Peptide-carrying vector system and use thereof

Abstract

A carrier system includes a nanocarrier and a peptide non-covalently bound to the nanocarrier. The peptide comprises an adaptor peptide sequence fused to the N-terminus of the target peptide, the adaptor peptide sequence being designed to facilitate binding to the nanocarrier. Also disclosed is a method of improving the immunogenicity of a peptide antigen by fusing the peptide antigen with an adaptor peptide sequence to form an immune peptide and contacting the immune peptide with a compatible nanocarrier. Further, a method of treating disease by immunizing with a peptide of interest that has been fused to an adaptor peptide sequence and thus bound to a nanocarrier is provided. The method induces an immune response to the peptide of interest to treat cancer, viral infection, bacterial infection, parasitic infection, autoimmunity, or an adverse immune response to a biological agent.

Description

Background Art

[0001] Customized cancer vaccines have been developed and have shown encouraging results in animal studies and early clinical trials. However, these studies and trials have shown that several key challenges need to be addressed before the potential of customized vaccines can be fully realized. For example, the stimulation of T cells against multiple anticancer peptide targets required for strong anticancer effects is a difficult task and requires new vaccine delivery technologies. Current clinical trial protocols include up to 10 booster vaccinations to elicit observable cellular immunity (see Sahin et al., Nature 547:222-226; Keskin et al., Nature 565:234-239; Hilf et al., Nature 565:240-245; and Ott et al., Nature 547:217-221), resulting in prolonged treatment and impaired therapeutic effects.

[0002] Synthetic nanocarriers have been tested as delivery vehicles for peptide antigens. It is believed that such nanocarriers shield peptides from the harsh extracellular environment after administration and promote their uptake by cells, thereby leading to enhanced efficacy. In addition, immunoadjuvants have been incorporated into nanocarriers to simultaneously deliver immune enhancement signals and peptides, which are ideal for eliciting immune responses (see Crouse, J. et al., Nature Rev. Immunol. 15: 231-42). However, this approach requires complex chemical reactions or the use of non-biocompatible materials (see Kuai, R. et al., Nature Materials 16: 489-496; Li, AW et al., Nature Materials 17: 528-534; Luo, M. et al., Nature Nanotechnol. 12: 648-654; and Liu, H. et al., Nature 507: 519-522), which raises logistical and safety concerns.

[0003] There is a need to develop a carrier system in which peptides with various physicochemical characteristics can be easily conjugated to nanocarriers without resorting to laborious chemical reactions. Such an approach will facilitate the formulation and delivery of peptides, thereby expanding the research and clinical application of peptide-based therapeutics. In particular, effective multi-antigen vaccine development requires a strategy to deliver various peptide antigens without compromising their immunogenicity. Carrier system technology is important for effective neoantigen vaccination and can also be used in the fields of infectious disease management and immune tolerance induction. Summary of the invention

[0004] In order to effectively deliver the target peptide as described above, a carrier system is provided, comprising a nanocarrier and a peptide non-covalently bound to the nanocarrier. The peptide is composed of a linker peptide sequence fused to the N-terminus of the target peptide. The nanocarrier has a core that can be hydrophobic or hydrophilic. The nanocarrier also has a surface, which can have a net negative charge, a net positive charge, or one or more functional groups. The linker peptide sequence is designed to non-covalently bind to the hydrophobic core, the hydrophilic core, the surface with a net negative charge, the surface with a net positive charge, or the surface with one or more functional groups.

[0005] A method for improving the immunogenicity of a peptide antigen is also provided. The method comprises the following steps: fusing the peptide antigen to a linker peptide sequence to form an immune peptide, and contacting the immune peptide with a nanocarrier, thereby stably and non-covalently binding the immune peptide to the nanocarrier. The target peptide is a class I MHC restricted antigenic determinant or a class II MHC restricted antigenic determinant, the nanocarrier has a hydrophilic core, and the linker peptide sequence comprises two or more hydrophilic amino acids selected from D, E, R, K and H.

[0006] Also disclosed is an immunization method for treating a disease of a subject. The target peptide is fused to a linker peptide sequence to form an immunization peptide, the immunization peptide is contacted with a nanocarrier, and the immunization peptide is stably non-covalently bound to the nanocarrier to form a carrier system, and the carrier system is administered to the subject, thereby increasing the immune response to the target peptide. The target peptide is a first class MHC restricted antigenic determinant or a second class MHC restricted antigenic determinant, and the method can be used to treat subjects suffering from cancer, viral infection, bacterial infection, parasitic infection, autoimmunity, or adverse immune response to biological agents.

[0007] The details of one or more embodiments are set forth in the following description and examples. Other features, objects, and advantages will become apparent from the detailed description, drawings, and appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The following description refers to the accompanying drawings, in which:

[0009] Figure 1 Schematic diagram of the peptide of the present invention. It includes a linker peptide sequence (a segment providing compatibility), an optional spacer fragment (a cleavable linker), and a target peptide. Each circle represents an amino acid.

[0010] Figure 2 Shown is for use with Figure 1 Schematic diagram of different nanocarriers in the peptide delivery system shown.

[0011] Figure 3 Shown are exemplary carrier systems of the invention, wherein the peptide is bound to the nanocarrier core via hydrophobic or hydrophilic interactions.

[0012] Figure 4 Shown are additional carrier systems encompassed by the present invention, wherein peptides interact with the surface charges of the nanocarriers.

[0013] Figure 5 Shown is a carrier system having functional groups, ie antibodies, on the surface of the nanocarrier, which are bound to the nanocarrier via a linker carrying the antigen and an antigenic determinant on the peptide.

[0014] Figure 6 Shown is another example of a carrier system having surface functional groups that interact with peptides.

[0015] Figure 7 Shown is a carrier system having self-assembling moieties on the surface of the nanocarrier and the same moieties fused to the target peptide.

[0016] Figure 8 Graph showing the absorbance versus retention time for HPLC analysis of hollow shell nanoparticles and hydrophilic peptides A (gp100; KVPRNQDWL-SEQ ID NO:1) and B (Trp1m; TAYRYHLL-SEQ ID NO:2) and unmodified tyrosinase-related protein 2 (Trp2; SVYDFFVWL-SEQ ID NO:3) (top) and control Trp2 peptide in DMSO (bottom).

[0017] Fig. 9 The absorbance versus retention time plot of the HPLC analysis of the following samples: hollow thin-shell nanoparticles encapsulating Trp2, N-terminal fusion peptide adapter / spacer sequence D of Trp2 3 G 3 (D 3 G 3 -Trp2; upper panel) and control D dissolved in DMSO 3 G 3 -Trp2 peptide (lower panel).

[0018] Fig.10 A bar graph showing the percentage of CD8 T cells producing interferon-γ (IFN-γ) after stimulation of splenocytes with Trp2 peptides. Splenocytes were isolated from mice vaccinated with the indicated Trp2 peptides embedded in hollow shell nanoparticles and the stimulator of interferon genes (STING) agonist cyclic di-GMP.

[0019] Fig.11A Figure 2 is the relationship between tumor size and days after inoculation of B16F10 mouse melanoma cells. 3 G 3 -Trp2 peptide hollow thin shell nanoparticles (NPs), (ii) modified D 3 G 3 -Trp2 peptide plus cyclic di-GMP (peptide+dcGMP), (iii) modified D 3 G 3 -Trp2 peptide plus poly(I:C) (peptide+poly(IC)), or PBS.

[0020] Fig. 11B Figure 2 is a graph showing the relationship between the survival rate of B16F10 mice with melanoma and the number of days after inoculation. Fig.11A as described in the instructions.

[0021] Fig.12 The absorbance and retention time relationship diagram of HPLC analysis of hollow thin-shell nanoparticles carrying three modified target peptides at the same time (above), the three modified target peptides are: D 3 G 3 Modified RalBP1-associated Eps domain-containing protein 1 (D 3 G 3 -Resp1), D 3 G 3 Modified ADP-dependent glucokinase (D 3 G 3 -Adpgk) and D 4 G 3 Modified dialkyl phosphate N-acetamido glucose phosphotransferase (D 4 G 3 -Dpagt1); and control peptide in DMSO (bottom three panels).

[0022] Fig.13A The bar graph shows the percentage of CD8 T cells producing IFN-γ after stimulating spleen cells with Resp1, Adpgk and Dpagt1 peptides. Splenocytes were isolated from mice vaccinated with: (i) three modified peptides D 3 G 3 -Resp1, D 3 G 3 -Adpgk and D 4 G 3-Hollow shell nanoparticles of Dpagt1 and the STING agonist cyclic di-GMP (nanoparticles), (ii) the three unmodified peptides plus cyclic di-GMP (peptide+cdGMP), and (iii) the three unmodified peptides plus poly(I:C) (peptide+poly(IC)).

[0023] Fig. 13B To inoculate MC38 mouse colon adenocarcinoma cells into Fig.13A A graph illustrating the relationship between tumor size and inoculation days after inoculation of the inoculated mice.

[0024] Fig.14 For HPLC analysis of D 3 G 3 - Relationship between absorbance and retention time of hollow thin-shell nanoparticles of Trp2, hydrophilic peptides C (gp100) and D (Trp1m).

[0025] Fig.15 A bar graph showing the ovalbumin antigen determinant OVA 257-264 The percentage of CD8 T cells producing IFN-γ after peptide stimulation of spleen cells. 257-264 The spleen cells were isolated from mice expressing OVA peptide. 257-264 The peptide is entrapped together with cyclic di-GMP in hollow thin-shell nanoparticles.

[0026] Fig.16A Included are bar graphs showing the percentage of IFN-γ producing CD8 T cells (upper half) and IFN-γ producing CD4 T cells (lower half) after stimulation of spleen cells with the indicated hydrophobic unmodified peptide antigens. Spleen cells were isolated from mice vaccinated with the indicated peptides entrapped in hollow thin-shell nanoparticles along with cyclic di-GMP.

[0027] Fig. 16B Included are bar graphs showing the percentage of CD8 T cells producing IFN-γ (upper panel) and CD4 T cells producing IFN-γ (lower panel) after stimulation of spleen cells with the indicated hydrophilic unmodified peptide antigens. Fig.16A Isolate from vaccinated mice as described in the instructions.

[0028] Fig.17 Schematic diagram showing a simple and unified approach for preparing customized cancer vaccines targeting neoantigenic epitopes.

[0029] Fig.18AFigure 2 is a plot of absorbance versus retention time for HPLC analysis of hollow thin-shell nanoparticles containing seven different B16 melanoma neoantigenic determinants, designated M05, M24, M27, M28, M30, M33, and M50 (Group I). Seven of the 21 neoantigenic determinants predicted using the IEDB consensus method version 2.5 were randomly combined to prepare nanoparticles.

[0030] Fig.18B Figure 2 is a plot of absorbance versus retention time for HPLC analysis of hollow shell nanoparticles containing seven different B16 melanoma neoantigenic determinants, designated M08, M12, M17, M21, M25, M29, and M44 (Group II).

[0031] Fig.18C Figure 2 is a plot of absorbance versus retention time for HPLC analysis of hollow thin-shell nanoparticles containing seven different B16 melanoma neoantigenic determinants, designated M20, M22, M36, M45, M46, M47, and M48 (Group III).

[0032] Fig.19A A bar graph showing the percentage of CD8 T cells producing IFN-γ after stimulation of splenocytes isolated from mice vaccinated with the modified neopeptides encapsulated with cyclic di-GMP in hollow shell nanoparticles. Prediction using IEDB consensus method version 2.5 Figures 18A-18C The new antigenic determinant candidates are listed in the description.

[0033] Fig.19B Graph showing the percentage of CD8 T cells producing IFN-γ after stimulating spleen cells with predicted neoantigenic epitopes in murine B16 melanoma using DeepHLApan. The spleen cells were isolated as Fig.19A as described in the instructions.

[0034] Fig. 20A A bar graph showing the percentage of CD8 T cells producing IFN-γ after stimulation of splenocytes isolated from human HLA-transgenic mice vaccinated with modified neopeptides encapsulated with cyclic di-GMP in hollow shell nanoparticles using DeepHLApan with predicted neoepitopes in a large colorectal cancer patient.

[0035] Fig. 20BFIG. 5 is a bar graph showing the percentage of CD8 T cells producing IFN-γ after stimulating spleen cells with predicted new antigenic determinants using DeepHLApan in a second colorectal cancer patient. The spleen cells were isolated as Fig. 20A as described in the instructions.

[0036] Fig.21A Schematic diagram showing the induction of tolerance to a peptide antigen by encapsulating a peptide modified with a peptide linker sequence together with an immunosuppressant in a nanocarrier.

[0037] Fig. 21B D 4 G 3 Modified OTII nanoparticles (D 4 G 3 -OTII; SEQ ID NO: 4) induced mice to resist OVA 323-339 timeline of tolerance.

[0038] Fig.22A Figure 1 is a flow cytometric analysis showing CD25 in spleen cells from mice vaccinated with the indicated aspirin / peptide formulations or control. + Foxp3 + T reg Percentage of population. NP = nanoparticle.

[0039] Fig. 22B is a bar graph showing the expression of CD25 in total CD4 T cells in mice vaccinated as shown. + Foxp3 + T reg The average percentage of

[0040] Fig. 22C is a bar graph showing the expression of CD25 in mice vaccinated as above. + Foxp3 + T reg The total number of cells.

[0041] Fig.22D Figure 1 is a flow cytometry analysis showing Foxp3 in OTII-tetramer-positive CD4 T cells in spleen cells of vaccinated mice. + T reg The percentage of cells.

[0042] Fig.22E A bar graph showing Foxp3 expression in OTII-tetramer positive CD4 T cells from mice vaccinated as shown. + T reg The average percentage of cells.

[0043] Fig.23 Schematic diagram illustrating the timeline and protocol used to evaluate the effects of nanoparticles on immune tolerance induction in vitro.

[0044] Fig.24 Included are bar graphs showing the percentage of JAWSII dendritic cells expressing CD80 (upper left graph), CD86 (upper right graph), MHC I (lower left graph), and MHC II (lower right graph) analyzed by flow cytometry after co-culture of cells with the indicated aspirin / peptide formulations. DETAILED DESCRIPTION

[0045] The carrier system of the present invention comprises a nanocarrier and a peptide non-covalently bound to the nanocarrier.

[0046] As described above, the peptide comprises a linker peptide sequence fused to the N-terminus of the target peptide. Figure 1 .

[0047] The adapter peptide sequence may include two or more hydrophilic amino acids selected from D, E, R, K and H. The adapter peptide sequence including the hydrophilic amino acids may be fused with the hydrophobic target peptide, thereby making the fusion peptide hydrophilic. The adapter peptide sequence may also be fused with the hydrophilic target peptide. The sequence of the adapter peptide sequence may be, but is not limited to, D n 、E n 、(DE) n , (DX) n or (EX) n , wherein n is an integer from 2 to 20, and X is any amino acid. In a specific embodiment, amino acids P, A, V, I, L, M, F, Y, and W are excluded from the adapter peptide sequences listed in this paragraph.

[0048] Other adaptor peptide sequences that may be used include two or more hydrophobic amino acids selected from A, V, I, L, P, F, W and M.

[0049] Additionally, adaptor peptide sequences having positively charged amino acids, such as K, R, and H, and adaptor peptide sequences having negatively charged amino acids, such as D and E, are also within the scope of the present invention.

[0050] In addition, the adaptor peptide sequence can be those sequences that bind to functional groups, such as FLAG tag (DYKDDDK-SEQ ID NO:5), HA tag (YPYDVPDYA-SEQ ID NO:6) and Myc tag (EQKLISEEDL-SEQ ID NO:7), each of which can bind to its own anti-tag antibody. Figure 5 .

[0051] Polyhistidine may also be included in the adaptor peptide sequence. Figure 6 .

[0052] Finally, if Figure 7 As shown, the adapter peptide sequence can be a self-assembling sequence (e.g., alpha helix, Q11 peptide, ion-complementary self-assembling peptide, and long-chain alkylated peptide). Additional self-assembling sequences are described in Sun et al., Int. J. Nanomedicine 2017: 73-86, and Li et al., Soft Matter, 15: 1704-1715.

[0053] The peptide in the disclosed vector system may include a spacer fragment fused between the target peptide and the adapter peptide sequence. The spacer fragment may contain two or more amino acid residues selected from G, A, S and P. An exemplary spacer fragment has an amino acid sequence of G n , wherein n is an integer from 1 to 15. The spacer fragment can be easily cleaved by the cellular machinery, so that after the nanocarrier delivers the peptide to the cell, the adapter peptide sequence can be cleaved from the target peptide.

[0054] Specific examples of the peptide include the adapter peptide sequence DDD (SEQ ID NO: 8) or DDDD (SEQ ID NO: 9) and the spacer fragment GGG (SEQ ID NO: 10). In the peptide, the adapter peptide sequence is fused to the N-terminus of the spacer fragment, which in turn is fused to the target peptide. Figure 1 .

[0055] As described above, the carrier system includes nanocarriers. The nanocarriers may be, but are not limited to: (i) hollow structures containing one or more aqueous cores for encapsulating hydrophilic drugs, (ii) solid or oil-based structures with hydrophobic cores for encapsulating hydrophobic drugs, (iii) carriers with positive electrostatic charge for carrying negatively charged drugs, (iv) carriers with negative electrostatic charge for carrying positively charged drugs, and (v) carriers with defined surface functional groups for binding to defined peptide sequences. Figure 2 .

[0056] In one embodiment, the nanocarrier is a hollow thin-shell nanoparticle having one or more aqueous cores, as described in Hu et al., Intl. Appl. Pub. No. 2017 / 165506, the contents of which are incorporated herein in their entirety.

[0057] The adapter peptide sequence can be selected based on the type of nanocarrier in the carrier system and the specific target peptide. For example, the adapter peptide sequence comprising the hydrophilic amino acids can be fused to the target peptide to increase its water solubility. The water-soluble peptide can be embedded in the inner aqueous core of the hollow polymer nanoparticle. Figure 3 Alternatively, a hydrophobic amino acid based adaptor peptide sequence can be fused to the target peptide for incorporation into the hydrophobic compartment of a solid or oil-based support.

[0058] Adaptor peptide sequences containing charged amino acids can be used to promote the binding between the target peptide and the nanocarrier with the opposite net charge. For example, an adaptor peptide sequence containing negatively charged aspartic acid or glutamic acid can be fused to the target peptide, so that the fused peptide binds to the positively charged nanocarrier. Figure 4 Similarly, an adaptor peptide sequence with positively charged amino acids, such as lysine, arginine, and histidine, can be fused to the target peptide, allowing it to be conjugated to a negatively charged carrier. See also Figure 4 .

[0059] The function of the carrier system can be used to give the nanocarrier a specific affinity for a specific amino acid sequence in the adapter peptide sequence. As described above, the adapter peptide sequence can include, for example, a FLAG tag, an HA tag, and a Myc tag. The target peptide fused to these adapter peptide sequences can be bound to the nanocarrier, which is bound to an antibody on its surface that binds to the tag. Figure 5 .

[0060] In another embodiment, the surface of the nanocarrier can be functionalized with a metal chelator, such as nitrosotriacetic acid, which has a strong affinity for polyhistidine in the presence of nickel or cobalt ions. A linker peptide sequence containing polyhistidine can be fused to the target peptide, thereby non-covalently binding the fusion peptide to the carrier surface. Figure 6 .

[0061] Moreover, a self-assembling amino acid sequence, such as an α-helix or a Q11 peptide, can be used as part of the adapter peptide sequence and can also be used to functionalize the surface of the nanocarrier. Due to the self-assembly ability of a specific sequence, the target peptide connected to the adapter peptide can be coupled to the nanocarrier. Figure 7 .

[0062] The carrier system disclosed herein may include a combination of the above-mentioned nanocarrier and a transfer peptide sequence-target peptide fusion. For example, an exemplary carrier system includes a nanocarrier having a hydrophilic core carrying two different peptides, each of which includes a transfer peptide sequence having hydrophilic amino acids.

[0063] The carrier system can be used to deliver any desired target peptide fused to an adapter peptide sequence. In one embodiment, the target peptide is a therapeutic peptide. In another embodiment, the nanocarrier can be detected in vivo, and the target peptide is used to localize the nanocarrier to a specific anatomical site.

[0064] In addition, the target peptide can be an MHC class I restricted antigenic determinant or an MHC class II restricted antigenic determinant. Such a target peptide is used together with the carrier system to enhance the response of T cells to the antigenic determinant.

[0065] In certain embodiments, the target peptide is a cancer neoantigen, a non-neoantigen cancer antigen, a bacterial antigen, a viral antigen, or a parasite antigen.

[0066] Specific examples of target peptides include Mycobacterium tuberculosis p25, influenza virus nucleoprotein NP311, and cancer-associated antigens Adpgk, Dpagt, Resp1, Trp1m, and gp100. Antigenic peptides from malaria parasites, HIV, HBV, and MERS-CoV are other examples of target peptides.

[0067] The carrier system including the antigen target peptide may also include an immunomodulator embedded in the nanocarrier together with the adapter peptide sequence / target peptide fusion. The immunomodulator may be an immune response stimulator, for example, a stimulator of the interferon gene (STING) agonist, such as cyclic di-GMP (cdGMP), CpG-ODN, R848 and poly (I: C). This carrier system can be used to enhance the immune response to the target peptide.

[0068] Alternatively, the carrier system can be used to suppress the immune response to the target peptide. In such a system, the immunomodulator embedded in the nanocarrier can be an immune response suppressor, such as rapamycin, aspirin, vitamin D, steroids and N-acetylcysteine.

[0069] A method for improving the immunogenicity of a peptide antigen also falls within the scope of the present invention. The method comprises the following steps: fusing the peptide antigen to a transfer peptide sequence to form an immune peptide, and contacting the immune peptide with a nanocarrier, thereby stably and non-covalently binding the immune peptide to the nanocarrier.

[0070] The immunogenicity of the peptide antigen is assessed by comparing the immune response of the peptide antigen with the immune response of the modified peptide antigen, i.e., the immune peptide. The immune response is characterized by measuring the percentage of the number of peptide-specific CD4+ or CD8+ T cells ("T cells") as a percentage of total T cells, i.e., frequency. Therefore, compared to the unmodified peptide antigen, an improved immune response can be defined as an increase of 1.2 to 250 times (e.g., 1.2, 1.5, 1.8, 2, 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225 and 250 times) of the frequency of peptide-specific T cells induced by the modified peptide antigen.

[0071] The target peptide is a class I MHC restricted antigenic determinant or a class II MHC restricted antigenic determinant, the nanocarrier has a hydrophilic core, and the adapter peptide sequence comprises two or more hydrophilic amino acids selected from D, E, R, K and H. The target peptide antigen, adapter peptide sequence, and nanocarrier have been described in detail above.

[0072] In a preferred embodiment, the immune peptide comprises the linker peptide sequence DDD (SEQ ID NO: 8) or DDDD (SEQ ID NO: 9), a spacer fragment GGG (SEQ ID NO: 10) fused to the C-terminus of the linker peptide sequence, and a peptide antigen fused to the C-terminus of the spacer fragment.

[0073] Also provided is an immunization method for treating a disease of a subject using the carrier system. The immunization method comprises the following steps: (i) fusing a target peptide to a linker peptide sequence to form an immunization peptide; (ii) contacting the immunization peptide with a nanocarrier so that the immunization peptide and the nanocarrier are stably non-covalently bound to form a carrier system; and (iii) administering the carrier system to the subject, thereby increasing the immune response to the target peptide.

[0074] In the method, the target peptide is a class I MHC restricted antigenic determinant or a class II MHC restricted antigenic determinant, and the disease is cancer, viral infection, bacterial infection, parasitic infection, autoimmunity, or adverse immune response to a biologic.

[0075] Without further elaboration, it is believed that one of ordinary skill in the art can utilize the disclosure to its maximum extent based on the disclosure herein. Therefore, the following specific examples should be interpreted as being merely illustrative and not limiting the remainder of the disclosure in any way. All publications cited herein are incorporated herein by reference in their entirety.

[0076] Example

[0077] Example 1. Delivery of a hydrophobic peptide

[0078] A hydrophobic peptide named Trp2 180-188 (Trp2; SVYDFFVWL-SEQ ID NO:3), modified by fusion with a peptide adapter sequence and embedded in the nanoparticle. Trp2 is a highly hydrophobic B16 mouse melanoma antigen determinant with immunodominance. The peptide is fused at its N-terminus with a hydrophilic adapter, namely D 3 G 3 The modified Trp2 peptide was fused to a peptide adapter sequence comprising three aspartic acid residues (D) and a spacer fragment of three glycine residues (G) to form a cleavable linker. The peptide was synthesized by conventional procedures. The sequence of the modified Trp2 peptide was DDDGGGSVYDFFVWL (D 3 G 3 -Trp2; SEQ ID NO: 11).

[0079] Hollow thin-shell nanoparticles with an aqueous core were prepared essentially as described in Hu et al.

[0080] To quantify the peptide loaded into the nanoparticles, HPLC analysis was performed as follows. The nanoparticles were lyophilized and then disrupted by adding 95% acetone. The acetone was removed by drying in a 60°C dry bath and the sample was resuspended in H 2 O and analyzed on an Agilent 1100 series HPLC system using a gradient HPLC method. In an exemplary method, the initial mobile phase consists of a 75:25 mixture of 0.1% trifluoroacetic acid in water and 0.1% trifluoroacetic acid in acetone. The second mobile phase is a 15:85 mixture of 0.1% trifluoroacetic acid in water and 0.1% trifluoroacetic acid in acetone for 20 minutes, followed by a 10-minute third phase wash with 0.1% trifluoroacetic acid in acetone. The standard calibration curve for quantification of peptides was determined by absorbance at a wavelength of 220 nm.

[0081] The unmodified Trp2 peptide is poorly soluble in water, with a maximum solubility of 0.06 mM. See Vasievich, EA et al., Molecular pharmaceutics, 2012, 9:261-8. As such, it cannot be embedded in the aqueous core of the hollow nanoparticles as shown by HPLC analysis. See Figure 8 In contrast, the two hydrophilic peptides, gp100 (peptide A) and Trp1m (peptide B), were easily incorporated into the nanoparticles. Figure 8 .

[0082] In contrast, D 3 G3 -Trp2 peptide has a solubility of >30 mM in water, which is more than 500 times higher than that of Trp2 peptide. 3 G 3 -Trp2 peptides are easily incorporated into the aqueous core of the nanoparticles. Fig. 9 .

[0083] Example 2. Immunogenicity of modified peptides

[0084] The immunogenicity of the modified Trp2 peptide was tested by encapsulating it in the aqueous core of hollow thin-shell nanoparticles together with a fixed amount of the STING agonist cyclic di-GMP (cdGMP) and injecting it into mice.

[0085] The modified Trp2 peptides are as follows: (i) D 4 -Trp2-D 5 (ii) Trp2-D 5 (iii) D 5 -Trp2, (iv)D 2 G 3 -Trp2-G 3 D 2 (v)Trp2-G 3 D 3 and (vi) D 3 G 3 Since Trp2 itself cannot be incorporated into the aqueous core due to its hydrophobicity, a longer Trp2 peptide, named Trp2 168-195 , embedded as positive control.

[0086] Nanoparticles each containing an equivalent dose of one of the Trp2 peptides and cdGMP were prepared as described in Example 1 above and administered to C57BL / 6 mice by subcutaneous injection at the base of the tail on days 0 and 21. On day 28, spleen cells from the inoculated mice were isolated and examined for Trp2-specific CD8 T cell immune responses. Briefly, spleen cells from each mouse were stimulated with Trp2 peptide, and the expression of IFN-γ in CD8 T cells was measured by intracellular cytokine staining and flow cytometric analysis. The results are shown in Figure 2. Fig.10 shown.

[0087] Compared with Trp2, all Trp2 peptides modified with the hydrophilic aspartic acid sequence showed a significant increase in water solubility, i.e., at least 30 mM. 3 G 3 -Trp2, in which the hydrophilic peptide linker sequence is combined with a cleavable spacer fragment fused to the N-terminus only, produced the highest amount of T cell stimulation, showing up to 4% of CD8 T cells producing interferon-γ. Fig.10 Surprisingly, the inclusion of a cleavable spacer segment and the positioning of the hydrophilic adapter and spacer segment at the N-terminus of the target peptide were both important for achieving maximal immunogenicity of the peptide.

[0088] In addition, the modified D 3 G 3 Immunization of mice with nanoparticles of -Trp2 resulted in significant protection against B16F10 melanoma challenge. More specifically, tumor growth was inhibited in mice immunized with these nanoparticles (see Fig.11A ), the survival rate is improved (see Fig. 11B ), which means D 3 G 3 The modification did not reduce the antitumor activity of the Trp2 peptide.

[0089] Without being bound by theory, the N-terminally fused D 3 G 3 Peptides are believed to be susceptible to processing by the cellular proteolytic machinery, resulting in an unhindered immune response to the peptide antigen.

[0090] In addition, Trp2 168-195 The immunogenicity of Trp2 168-195 It is a long peptide that contains the amino acid sequence flanking the target antigenic determinant, i.e., amino acids 180-188. Trp2 168-195 The water solubility is better than Trp2 180-188 , which would allow longer peptides to be incorporated into the aqueous core. 3 G 3 -Trp2,Trp2 168-195 Peptide-induced CD8 + The T cell response is about 20 times weaker. Fig.10 .

[0091] Example 3. Vaccination with cancer neoantigen epitopes

[0092] The above modification strategy was also tested on three cancer neoantigenic determinants derived from the Resp1, Adpgk and Dpagt1 genes in MC38 mouse colon adenocarcinoma cells. See Yadav, M. et al., Nature 515:572-576. Each of these three neoantigenic determinants contains a high proportion of hydrophobic amino acids and is therefore inherently insoluble in water. 3 G 3 After the peptides were fused to their N-termini, the proportion of hydrophobic amino acids in the fusion peptides was reduced to less than 40%, and their solubility in water was increased to more than 30 mM.

[0093] Three modified peptides, namely D 3 G3 -Resp1, D 3 G 3 -Adpgk and D 3 G 3 -Dpagt, and co-encapsulated in hollow PLGA-based nanoparticles prepared using a double emulsion process as described in Example 1. Analysis of the nanoparticles by HPLC confirmed that all three peptides were co-encapsulated. Fig.12 .

[0094] Mice were vaccinated with: (i) a vaccine containing three D 3 G 3 Nanoparticles of modified neoepitope peptides and a STING agonist adjuvant, or (ii) unmodified neoepitope peptides and a poly(I:C) adjuvant as described in Example 2. The immune responses elicited by the different vaccinations were examined by CD8 T cell cytokine production and tumor cell attack.

[0095] As described above, the percentage of IFN-γ-producing CD8 T cells was measured in spleen cells stimulated with each unmodified neoepitope peptide. The results are shown in Fig.13A In the present study, 5% to 12% of CD8 T cells were shown to produce IFN-γ after neoepitope stimulation. No measurable CD8 T cell responses were observed in spleen cells of mice vaccinated with free, unmodified neoepitope peptides.

[0096] Turning to attacking tumor cells, MC38 cells were subcutaneously injected into mice previously immunized with: (i) PBS, (ii) a mixture of three unmodified neoantigenic determinant peptides and poly(I:C) adjuvant, (iii) a mixture of three unmodified neoantigenic determinant peptides and the STING agonist cyclic di-GMP, or (iv) nanoparticles containing all three modified neoantigenic peptides and the STING agonist. The results are shown in Fig. 13B Nanoparticle vaccination provided significant protective immunity against subcutaneous challenge of MC38 tumor cells, as demonstrated by inhibition of tumor growth. In contrast, vaccination with three free neoantigenic determinant peptides plus cyclic di-GMP or poly(I:C) adjuvants was significantly less effective in slowing tumor growth.

[0097] The above results clearly show that hydrophilic peptide linkers such as D 3 G 3 , to unify the physicochemical properties of multiple peptides. Through this strategy, different peptides can be simultaneously encapsulated in hollow thin-shell nanoparticles regardless of their original properties.

[0098] In another embodiment of co-embedding, D 3 G 3 -Trp2 has been successfully co-encapsulated with two hydrophilic peptides, gp100 and Trp1m. Fig.14 .

[0099] Example 4. Modification of water-soluble peptide antigenic determinants

[0100] Modification of the ovalbumin peptide antigenic determinant OVA 257-264 (OVA; SIINFEKL-SEQ ID NO: 12) and incorporated into nanoparticles to test the effect of hydrophilic peptide adapters on the immunogenicity of water-soluble peptide epitopes. The modified OVA peptides were as follows: (i) D 4 -OVA-D 5 (ii) OVA-D 4 (iii) D 4 -OVA, (iv)D 2 G 3 -OVA-G 3 D 2 (v) OVA-G 3 D 4 and (vi) D 3 G 3 -OVA.

[0101] By incorporating a hydrophilic peptide adapter, D 3 G 3 Fusion to its N-terminus, OVA 257-264 The water solubility of 2mM was increased to 50mM. 3 G 3 Fusion to OVA 257-264 C-terminus, and by inserting D 4 G 3 Similar solubility improvements can be obtained by fusion to its N-terminus or C-terminus.

[0102] The immunogenicity of OVA and each modified OVA peptide was tested as described above. Fig.15 As shown, the results show that when D 3 G 3 When the peptide was transferred, the OVA peptide-specific response in spleen cells from immunized mice was increased, while the other modifications led to a slight decrease in immunogenicity.

[0103] Example 5. Modification of Class I MHC and Class II MHC Epitopes

[0104] The broad applicability of the above peptide linker modification strategy was tested by preparing the fusion peptides shown in Table 1 below.

[0105] Table 1. Modified peptide antigens

[0106]

[0107] As described above, each fusion peptide was loaded into the aqueous core of hollow thin-walled nanoparticles along with 1000 molecules of cdGMP. All modified peptides as well as unmodified hydrophilic peptide epitopes were readily incorporated into the aqueous core of the nanoparticles.

[0108] Mice were vaccinated with nanoparticles as described above, and T cell responses were measured by intracellular cytokine staining of CD8 or CD4 T cells after stimulation of splenocytes isolated from vaccinated mice with unmodified peptide epitopes. The results are shown in Fig.16A and 16B .

[0109] All modified peptide antigens resulted in an increase in immune responses in immunized mice compared to mice vaccinated with unmodified peptide antigens. Such increases were shown for both CD8 and CD4 T cells, and this was applicable to the antigens tested. Clearly, the above peptide antigen modification strategy can be applied to many different peptide antigen sequences, regardless of their inherent hydrophobicity and hydrophilicity.

[0110] Furthermore, the above results surprisingly show that, despite being delivered by the same vector system, all D 3 G 3 or D 4 G 3 The immunogenicity of the modified hydrophilic peptide antigens was higher than that of the corresponding unmodified hydrophilic peptide antigens. Fig. 16B This surprising result suggests that peptide adapters can not only unify the physicochemical properties of different peptide antigens but also improve antigen processing and antigen presentation.

[0111] Furthermore, it is necessary to repeat the immune response measured in spleen cells stimulated with unmodified peptide antigens. The fact that spleen cells from mice vaccinated with modified peptide antigens reacted to unmodified peptide antigens shows that the modifications do not affect the specificity of T cells for the desired antigen sequence.

[0112] Example 6. Identification of modified peptides encapsulated in thin-shell nanoparticles and immunization of new antigens

[0113] Individual cancer patients' unique mutations, called neoantigens, have been studied for their potential to trigger tumor-specific immune responses. This customized cancer vaccine approach can overcome issues such as tumor heterogeneity and patient-specific HLA haplotype differences to maximize anti-tumor efficacy for each patient.

[0114] The peptide linker modifications described above are ideal for unifying the physicochemical properties of different peptides, such as neoepitopes, so that they can be co-encapsulated in thin-shell nanoparticles in a streamlined process. Fig.17 .

[0115] The feasibility of the method was tested by predicting two sets of 21 new murine B16 melanoma antigenic determinants using (i) the Immune Epitope Database (IEDB) consensus method version 2.5 and (ii) DeepHLApan software (see Wu et al., 2019, Front. Immunol. 10:2559). Each peptide was synthesized and linked with a hydrophilic adapter D 4 G 3 The peptides were modified and then incorporated into thin-shell nanoparticles in groups of seven.

[0116] HPLC analysis showed that all 21 neoantigenic determinants predicted by the IEDB consensus method were successfully embedded into the hollow nanoparticles of the seven modified peptide groups. Figures 18A-18C Similar results were obtained with the 21 antigenic determinants predicted by DeepHLApan (data not shown).

[0117] As described above, mice were primed and boosted with modified peptides loaded into nanoparticles with cdGMP. Strong CD8+ T cell responses were detected for 6 IEDB consensus predicted neoantigenic determinants (M33, M21, M28, M47, M05, and M45). 3 DeepHLApan predicted neoantigenic determinants (N22, N8, and N14; see Fig.19B Among the predicted neoantigenic determinants of mouse B16 melanoma, M28, M45, N22, N8, and N14 were newly discovered.

[0118] Furthermore, compared to previous literature, which reported unexpected dominant CD4+ T cell responses when mice were vaccinated with long synthetic peptides (see Kreiter et al., 2015, Nature 520(7549):692-696), no significant CD4+ T cell responses were observed in the present invention. Apparently, peptide hydrophilic transfer modification not only facilitates the production of customized cancer vaccines, but also facilitates precise neoantigen determinant-specific immunity.

[0119] Example 7. Identification and immunization of human cancer neoantigens using modified peptides embedded in thin-shell nanoparticles

[0120] The peptide adapters described above were modified for patient-derived neoantigenic determinants. Tumor samples were collected from two colorectal cancer patients and subjected to next generation sequencing to identify tumor-specific mutations. Using DeepHLApan, a set of 9 and 21 neoantigenic determinants were predicted and linked to the hydrophilic adapter D 3 G 3 synthesized under the condition of .

[0121] Transgenic mice carrying patient-specific HLA haplotypes were immunized with modified nanoparticle vaccines containing new antigenic determinants. The results showed that three antigenic determinants for one patient (see Fig. 20A ) and 5 antigenic determinants from another patient (see Fig. 20B ) stimulated different CD8+ T cell responses.

[0122] These results suggest that peptide adapter design is a viable strategy for aligning various peptide properties to facilitate the co-delivery of new antigenic determinants with thin-shell nanoparticles. In addition, this approach together with human HLA-transgenic mice can accelerate the identification and validation of immunogenic antigenic determinants. This provides a simple and effective platform for the development of customized new antigen vaccines.

[0123] Example 8. Induction of Treg cells with modified peptide antigens

[0124] A peptide linker modification strategy was performed to prepare tolerance-inducing nanoparticles by co-encapsulating the modified peptide antigen with an immunosuppressant, aspirin, in hollow polymer nanoparticles. Aspirin is a compound that can induce a tolerance phenotype in dendritic cells. Binding the compound to a specific antigen can induce antigen-specific regulatory T cells (Tregs). Such Treg cells can be used to treat autoimmune diseases and reduce immune responses to therapeutic biologics. See Fig.21A .

[0125] Aspirin and D 4 G 3 -Modified OTII peptide antigen (D 4 G 3 -OTII) are co-encapsulated in the nanoparticles. Fig. 21B As shown in Figure 1, tolerance was induced using nanoparticles. Nanoparticles were injected intravenously three times at weekly intervals. Seven days after the last injection, mice were stimulated with OTII peptide mixed with resiquimod (also known as R848) to simulate an immune stimulation event. Control mice were injected with PBS or free D 4 G 3 -OTII with aspirin.

[0126] The results showed that compared with mice treated with PBS and free aspirin / peptide, mice vaccinated with nanoparticles produced more CD25 + Foxp3 + Treg cells are 7 to 10 times more numerous. Figures 22A-22C .

[0127] The generated Treg cells were further analyzed by checking antigen specificity by binding to OTII tetramers. 4 G 3 In the spleen cells isolated from mice vaccinated with OTII and aspirin-loaded nanoparticles, OTII-specific CD4 T cells were also Foxp3 + , the proportion is as high as 15%, compared with the inoculated free D 4 G 3 -OTII was at least 9 times higher in mice treated with aspirin. Fig.22D and 22E .

[0128] Example 9. Mechanism of tolerance induction

[0129] Tolerogenic dendritic cells typically exhibit a phenotype with characteristic low expression of MHC molecules (eg, MHC class I and MHC class II) and co-stimulatory molecules (eg, CD80 and CD86) on their surface.

[0130] The expression of surface markers of DCs was examined in an in vitro system to determine how the tolerance-inducing nanoparticles skew DCs toward a tolerogenic phenotype.

[0131] Dendritic cells co-encapsulated with nanoparticles containing transferrin-modified OTII peptides were treated with aspirin or transferrin-modified peptides for only 6 hours and then stimulated with low-dose lipopolysaccharide (LPS). Dendritic cell phenotypes were observed after 24 hours. Fig.23 The results are shown in Fig.24 shown.

[0132] As expected, LPS treatment resulted in increased expression of CD80, CD86, MHC class I, and MHC class II on the surface of treated DCs compared to vehicle controls. Fig.24 , black columns. Nanoparticles encapsulating the adapter-modified OTII peptide had little effect on LPS-induced expression of CD80, CD86, class I MHC, and class II MHC. Fig.24, the fifth column from the left in each figure. Surprisingly, nanoparticle co-encapsulation of the adapter-modified OTII peptide and aspirin inhibited LPS-induced expression of CD80, CD86, class I MHC, and class II MHC. Fig.24 , the rightmost column in each graph. These data suggest that the adaptor modification strategy can be used to prepare tolerogenic nanoparticles to convert dendritic cells into tolerogenic dendritic cells by reducing the presentation of MHC molecules and co-stimulatory molecules on the cells.

[0133] The above results clearly indicate that the peptide-transfer modification strategy allows the preparation of tolerogenic nanoparticles by facilitating antigen / nanocarrier coupling without compromising the antigenic determinant characteristics of the modified peptides.

[0134] The above examples show that peptide transfers can be universally applied to peptide targets to enable the conjugation of multiple peptide targets to a selected carrier system. In addition, the fusion of the peptide transfer to the target peptide surprisingly does not reduce the immunogenicity or specificity of the target peptide. This is particularly important in the production of customized cancer vaccines against new antigenic determinants. Once tumor-specific new antigenic determinants are identified, they can be synthesized with peptide transfers attached to their N-termini without the need for detailed analysis of the characteristics of the antigenic determinant.

[0135] Other specific embodiments

[0136] All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature having the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, each feature disclosed is merely an example of a series of equivalent or similar features.

[0137] Based on the above description, those skilled in the art can easily determine the basic characteristics of the present invention, and can make various changes and modifications to the present invention to adapt it to various uses and conditions without departing from the spirit and scope of the present invention. Therefore, other embodiments are also within the scope of the scope of the application. Sequence Listing <110> Zhou Meiyin <120> Peptide-carrying carrier system and its use <130> GCI22TW0071 <140> 202080054080.2 <141> 2020-07-29 <150> US 62 / 880,421 <151> 2019-07-30 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 1 Lys Val Pro Arg Asn Gln Asp Trp Leu 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 2 Thr Ala Tyr Arg Tyr His Leu Leu 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 3 Ser Val Tyr Asp Phe Phe Val Trp Leu 1 5 <210> 4 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 4 Asp Asp Asp Asp Gly Gly Gly Ile Ser Gln Ala Val His Ala Ala His 1 5 10 15 Ala Glu Ile Asn Glu Ala Gly Arg 20 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 5 Asp Tyr Lys Asp Asp Asp Lys 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 6 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 7 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 <210> 8 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 8 Asp Asp Asp 1 <210> 9 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 9 Asp Asp Asp Asp 1 <210> 10 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 10 Gly Gly Gly 1 <210> 11 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 11 Asp Asp Asp Gly Gly Gly Ser Val Tyr Asp Phe Phe Val Trp Leu 1 5 10 15 <210> 12 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 12 Ser Ile Ile Asn Phe Glu Lys Leu 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 13 Ala Ser Met Thr Asn Met Glu Leu Met 1 5 <210> 14 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 14 Ser Ile Ile Val Phe Asn Leu Leu 1 5 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 15 Ala Gln Leu Ala Asn Asp Val Val Leu 1 5 <210> 16 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 16 Phe Gln Asp Ala Tyr Asn Ala Ala Gly Gly His Asn Ala Val Phe 1 5 10 15 <210> 17 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 17 Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly 1 5 10 15 Arg <210> 18 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 18 Gln Val Tyr Ser Leu Ile Arg Pro Asn Glu Asn Pro Ala His Lys 1 5 10 15

Claims

1. A carrier system comprising a nanocarrier and a peptide non-covalently bound to the nanocarrier, wherein the peptide comprises a linker peptide sequence fused to the N-terminus of a target peptide, the nanocarrier having a core and a surface, wherein the core is hydrophobic or hydrophilic, the surface has a net negative charge, has a net positive charge or carries one or more functional groups, and the linker peptide sequence facilitates the non-covalent binding of the peptide to the core or surface of the nanocarrier.

2. The vector system according to claim 1, wherein the linker peptide sequence comprises two or more hydrophilic amino acids selected from D, E, R, K and H, or the linker peptide sequence comprises two or more hydrophobic amino acids selected from A, V, I, L, P, F, W and M.

3. The carrier system of claim 2, wherein the core is hydrophilic and the adapter peptide sequence is Dn, En, (DE)n, (DX)n or (EX)n, wherein n is an integer from 2 to 20, and X is any amino acid.

4. The vector system according to claim 3, further comprising a spacer fragment fused between the target peptide and the adapter peptide sequence, wherein the spacer comprises two or more amino acid residues selected from G, A, S and P.

5. The vector system according to claim 4, wherein the spacer fragment is Gn, wherein n is an integer from 1 to 15.

6. The vector system according to claim 5, wherein the adapter peptide sequence is DDD (SEQ ID NO: 8) or DDDD (SEQ ID NO: 9), the spacer fragment is GGG (SEQ ID NO: 10), and the target peptide is fused to the C-terminus of the spacer fragment.

7. The vector system according to claim 6, wherein the target peptide is a class I MHC restricted antigenic determinant or a class II MHC restricted antigenic determinant.

8. The vector system of claim 7, further comprising an immune response stimulator selected from the group consisting of interferon gene (STING) agonist, CpG-ODN, R848 and poly(I:C).

9. The carrier system according to claim 7, further comprising an immune response suppressor selected from the group consisting of rapamycin, aspirin, vitamin D, steroids and N-acetylcysteine.

10. The carrier system of claim 8, wherein the nanocarrier is a hollow polymer nanoparticle.

11. The carrier system of claim 9, wherein the nanocarrier is a hollow polymer nanoparticle.

12. A method for improving the immunogenicity of a peptide antigen, the method comprising fusing the peptide antigen to a linker peptide sequence to form an immune peptide, and contacting the immune peptide with a nanocarrier so that the immune peptide is stably non-covalently bound to the nanocarrier, wherein the target peptide is a class I MHC restricted antigenic determinant or a class II MHC restricted antigenic determinant, the nanocarrier has a hydrophilic core, and the linker peptide sequence comprises two or more hydrophilic amino acids selected from D, E, R, K and H.

13. The method of claim 12, wherein the adaptor peptide sequence is Dn, En, (DE)n, (DX)n or (EX)n, wherein n is an integer from 2 to 20, and X is any amino acid.

14. The method according to claim 13, further comprising fusing a spacer fragment between the peptide antigen and the adapter peptide sequence, wherein the spacer comprises two or more amino acid residues selected from G, A, S and P.

15. The method of claim 14, wherein the spacer fragment is Gn, wherein n is an integer from 1 to 15.

16. The method of claim 15, wherein the adapter peptide sequence is DDD (SEQ ID NO: 8) or DDDD (SEQ ID NO: 9), the spacer fragment is GGG (SEQ ID NO: 10), and the peptide antigen is fused to the C-terminus of the spacer fragment.

17. An immunization method for treating a disease in a subject, the method comprising fusing a target peptide to a linker peptide sequence to form an immune peptide, contacting the immune peptide with a nanocarrier, thereby stably and non-covalently binding the immune peptide to the nanocarrier to form a carrier system, and administering the carrier system to the subject to increase the immune response to the target peptide, wherein the target peptide is a class I MHC restricted antigenic determinant or a class II MHC restricted antigenic determinant, and the disease is cancer, viral infection, bacterial infection, parasitic infection, autoimmunity, or an adverse immune response to a biologic.

18. The method according to claim 17, wherein the immunizing peptide further comprises a spacer fragment, wherein the spacer fragment is fused between the C-terminus of the adapter peptide sequence and the N-terminus of the target peptide.

19. The method of claim 18, wherein the adapter peptide sequence is DDD (SEQ ID NO: 8) or DDDD (SEQ ID NO: 9), and the spacer fragment is GGG (SEQ ID NO: 10).

20. The method of claim 17, further comprising incorporating the nanocarrier into an immune response stimulator selected from the group consisting of an interferon gene (STING) agonist, CpG-ODN, R848, and poly(I:C), wherein the target peptide is a cancer antigen, a viral antigen, a bacterial antigen, or a parasite antigen.

21. The method of claim 17, further comprising incorporating into the nanocarrier an immune response inhibitor selected from rapamycin, aspirin, vitamin D, steroids, and N-acetylcysteine, wherein the target peptide is a self-antigen and administration of the carrier system induces tolerance to the self-antigen.

22. The method of claim 20, wherein the nanocarrier is a hollow polymer nanoparticle.

23. The method of claim 21, wherein the nanocarrier is a hollow polymer nanoparticle.

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