpyCatcher002 pair, (vi) RrgATag, RrgATag2 or DogTag:RrgACatcher pair, (vii) IsopepTag-N:Pilin-N pair, (viii) PsCsTag:PsCsCatcher pair; Preferably, the protein-protein binding pair is a SpyTag:SpyCatcher pair; for example, the SpyTag comprises the sequence shown in SEQ ID NO:9; for example, the SpyCatcher comprises the sequence shown in SEQ ID NO:

10.

5. The complex according to claim 4, wherein The protein component is formed by connecting a first protein and a second protein via an isopeptide bond, wherein: The first protein comprises the carrier peptide and a first member of the SpyTag:SpyCatcher pair, wherein the first member is optionally connected to the carrier peptide via a first linker (eg, connected to the C-terminus of the carrier peptide); The second protein comprises the cargo peptide and a second member of the SpyTag:SpyCatcher pair, wherein the second member is optionally linked to the cargo peptide via a second linker (eg, linked to the N-terminus of the cargo peptide); Preferably, the first protein comprises the carrier peptide and SpyCatcher, and the second protein comprises the cargo peptide and SpyTag.

6. The complex according to claim 1 or 2, wherein The carrier peptide in the protein component is optionally connected to the first member of the specific binding pair through a linker to form a first protein, and the cargo peptide is optionally connected to the second member of the specific binding pair through a linker to form a second protein; wherein the first member and the second member are bound by a non-covalent bond formed by the specific binding; Preferably, the specific binding pair is selected from the group consisting of: an antibody and its antigen, streptavidin and biotin.

7. The complex according to any one of claims 1 to 6, wherein The multimerization domain is a dimerization domain, a trimerization domain, a tetramerization domain or any higher order multimerization domain; Preferably, the multimerization domain is selected from the group consisting of: leucine zipper, NOE, GCN4-P1, Delta, and any combination thereof; Preferably, the multimerization domain is selected from leucine zippers; Preferably, the multimerization domain sequence comprises the sequence shown in SEQ ID NO:36 or 37.

8. The complex according to any one of claims 1 to 7, wherein The cell-penetrating peptide is selected from the group consisting of penetratin, Tat-derived peptide (e.g., Tat (48-60) or Tat (47-57)), Rev (34-50), VP22, transportan, Pep-1, Pep-7, and any combination thereof; Preferably, the cell-penetrating peptide comprises a Tat-derived peptide, such as Tat(48-60); Preferably, the cell-penetrating peptide comprises the sequence shown in SEQ ID NO:

1.

9. The complex according to any one of claims 1 to 8, wherein The pH-sensitive fusion peptide is selected from influenza virus HA2 or its mutants (such as INF7, KALA or GALA), melittin, and any combination thereof; Preferably, the pH-sensitive fusion peptide comprises INF7; Preferably, the pH-sensitive fusion peptide comprises the sequence shown in SEQ ID NO:

46.

10. The complex according to any one of claims 1 to 9, wherein The protease recognition sequence comprises a furin recognition sequence and / or a cathepsin L recognition sequence; Preferably, the furin recognition sequence comprises R-X1-X2-R (SEQ ID NO: 41), wherein X1 is any amino acid, and X2 is K or R; Preferably, the furin recognition sequence comprises RR-X1-X2-R (SEQ ID NO: 42); Preferably, the furin recognition sequence comprises the sequence shown in SEQ ID NO:43; Preferably, the furin recognition sequence comprises the sequence shown in SEQ ID NO:44; Preferably, the cathepsin L recognition sequence comprises the sequence shown in SEQ ID NO:45; Preferably, the protease recognition sequence comprises SEQ ID NO:43 and SEQ ID NO:45; Preferably, the protease recognition sequence comprises SEQ ID NO:44 and SEQ ID NO:

45.

11. The complex according to any one of claims 1 to 10, wherein The carrier peptide comprises, from the N-terminus to the C-terminus: the cell-penetrating peptide, the pH-sensitive fusion peptide, and the protease recognition sequence, or, from the N-terminus to the C-terminus: the pH-sensitive fusion peptide, the cell-penetrating peptide, and the protease recognition sequence; and the multimerization domain sequence is located at the N-terminus or the C-terminus of the carrier peptide, or between any two adjacent domains mentioned above; Preferably, the protease recognition sequence comprises the furin recognition sequence and the cathepsin L recognition sequence from the N-terminus to the C-terminus, or comprises the cathepsin L recognition sequence and the furin recognition sequence from the N-terminus to the C-terminus; Preferably, the carrier peptide comprises, from N-terminus to C-terminus: the multimerization domain sequence, the cell-penetrating peptide, the pH-sensitive fusion peptide, and the protease recognition sequence; Preferably, the carrier peptide comprises, from N-terminus to C-terminus: the multimerization domain sequence, the pH-sensitive fusion peptide, the cell-penetrating peptide, and the protease recognition sequence.

12. The complex according to any one of claims 1 to 11, wherein Any two adjacent domains contained in the carrier peptide are optionally connected by a peptide linker; Preferably, the peptide linker is (G m S) n , wherein m is selected from an integer of 1-4, and n is selected from an integer of 1-3.

13. The complex according to any one of claims 1 to 12, wherein The carrier peptide comprises the sequence shown in SEQ ID NO:

3.

14. The complex according to any one of claims 1 to 13, wherein The cargo peptide comprises an antigenic peptide derived from a tumor antigen; Preferably, the tumor antigen is selected from tumor-associated antigen (TAA), tumor-specific antigen (TSA) or tumor neoantigen (neoantigen); Preferably, the tumor antigen is a solid tumor antigen, such as a melanoma antigen, a colorectal cancer antigen, a breast cancer antigen, a prostate cancer antigen, a bladder cancer antigen, a lung cancer antigen, an ovarian cancer antigen, a brain cancer antigen, a gastric cancer antigen, a renal cell carcinoma antigen, a pancreatic cancer antigen, a liver cancer antigen, an esophageal cancer antigen, or a head and neck cancer antigen; Preferably, the tumor antigen is a blood tumor antigen, such as a lymphoma antigen, a myeloma (such as multiple myeloma or plasma cell myeloma) antigen, an acute lymphocytic leukemia antigen, a chronic myeloid leukemia antigen or an acute myeloid leukemia antigen; Preferably, the tumor antigen is selected from RepS1, Adpgk, HER2, GP100, TRP1, TRP2, M27, M30, WT1, CEA, HVP16 E7, Kras, P53, LCMV gp33, LCMV NP369, Influenza NP, HIV gag, MuLV gp70, MCMVIE1, ​​VSV NP, HSV-1gB, and HBc helper peptide.

15. The complex according to any one of claims 1 to 13, wherein The cargo peptide comprises an antigenic peptide derived from a pathogen antigen; Preferably, the pathogen is selected from bacteria, fungi, viruses or parasites.

16. The complex according to any one of claims 1 to 15, wherein The cargo peptide optionally further comprises an additional functional peptide linked to the antigenic peptide; Preferably, the additional functional peptide is a lytic tag, such as protein phosphatase 1B (Ppm1b).

17. The complex according to any one of claims 1 to 16, wherein The protein component is a polymer; Preferably, the multimer is a dimer, trimer or tetramer; Preferably, the multimer is a homomultimer.

18. A kit comprising: a protein component and a nucleic acid component of the complex according to any one of claims 1 to 17 packaged in separate containers.

19. The kit according to claim 18, wherein The protein component is as defined in claim 3, and the kit comprises: a first container containing the protein component and a second container containing the nucleic acid component.

20. The kit according to claim 18, wherein The protein component is as defined in claims 4-6, and the kit comprises: a first container containing a first protein in the protein component, a second container containing a second protein in the protein component, and a third container containing the nucleic acid component.

21. A kit comprising: (i) a nucleic acid molecule or a vector comprising a nucleotide sequence encoding a protein component in the complex according to any one of claims 1 to 17; and, (ii) CpG adjuvant.

22. The kit according to claim 21, wherein The protein component is as defined in claim 3, wherein (i) the nucleic acid molecule or vector comprises a nucleotide sequence encoding the protein component; Preferably, the kit comprises: a first container containing (i) the nucleic acid molecule or vector, and a second container containing a CpG adjuvant.

23. The kit according to claim 21, wherein The protein component is defined in any one of claims 4 to 6, wherein the nucleic acid molecule or vector (i) comprises: a first nucleotide sequence encoding the first protein, and a second nucleotide sequence encoding the second protein; Preferably, the first nucleotide sequence and the second nucleotide sequence are present on the same or different nucleic acid molecules or vectors; Preferably, the kit comprises: a first container comprising a vector comprising the first nucleotide sequence, a second container comprising a vector comprising the second nucleotide sequence, and a third container comprising a CpG adjuvant.

24. A method for preparing the composite of any one of claims 1 to 17, comprising: (1) providing a protein component and a nucleic acid component in the complex; (2) Mixing the protein component and the nucleic acid component to form a complex.

25. The method of claim 24, wherein: The protein component is defined in claim 3, and step (1) comprises: introducing a nucleic acid molecule or a vector encoding a nucleotide sequence of the protein component into a host cell, culturing the host cell under conditions allowing protein expression, and recovering the protein component from the cultured host cell culture, wherein the protein component optionally exists in a polymeric form.

26. The method of claim 24, wherein: The protein component is as defined in any one of claims 4 to 6, and step (1) comprises: (a) providing the first protein and the second protein; (b) mixing the two to form a ligation product, thereby providing a protein component; Preferably, a first nucleotide sequence encoding the first protein or a nucleic acid molecule or vector comprising the first nucleotide sequence is introduced into a host cell, the host cell is cultured under conditions allowing protein expression, and the first protein is recovered from the cultured host cell culture, wherein the first protein is optionally present in a polymeric form; Preferably, the second nucleotide sequence encoding the second protein or a nucleic acid molecule or vector comprising the second nucleotide sequence is introduced into a host cell, the host cell is cultured under conditions allowing protein expression, and the second protein is recovered from the cultured host cell culture.

27. A pharmaceutical composition comprising the complex according to any one of claims 1 to 17; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition is a vaccine.

28. Use of the complex according to any one of claims 1 to 17 or the pharmaceutical composition according to claim 27 in the preparation of a preparation for inducing an immune response in a subject (eg, a human); Preferably, the formulation is used to induce an antigen-specific immune response in a subject (e.g., a human); Preferably, the antigenic peptide contained in the complex is derived from a tumor antigen, and the preparation is used to induce an anti-tumor immune response in a subject (e.g., a human); Preferably, the antigenic peptide contained in the complex is derived from a pathogen antigen, and the preparation is used to induce an immune response against the pathogen in a subject (eg, a human).

29. Use of the complex according to any one of claims 1 to 17 or the pharmaceutical composition according to claim 27 in the preparation of a preparation for preventing and / or treating a tumor in a subject (eg, a human); Preferably, the tumor is a solid tumor, such as melanoma, colorectal cancer, breast cancer, prostate cancer, bladder cancer, lung cancer, ovarian cancer, brain cancer, gastric cancer, renal cell carcinoma, pancreatic cancer, liver cancer, esophageal cancer, head and neck cancer; Preferably, the tumor is a blood tumor, such as lymphoma, myeloma (such as multiple myeloma or plasma cell myeloma), acute lymphocytic leukemia, chronic myeloid leukemia or acute myeloid leukemia; Preferably, the tumor is a metastatic tumor; Preferably, the subject is a subject without a tumor or a subject with a tumor; Preferably, the complex or pharmaceutical composition is administered alone or in combination with another therapeutic agent (anti-tumor agent); Preferably, the complex or pharmaceutical composition is administered in combination with an immune checkpoint inhibitor (eg, PD-L1 antibody, PD-1 antibody, CTLA-4 antibody).

30. Use of the complex according to any one of claims 1 to 17 or the pharmaceutical composition according to claim 27 in the preparation of a preparation for preventing and / or treating infection in a subject (eg, a human); Preferably, the infection is selected from a viral infection, a bacterial infection, a fungal infection or a parasitic infection; Preferably, the subject is a subject not suffering from an infection or a subject suffering from an infection.

31. Use of the complex of any one of claims 1 to 17 or the pharmaceutical composition of claim 27 in the preparation of a formulation for targeted delivery of an active agent to a lymphoid organ in a subject; Preferably, the lymphoid organ is a lymph node; Preferably, the preparation is used to induce an immune response, prevent and / or treat a tumor, or prevent and / or treat an infection in a subject (eg, a human).

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