Tumor immunotherapy

Abstract

A composition comprising a closed loop peptide containing at least two methionines and at least two sulfonium centers, and a conjugate formed by a negatively charged immune adjuvant and a polypeptide.

Description

Detailed Description of the Invention

[0001] [Field of the Invention] The present disclosure relates generally to the field of medicine, and more specifically to the field of tumor immunotherapy.

[0002] [background] Tumor immunotherapy is known as the third revolution in cancer treatment. Different from surgical resection, chemotherapy, and radiotherapy, which act directly on tumor sites, tumor immunotherapy can generate specific immune responses (e.g., activation of killer T cells) by activating the host immune system, indirectly target tumor cells precisely, and form long-term memory, thus effectively preventing tumor recurrence.

[0003] Neoantigens are generated by somatic mutations and are expressed only in tumor cells, effectively evading host central thymic tolerance restrictions, and have high tumor specificity and immunogenicity. Therefore, targeting them can induce the host immune system to precisely evade normal cells, allowing for precise individualization of immunotherapy in tumors.

[0004] [overview] In one aspect, the present disclosure relates to a composition comprising a closed-loop peptide comprising at least two methionines and at least two sulfonium centers, and a conjugate formed by a negatively charged immune adjuvant and the peptide.

[0005] In another aspect, the present disclosure relates to a process for preparing a composition comprising dissolving a linear peptide comprising at least two methionines in a weakly acidic polar solution and adding a closed-loop reagent to obtain a closed-loop peptide, mixing an immune adjuvant with a sulfonium peptide under basic conditions to obtain a conjugate by addition reaction, and mixing the conjugate with the closed-loop peptide to obtain a composition, wherein the composition comprises a closed-loop peptide comprising at least two methionines and at least two sulfonium centers, and a conjugate formed by the negatively charged immune adjuvant and the peptide.

[0006] In a further aspect, the present disclosure relates to a method of treating or preventing a tumor comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a composition, wherein the composition comprises a closed-loop peptide comprising at least two methionine and at least two sulfonium centers, and a conjugate formed by a negatively charged immune adjuvant and the peptide.

[0007] In yet another aspect, the present disclosure relates to a method for activating immunity comprising administering to a subject in need thereof an effective amount of a composition, wherein the composition comprises a closed loop peptide comprising at least two methionine and at least two sulfonium centers, and a conjugate formed by a negatively charged immune adjuvant and the peptide.

[0008] In another aspect, the present disclosure relates to a nanoparticle comprising a closed-loop peptide comprising at least two methionines and at least two sulfonium centers, and a conjugate formed by a negatively charged immune adjuvant and the peptide, wherein the closed-loop peptide self-assembles with the conjugate to form the nanoparticle.

[0009] In a further aspect, the present disclosure relates to a composition for treating or preventing tumors comprising a closed-loop peptide comprising at least two methionines and at least two sulfonium centers, and a conjugate formed by a negatively charged immune adjuvant and the peptide.

[0010] In yet another aspect, the present disclosure relates to a composition for immune activation comprising a closed-loop peptide comprising at least two methionines and at least two sulfonium centers, and a conjugate formed by a negatively charged immune adjuvant and the peptide.

[0011] In another aspect, the present disclosure relates to the use of a composition in the preparation of a medicament for treating or preventing a tumor, wherein the composition comprises a closed-loop peptide comprising at least two methionine and at least two sulfonium centers, and a conjugate formed by a negatively charged immune adjuvant and the peptide.

[0012] In a further aspect, the present disclosure relates to the use of a composition in the preparation of a medicament for immune activation, wherein the composition comprises a closed-loop peptide comprising at least two methionine and at least two sulfonium centers, and a conjugate formed by a negatively charged immune adjuvant and the peptide. [Brief description of the drawings]

[0013] [Figure 1] The preparation and mass spectrometry identification results of M-CP closed loop peptide are shown, where (a) shows a schematic diagram of the preparation of M-CP closed loop peptide, (b) shows the structure and mass spectrometry results of linear M-CP peptide, where P2, P3, and P4 represent the peak positions in mass spectrometry when the peptide has 2, 3, and 4 electrons, respectively, and (c) shows the structure and mass spectrum results of the peptide after closing (peptide sequence information: M-CP: Fmoc-RRMEHRMEW, SEQ ID NO1). [Diagram 2] The results of covalent bonding between CpG adjuvant and antigen peptide OVA are shown, where (a) shows a schematic diagram of the covalent bond between CpG and OVA, (b) shows the structure and MS identification results of FAM-OVA with bromopropyne attached, and (c) shows the electrophoretic verification results of the covalent bond (peptide sequence information: FAM-OVA: Ac-WMβASIINFEK(FAM)L, SEQ ID NO3). [Diagram 3] LC-MS identification results of the covalent bond between CpG adjuvant and antigen peptide OVA are shown, where (a) shows the LC-MS identification results of CpG, and (b) shows the LC-MS identification results of the CpG-OVA covalent conjugate (peptide sequence information: FAM-OVA: Ac-MβASIINFEK(FAM)L, SEQ ID NO3; CpG sequence information: 5'-3', HS-TCCATGACGTTCCTGACGTT, SEQ ID NO6). [Figure 4] 1 shows the results of verification of reversible reduction of CpG-OVA covalent conjugate by GSH, (a) shows the reduction of CpG-OVA by GSH, and (b) shows the electrophoretic verification map of the reduction product. [Diagram 5] Electrophoretic results of self-assembly of M-CP closed-loop peptide (referred to as MCN-NP) induced by CpG-OVA conjugate, where (a) shows self-assembly of M-CP closed-loop peptide induced by CpG-OVA conjugate, (b) shows electrophoretic results of composite with different substrate ratios, (c) shows self-assembly results of composite under different buffer conditions, and (d) shows electrophoretic results of nanocomposites prepared with different mixing times. [Figure 6] 1 shows DLS analysis results of MCN-NPs, where (a) shows the particle size distribution of the nanocomposite, (b) shows the potential profile of the nanocomposite, and (c) shows the TEM electron microscope results of the nanocomposite. [Figure 7] 1 shows the stability analysis results of MCN-NPs in different buffer solutions, where (a) shows the particle size change statistics and (b) shows the potential change statistics. [Figure 8] Figure 1 shows the results of proliferation inhibition of RAW264.7 cells by MCN-NPs based on MTT assay analysis (with respect to the concentration of OVA). [Figure 9] FIG. 1 shows the hemolysis evaluation results of MCN-NPs (measured by M-CP), where (a) shows a photograph of the supernatant after centrifugation, and (b) shows the absorbance of the supernatant at 570 nm. [Figure 10] The results of evaluation of the membrane permeability of MCN-NP are shown, where (a) shows the statistical results of the amount of fluorescence of MCN-NP in RAW264.7 cells 4 hours after administration, and (b) shows the fluorescence localization of MCN-NP in RAW264.7 cells 4 hours after administration. [Figure 11] Statistical results of the fluorescence amount of MCN-NP in DC2.4 cells are shown, where (a) shows the statistical results of the fluorescence amount of MCN-NP in DC2.4 cells 4 hours after administration, (b) shows the median axis value statistics of the fluorescence amount of MCN-NP in DC2.4 cells 4 hours after administration, and (c) shows the fluorescence localization of MCN-NP in DC2.4 cells 4 hours after administration. [Figure 12] Fluorescence quantitative analysis results of MCN-NP in BMDC cells are shown, where (a) shows the statistical results of the fluorescence amount of MCN-NP in BMDC cells 4 hours after administration, (b) shows the median axis value statistics of the fluorescence amount of MCN-NP in BMDC cells 4 hours after administration, and (c) shows the distribution of MCN-NP in BMDC cells 4 hours after administration. [Figure 13] The results of MCN-NP uptake by BMDC cells at different time periods are shown. [Figure 14] 1 shows secretion of tumor-associated factors TNF-α, interleukin IL-6, and interleukin IL-12 by RAW264.7 cells stimulated with MCN-NPs, where (a) shows the ELISA analysis results of secretion of tumor-associated factor TNF-α by RAW264.7 cells, (b) shows the ELISA analysis results of secretion of interleukin IL-6 by RAW264.7 cells, and (c) shows the ELISA analysis results of secretion of interleukin IL-12 by RAW264.7 cells. [Figure 15] 1 shows the enrichment of MCN-NPs in lymph nodes, where (a) shows lymph node size comparison and fluorescence imaging of Cy5.5 in lymph nodes 24 hours after subcutaneous administration, and (b) shows the statistics of fluorescence intensity for Cy5.5. [Figure 16] Figure 1 shows the apparent evaluation results in lymph nodes after three immunizations, where (a) shows the immunization dosing regimen, (b) shows the size comparison of lymph nodes in each mouse group on day 7 after the final administration of MCN-NP, (c) shows the lymph node weight statistics corresponding to (b), and (d) shows the total cell count in lymph nodes corresponding to (b). [Figure 17] Figure 1 shows the proliferation of each immune cell in lymph nodes after three immunizations, where (a) shows the percentage of DC cell population in lymph nodes, (b) shows the percentage of B cell population in lymph nodes, and (c) shows the percentage of macrophages in lymph nodes. [Figure 18] 1 shows changes in T cell populations in the blood of mice after three immunizations, where (a) shows the change in the proportion of the total T cell population in the blood 7 days after the final immunization, (b) shows the change in the proportion of CD8+ T cells in the blood 7 days after the final immunization, (c) shows the proportion of OVA-specific cells among CD8+ T cells 1 day and 7 days after the final immunization, and (d) shows the zoning results of OVA+CD8+ T cells in the blood of each mouse group 7 days after the final immunization. [Figure 19] 1 shows the inhibition of growth following administration of subcutaneously inoculated B16-OVA melanoma, where (a) shows the dosing regimen for tumor prevention, (b) shows statistical data of tumor volume in mice within 24 days after tumor inoculation, (c) shows monitoring of tumor growth in individual mice per group, (d) shows effective inhibition rate of tumor in mice 24 days after inoculation, and (e) shows statistics of mouse life cycle. [Figure 20] 1 shows the change in body weight after administration in mice bearing subcutaneously inoculated B16-OVA melanoma. [Figure 21]1 shows lung metastasis after administration of B16-OVA melanoma tumor cells 20 days after injection via tail vein, where (a) shows the administration schedule of the lung metastasis model, (b) shows statistical data of lung nodules, (c) shows a photo of mouse lungs, and (d) shows H&E staining results in mouse lung sections. [Figure 22] H&E staining of major tissue organs after administration in mice 20 days after injection of B16-OVA melanoma via the tail vein is shown. [Figure 23-1] 1 shows inhibition of tumor growth of B16-OVA seeded prior to dosing, where (a) shows the dosing regimen for the tumor treatment model, (b) shows statistical data of tumor volume growth, (c) shows statistics of mouse life cycle, and (d) shows statistics of mouse weight. [Figure 23-2] Inhibition of tumor growth of B16-OVA inoculated prior to dosing is shown, where (e) shows monitoring of individual tumor growth in each group of mice. [Figure 24-1] Figure 2 shows inhibition of MC-38 tumor growth by MCN-NPs (Adpgk), where (a) shows the tumor growth curve, (b) shows the weight statistics of each tumor group on day 34, and (c) shows the comparison plot of each tumor group on day 34 (peptide sequence information: Adpgk:Ac-ELASMTNMELMSS, SEQ ID NO4). [Figure 24-2] Figure 2 shows the inhibition of MC-38 tumor growth by MCN-NPs (Adpgk), where (d) shows the growth curves of individual tumors in each mouse group, and (e) shows the tumor inhibition rate in each mouse group (peptide sequence information: Adpgk:Ac-ELASMTNMELMSS, SEQ ID NO4). [Diagram 25] The changes in mouse body weight in each group (MC-38 tumor group) are shown. [Figure 26] FIG. 1 is a schematic diagram of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] [Detailed Description] In the following description, certain specific details are included to provide a thorough understanding of various disclosed embodiments, however, one of ordinary skill in the art will recognize that an embodiment may be practiced without one or more of these specific details, but using other methods, components, materials, etc.

[0015] Unless otherwise stated in this application, throughout the specification and the appended claims, the terms "comprise," "comprising," "contain," and "having" are to be interpreted in their open and inclusive sense, i.e., "including, but not limited to."

[0016] As used in this disclosure and the appended claims, singular references without a quantity include plural references unless the context clearly dictates otherwise.

[0017] References throughout this specification to "one embodiment" or "an embodiment" or "in other embodiments" or "in some embodiments" mean that the particular referent feature or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment," "in an embodiment," "in other embodiments," or "in some embodiments" in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0018] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a composition that includes "a closed-loop peptide comprising at least two methionines and at least two sulfonium centers" includes one closed-loop peptide that comprises at least two methionines and at least two sulfonium centers, or two or more closed-loop peptides that comprise at least two methionines and at least two sulfonium centers.

[0019] [Definition] As used herein, the term "sulfonium" refers to a compound that contains quadrivalent sulfur. The term "conjugate" as used herein refers to a novel compound formed by two different types of compounds through a covalent bond. As used herein, the term "room temperature" refers to a temperature of 25° C. at standard atmospheric pressure. As used herein, the term "M-CP" refers to SEQ ID NO:1, in which the sequence (N→C) is Fmoc-RRMEHRMEW. As used herein, the term "OVA" refers to SEQ ID NO2, whose sequence (N→C) is Ac-WMβASIINFEKL. As used herein, the term "FAM-OVA" refers to SEQ ID NO3, whose sequence (N→C) is Ac-MβASIINFEK(FAM)L. As used herein, the term "Adpgk" refers to SEQ ID NO:4, whose sequence is Ac-ELASMTNMELMSS. As used herein, the term "CpG" refers to SEQ ID NO:6, the sequence (5'→3') of which is HS-TCCATGACGTTCCTGACGTT. As used herein, the term "Ac" refers to an amide group. As used herein, the term "βA" refers to an isomer of alanine (A). The term "mammal" as used herein refers to animals including, for example, dogs, cats, cows, sheep, horses, humans, etc. In some embodiments, a mammal includes a human. As used herein, the term "patient" refers to animals (e.g., humans), companion animals (such as dogs, cats, or horses), and livestock (such as cows, pigs, and sheep). In some embodiments, the patient is a mammal, including males and females. In some embodiments, the patient is a human. As used herein, "treating" or "treatment" encompasses the treatment of a disease or condition of interest in a mammal, such as a human, having the disease or disorder of interest, and includes: (i) preventing the development of a disease or condition in a mammal, particularly where the mammal is susceptible to the condition but has not yet been diagnosed as afflicted; (ii) inhibiting the disease or condition, i.e., arresting its progression; or (iii) relieving the disease or condition, i.e. causing regression of the disease or condition.

[0020] [Specific embodiment] In one aspect, the present disclosure relates to a composition comprising a closed-loop peptide comprising at least two methionines and at least two sulfonium centers, and a conjugate formed by a negatively charged immune adjuvant and the peptide.

[0021] In some embodiments, illustrative examples of negatively charged immune adjuvants that can be used in the present disclosure include, but are not limited to, nucleic acid immune adjuvants, activators of immune pathways, and adjuvants that have the effect of blocking immune suppressive pathways.

[0022] In some embodiments, an illustrative example of a negatively charged immune adjuvant that can be used in the present disclosure is CpG to silence immune suppressive pathways or activate immune pathways. 1826, poly(I:C), and siRNA / shRNA.

[0023] In some embodiments, a negatively charged immunoadjuvant that can be used in the present disclosure comprises at least 20 nucleotides.

[0024] In some embodiments, illustrative examples of negatively charged immune adjuvants that can be used in the present disclosure include, but are not limited to, nucleic acid immune adjuvants comprising the sequence of SEQ ID NO:6.

[0025] In some embodiments, an illustrative example of a negatively charged immune adjuvant that can be used in the present disclosure includes, but is not limited to, a nucleic acid immune adjuvant represented by the sequence of SEQ ID NO:6.

[0026] In some embodiments, illustrative examples of peptides that can be used in the present disclosure include, but are not limited to, antigenic peptides.

[0027] In some embodiments, illustrative examples of antigenic peptides that can be used in the present disclosure include, but are not limited to, melanoma B16-OVA (OVA257-264:Ac-SIINFEKL) and the neo-antigenic peptide of MC-38 tumor cells (Adpgk:Ac-ELASMTNMELMSS).

[0028] In some embodiments, illustrative examples of peptides that can be used in the present disclosure include, but are not limited to, sulfonium peptides.

[0029] In some embodiments, illustrative examples of peptides that can be used in the present disclosure include, but are not limited to, a peptide comprising the sequence of SEQ ID NO2, a peptide comprising the sequence of SEQ ID NO3, and a peptide comprising the sequence of SEQ ID NO4.

[0030] In some embodiments, illustrative examples of peptides that can be used in the present disclosure include, but are not limited to, a peptide represented by the sequence of SEQ ID NO2, a peptide represented by the sequence of SEQ ID NO3, and a peptide represented by the sequence of SEQ ID NO4.

[0031] In some embodiments, illustrative examples of peptides that can be used in the present disclosure include, but are not limited to, closed loop peptides containing at least 9 amino acids.

[0032] In some embodiments, illustrative examples of closed loop peptides that can be used in the present disclosure include, but are not limited to, closed loop peptides comprising the sequence of SEQ ID NO1.

[0033] In some embodiments, the structure of a closed loop peptide that can be used in the present disclosure is as follows:

[0034] [ka]

[0035] In some embodiments, illustrative examples of conjugates that can be used in the present disclosure include, but are not limited to, reversible covalent conjugates.

[0036] In some embodiments, the particle size of the composition is about 50 nm to 200 nm.

[0037] In some embodiments, the codelivery system of the present disclosure is capable of rapid self-assembly and may simultaneously incorporate immune adjuvants, tumor neo-antigens, and tumor-associated antigens, which overcomes the problems of long sequences, high cost, and limited stability of peptide-based nanoparticle supports.

[0038] In another aspect, the present disclosure relates to a process for preparing a composition, comprising dissolving a linear peptide comprising at least two methionines in a weakly acidic polar solution, adding a closed-loop reagent to obtain a closed-loop peptide, mixing an immune adjuvant with a sulfonium peptide under basic conditions to obtain a conjugate by addition reaction, and mixing the conjugate with the closed-loop peptide to obtain a composition, wherein the composition comprises a closed-loop peptide comprising at least two methionines and at least two sulfonium centers, and a conjugate formed by the negatively charged immune adjuvant and the peptide.

[0039] In some embodiments, illustrative examples of immune adjuvants that can be used in the processes of the present disclosure include, but are not limited to, nucleic acid adjuvants, activators of immune pathways, and adjuvants that have the effect of blocking immune suppressive pathways.

[0040] In some embodiments, illustrative examples of immune adjuvants that can be used in the processes of the present disclosure include, but are not limited to, sulfhydryl-terminated immune adjuvants.

[0041] In some embodiments, illustrative examples of immune adjuvants that can be used in the processes of the present disclosure include, but are not limited to, immune adjuvants comprising the sequence of SEQ ID NO:6.

[0042] In some embodiments, illustrative examples of immune adjuvants that can be used in the processes of the present disclosure include, but are not limited to, the immune adjuvant represented by the sequence of SEQ ID NO:6.

[0043] In some embodiments, illustrative examples of sulfonium peptides that can be used in the processes of the present disclosure include, but are not limited to, alkynyl-terminated sulfonium peptides and alkenyl-terminated sulfonium peptides.

[0044] In some embodiments, illustrative examples of sulfonium peptides that can be used in the processes of the present disclosure include, but are not limited to, a sulfonium peptide comprising the sequence of SEQ ID NO2, a sulfonium peptide comprising the sequence of SEQ ID NO3, and a sulfonium peptide comprising the sequence of SEQ ID NO4.

[0045] In some embodiments, illustrative examples of sulfonium peptides that can be used in the processes of the present disclosure include, but are not limited to, a sulfonium peptide represented by the sequence of SEQ ID NO2, a sulfonium peptide represented by the sequence of SEQ ID NO3, and a sulfonium peptide represented by the sequence of SEQ ID NO4.

[0046] In some embodiments, the molar ratio of immune adjuvant to sulfonium peptide is about 1:2 to 1:10.

[0047] In some embodiments, the molar ratio of immune adjuvant to closed-loop peptide is about 1:50 to 1:400.

[0048] In some embodiments, illustrative examples of linear peptides containing at least two methionines that can be used in the processes of the present disclosure include, but are not limited to, the sequence of SEQ ID NO1, a peptide containing the sequence of SEQ ID NO1, the sequence of SEQ ID NO1 with one or more amino acid mutations, and a peptide containing the sequence of SEQ ID NO1 with one or more amino acid mutations.

[0049] In some embodiments, illustrative examples of linear peptides containing at least two methionines that can be used in the processes of the present disclosure include, but are not limited to, the linear peptide represented by the sequence of SEQ ID NO1.

[0050] In some embodiments, a linear peptide containing at least two methionines that can be used in the process of the present disclosure has the following structure:

[0051] [ka]

[0052] In some embodiments, illustrative examples of weakly acidic polar solutions that can be used in the processes of the present disclosure include, but are not limited to, a mixture of formic acid / acetonitrile / water.

[0053] In some embodiments, the volume ratio of formic acid, acetonitrile and water in the mixed solution of formic acid / acetonitrile / water that can be used in the process of the present disclosure is about 1:(1-5):(1-4).

[0054] In some embodiments, illustrative examples of closed loop reagents that can be used in the present disclosure include, but are not limited to, o-dibromobenzyl, 1,3-bis(bromomethyl)benzene, 1,4-bis(bromomethyl)benzene, 1,4-dibromobutene, 2,3-bis(bromomethyl)quinoxaline, and 4,4′-dibromomethylbiphenyl.

[0055] In some embodiments, the concentration of the closed-loop reagent is about 6 mg / mL to 8 mg / mL.

[0056] In some embodiments, the conjugate self-assembles with the closed loop peptide to obtain the composition.

[0057] In some embodiments, the immune adjuvant is mixed with the sulfonium peptide under basic conditions at about 4° C. to 28° C., and the conjugate is obtained by addition reaction.

[0058] In some embodiments, the immune adjuvant is mixed with the sulfonium peptide at room temperature under basic conditions to obtain the conjugate by addition reaction.

[0059] In some embodiments, a linear peptide containing at least two methionines is dissolved in a mixture of formic acid / acetonitrile / water, and a closed-loop reagent is added and reacted for at least 8 hours to obtain a closed-loop peptide.

[0060] In some embodiments, the processes of the present disclosure are simple and efficient.

[0061] In a further aspect, the present disclosure relates to a method of treating or preventing a tumor, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a composition, wherein the composition comprises a closed-loop peptide comprising at least two methionine and at least two sulfonium centers, and a conjugate formed by a negatively charged immune adjuvant and the peptide.

[0062] In some embodiments, illustrative examples of tumors that can be used in the present disclosure include, but are not limited to, melanoma and colon cancer.

[0063] In some embodiments, illustrative examples of subjects that can be used with the present disclosure include, but are not limited to, mammals.

[0064] In some embodiments, illustrative examples of subjects that can be used with the present disclosure include, but are not limited to, humans.

[0065] In some embodiments, the disclosed methods for treating or preventing a tumor further comprise administering to the subject another agent for preventing or treating the tumor.

[0066] In some embodiments, illustrative examples of agents for preventing or treating tumors that can be used in the present disclosure include, but are not limited to, chemotherapeutic agents, biologic agents, and any combinations that can be used in combination.

[0067] In some embodiments, illustrative examples of chemotherapeutic agents for preventing or treating tumors that can be used in the present disclosure include, but are not limited to, small molecule chemotherapeutic agents.

[0068] In some embodiments, illustrative examples of small molecule chemotherapeutic agents for preventing or treating tumors that can be used in the present disclosure include, but are not limited to, paclitaxel.

[0069] In some embodiments, illustrative examples of biologic agents for preventing or treating tumors that can be used in the present disclosure include, but are not limited to, tumor neoantigen peptide vaccines, peptide drugs, and antibody drugs.

[0070] In some embodiments, illustrative examples of antibody agents for preventing or treating tumors that can be used in the present disclosure include, but are not limited to, PD-1 monoclonal antibodies and PD-L1 monoclonal antibodies.

[0071] In some embodiments, illustrative examples of PD-1 monoclonal antibodies for preventing or treating tumors that can be used in the present disclosure include, but are not limited to, nivolumab, pembrolizumab, and tislelizumab.

[0072] In some embodiments, illustrative examples of PD-L1 monoclonal antibodies for preventing or treating tumors that can be used in the present disclosure include, but are not limited to, atezolizumab, durvalumab, and avelumab.

[0073] In yet another aspect, the present disclosure relates to a method for immune activation comprising administering to a subject in need thereof an effective amount of a composition comprising a closed loop peptide comprising at least two methionine and at least two sulfonium centers, and a conjugate formed by a negatively charged immune adjuvant and the peptide.

[0074] In some embodiments, the immunity is tumor immunity. In some embodiments, the immunity is cellular immunity.

[0075] In another aspect, the present disclosure relates to a nanoparticle, comprising a closed-loop peptide comprising at least two methionine and at least two sulfonium centers, and a conjugate formed by a negatively charged immune adjuvant and the peptide, wherein the closed-loop peptide self-assembles with the conjugate to form a nanoparticle.

[0076] In certain embodiments, the nanoparticles of the present disclosure have a particle size of about 50 nm to 200 nm.

[0077] In a further aspect, the present disclosure relates to a composition for treating or preventing a tumor, comprising a closed-loop peptide comprising at least two methionine and at least two sulfonium centers, and a conjugate formed by a negatively charged immune adjuvant and the peptide.

[0078] In yet another aspect, the present disclosure relates to a composition for immune activation, comprising a closed-loop peptide comprising at least two methionine and at least two sulfonium centers, and a conjugate formed by a negatively charged immune adjuvant and the peptide.

[0079] In another aspect, the present disclosure relates to the use of a composition in the preparation of a medicament for treating or preventing a tumor, wherein the composition comprises a closed-loop peptide comprising at least two methionine and at least two sulfonium centers, and a conjugate formed by a negatively charged immune adjuvant and the peptide.

[0080] In a further aspect, the present disclosure relates to the use of a composition in the preparation of a medicament for immune activation, wherein the composition comprises a closed-loop peptide comprising at least two methionine and at least two sulfonium centers, and a conjugate formed by a negatively charged immune adjuvant and the peptide.

[0081] The present disclosure will be described in detail below in order to better understand the aspects and advantages of the present application. However, it should be understood that the following examples are not limiting and merely illustrate certain embodiments of the present disclosure.

[0082] [Example] Reagents and equipment used in the embodiments of the present disclosure are conventional and commercially available, for example:

[0083] [Table 1-1]

[0084] [Table 1-2]

[0085] [Table 1-3]

[0086] [Table 1-4]

[0087] [Example 1] Synthesis, isolation and purification of linear peptides

[0088] The desired peptide was obtained using conventional solid phase peptide synthesis techniques.

[0089] [Table 2]

[0090] The specific operation steps are as follows: (1) Solid-phase synthesis of peptides: 500 mg of Rink amide MBHA resin was weighed into a 10 mL peptide tube, dichloromethane (DCM) was added, and nitrogen was bubbled through for 30 min. A solution of 50% (v / v) morpholine in N,N-dimethylformamide (DMF) was added, and nitrogen was bubbled through for 30 min to remove the Fmoc protecting group from the resin. The resin was washed alternately six times with DMF and DCM, and the above step was repeated twice to remove Fmoc. The resin was washed alternately six times with DMF and DCM, and then a DMF solution containing amino acid (5 eq, 0.4 M, DMF solvent), 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) (5 eq, 0.38 M, DMF solvent), and N,N-diisopropylethylamine (DIPEA) (10 eq) was added by bubbling through the resin for 2 h. The reaction solution was removed and the resin was washed six times alternately with DMF and DCM, after which the Fmoc was removed in the same manner as described above, followed by the addition of the next amino acid until all amino acid couplings were completed. (2) Peptide terminal amidation treatment: In order to improve the stability of peptides, the N-terminal amino group was generally acetylated. The specific steps are as follows: after the final amino acid was coupled, the Fmoc group was removed according to the method in step (1), and then 2.5 mL of amidation reagent (containing acetic anhydride: DIPEA: DMF (v / v / v) = 1: 3: 16) was added, and after 1 hour of nitrogen bubbling, the resin was washed alternately with DMF and DCM 10 times. The above procedure was repeated again to complete the amidation protection of the N-terminal NH2. (3) Cleavage of peptides: The peptides were cleaved from the resin using a mixed cleavage solution containing trifluoroacetic acid (TFA), triisopropylsilane (TIPS), and water (TFA:TIPS:water (v:v:v)=95:2.5:2.5), and the cleavage solution was removed by nitrogen purging. Then, the peptides were precipitated with ice ether to complete the pre-purification of the peptides and obtain the crude products of M-CP, FAM-OVA, OVA, or Adpgk peptides. (4) The pre-purified sample was separated and purified using a C18 high performance liquid chromatography column and identified by LC-MS. The resulting product was lyophilized using a freeze dryer to obtain a peptide pure product with a purity of more than 95%.

[0091] [Example 2] M-CP peptide closure, isolation, and purification steps The HPLC-purified M-CP peptide solid was dissolved in a mixed solution containing formic acid, acetonitrile, and water (formic acid:acetonitrile:water (v:v:v) = 1:5:4), and 10 eq of o-dibromobenzyl was added. After the reaction was performed on a shaker overnight, the reaction mixture was purified again by HPLC and lyophilized to obtain the closed-loop product of M-CP (Figure 1a). The introduction of o-dibromobenzyl added two positively charged "sulfonium" centers to the original linear peptide with two methionines, resulting in an increase in the overall positively charged nature of the peptide, which provided a stronger electrostatic driving force for nucleic acid-induced self-assembly. Furthermore, the active "sulfonium" center site can be reduced by a reducing agent such as intracellular GSH, thus releasing the internal drug. Rapid in vitro self-assembly and in vivo reversible reductive release in the closed-loop peptide can be realized by a simple closing step. Figure 1b and Figure 1c show that the two methionines M in the M-CP peptide were immobilized after the addition of o-dibromobenzyl, at which point the molecular weight of the peptide increased from the original 1551.8 Da to 1655.9 Da. The MS identification results were consistent with the theoretical molecular weight, indicating that the closed-loop peptide was successfully obtained.

[0092] [Example 3] Preparation, isolation, and purification of OVA bearing a sulfonium center In order to covalently bind the tumor antigen peptide to the nucleic acid immune adjuvant, a methionine was added to the end of the OVA peptide. An alkynyl group with a sulfonium center was introduced at the methionine position under acidic conditions. At this point, the highly active alkynyl group can be added to the sulfhydryl group at the end of the nucleic acid under the driving force of the positively charged "sulfonium" center to realize the covalent bond between the peptide and the nucleic acid. The "sulfonium" center in the conjugate can provide an active site for reversible cleavage in a reducing agent (such as GSH or mercaptopyridine), achieving efficient conjugation in vitro and reversible release of the antigen peptide-adjuvant in vivo. (1) The OVA peptide solid was dissolved in a mixed solution containing formic acid, acetonitrile, and water (formic acid:acetonitrile:water (v:v:v) = 1:5:4), and 10 eq of tribromopropyne was added. The reaction was carried out overnight on a shaker to introduce an alkynyl group having a "sulfonium" center at the methionine position of the peptide (Figure 2a). (2) The reacted sample was separated and purified using a C18 high performance liquid chromatography column and identified by LC-MS. Figure 2b shows that the molecular weight of the FAM-OVA peptide after the introduction of the "sulfonium" center was 1603.8 Da, which was consistent with the theoretical data. (Figure 2b is a schematic structural diagram and mass spectrometry identification of the FAM-OVA peptide.)

[0093] [Example 4] Preparation, isolation, and purification of CpG-OVA covalent conjugates

[0094] [Table 3]

[0095] (1) Aligned sulfhydryl-terminated DNA may partially dimerize during transportation or due to the influence of temperature, which may result in a decrease in the covalent conjugation efficiency of nucleic acid peptides. Therefore, pretreatment of aligned DNA before ligation is first required to open the disulfide bonds formed by DNA self-aggregation. The specific procedure is as follows: 200 eq of TCEP was added to 50 μL of 100 μM DNA aqueous solution, reacted at room temperature for 4 hours, and then 0.1 eq of sodium acetate (pH 5.2, 3 M) and 2.5 eq of ice ethanol were added. After centrifugation at 12000 rpm for 30 minutes, the supernatant was removed to remove excess TCEP. Then, 200 μL of ice ethanol was added, washed once, and centrifuged at 12000 rpm for 30 minutes, and the supernatant was removed. The precipitate was dried on ice for 10 minutes, dissolved in ultrapure water, and quantified using Nanodrop. (2) According to the protocol shown in Figure 2a, 2eq to 10eq of FAM-OVA peptide was added to the CpG system, and the reaction was carried out in 10mM (NH4)2CO3 solution at 4℃ overnight, after which the reaction product was isolated and purified by C18 high performance liquid chromatography column. The ligation result was detected by PAGE gel electrophoresis. The results in Figure 2c showed that after adding FAM-OVA to CpG, a new product with a delayed band position was generated in the lane. Further analysis by FAM fluorescence channel revealed that the product in the CpG-OVA lane was different from FAM-OVA, which demonstrated that the OVA peptide with a sulfonium center could be covalently linked to the sulfhydryl-terminated CpG. (3) To further determine the covalent conjugation of CpG to OVA, the final conjugated product was examined by LC-MS, and the molecular weight of the DNA-peptide covalent conjugate product was determined to be 7858 Da (Figure 3b), which was equal to the sum of the molecular weights of CpG (6255 Da, Figure 3a) and FAM-OVA peptide (1603 Da, Figure 2b).

[0096] [Example 5] Reverse reduction of the CpG-OVA covalent conjugate product To confirm that the CpG-OVA covalent conjugate prepared according to the present disclosure can be re-reduced and cleaved to release the OVA peptide and the immune adjuvant CpG, and is capable of efficient in vitro assembly and in vivo reversible release in nanocomposites, this example demonstrated the reduction of the CpG-OVA covalent conjugate by glutathione GSH. 100 eq of GSH reducing agent was added to the CpG-OVA covalent conjugate product at a final concentration of 40 μM, and the reaction was carried out at 37° C. in PBS buffer for 0, 4, 8, 12, 24, 36, 48, and 72 hours. The reduction of the covalent conjugate product was analyzed by 10% PAGE gel electrophoresis. The results in FIG. 4 showed that as the amount of CpG substrate gradually increased, the reaction time was extended, and the band of the initially conjugated product gradually became darker. The covalent conjugate was determined to be reductively cleavable by the reducing agent.

[0097] [Example 6] Rapid self-assembly of M-CP closed-loop peptides induced by CpG-OVA (1) Two positively charged "sulfonium" centers were further introduced into the linear M-CP peptide with two methionines after closing, providing sufficient electrostatic interaction sites for the preparation of nanocomposites. The negatively charged nucleic acid immune adjuvant CpG in this disclosure acted as a trigger for self-assembly in the peptides, inducing the rapid assembly of the positively charged M-CP closed-loop peptides (Fig. 5a, the nanocomplex was abbreviated as MCN-NP). It was worth mentioning that the active "sulfonium" centers in both the closed-loop peptides and the CpG-OVA covalent conjugates could both provide action sites for the reduction of intracellular GSH and the degradation of the nanocomposite. Efficient, rapid self-assembly in the nanocomposite in vitro and reversible reduction release in vivo were finally achieved. The results in Figure 5b showed that the CpG-OVA band disappeared as the concentration of M-CP closed-loop peptide increased, and complete encapsulation of CpG-OVA was achieved with the addition of 100 equivalents of M-CP closed-loop peptide. In addition to aqueous environments, self-assembly of the composites can also be achieved in opti-MEM cell culture medium and cell culture medium containing 10% serum in 10 min (Figures 5c and 5d). (2) The microscopic parameters of MCN-NP were characterized by dynamic nanoparticle particle size meter DLS. The results in Figure 6a showed that the nanocomposite had a particle size of 54.4 ± 0.9 nm and a PDI index of 0.19 ± 0.02 at CpG-OVA:M-CP = 1:300 (the PDI index is used to evaluate the homogeneity of the nanocomposite, the numerical range is 0-1, the smaller the numerical range, the higher the particle homogeneity, generally less than 0.3). The results in Figure 6b showed that with the addition of M-CP, the charge of the composite system gradually changed from the original negative charge to a positive charge, again confirming that the negatively charged nucleic acid induced the self-assembly of the positively charged M-CP closed-loop peptide. (3) Furthermore, in this study, the micromorphology of MCN-NPs was observed by transmission electron microscopy (TEM). The electron microscopy results in Figure 6c show that the nanocomposites were uniform spheres with particle sizes consistent with the DLS results.

[0098] [Example 7] Stability assessment of MCN-NPs Due to the stability of the peptide, it is difficult to effectively activate the host's immune system when the tumor antigen peptide is administered alone, and as a result, clinical trials on new tumor antigens have been insufficient. Encapsulation of antigen peptides by nanocomposites played an important role in enhancing the stability and efficacy of antigen peptides. In this disclosure, the stability of MCN-NPs in aqueous phase and culture medium containing 10% serum RPMI1640 cells was evaluated. After incubation at 4°C for 24 hours, DLS analysis showed that the particle size and potential of MCN-NPs did not increase significantly (Figure 7), indicating that the nanocomposite prepared according to this disclosure had high stability.

[0099] [Example 8] Safety evaluation of MCN-NPs The safety and suitability of the tumor antigen delivery system were prerequisites for its clinical application and promotion. To this end, the present disclosure evaluated the biological safety of MCN-NPs using the classical MTT method and hemolysis assay. (1) For the MTT assay, RAW264.7 cells were pre-plated in a 96-well plate and administered with a maximum dose of 1 μm of CpG-OVA (where the M-CP dose was normalized to the OVA dose) according to CpG-OVA:M-CP=1:300, followed by serial dilutions in a gradient fashion. 24 hours after administration, 20 μL of MTT (3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide) was added to each well and incubated for another 4 hours at 37°C. After carefully aspirating the cell supernatant, 100 μL of methadone crystals, generated by lysis of live cells lysed with DMSO, were added to each well. The absorbance was measured at 490 nm for each well using full-function enzyme calibration. The cell proliferation inhibition rate was calculated, and the cytotoxicity of MCN-NPs was evaluated using drug-free cells as a control group. The results in FIG. 8 showed that the cells in the MCN-NP group had nearly 100% cell viability even at the maximum dose, and MCN-NP had a weak proliferation-promoting effect at low drug concentrations, suggesting that MCN-NP did not have significant cytotoxicity. (2) Furthermore, the biological safety of MCN-NPs was evaluated by a cell hemolysis assay. Fresh mouse whole blood was removed by centrifugation at 1200 rpm for 5 min, and the cells were washed with PBS until the supernatant was colorless. Subsequently, MCN-NPs with different concentrations (maximum dose of 1 μM, calculated as OVA concentration) were administered in 10 5The MCN-NPs were mixed with 100 μl of red blood cells. The mixture was incubated at 37° C. for 1 h. The supernatant was then centrifuged at 1200 rpm for 5 min, and 100 μl of the supernatant was collected into a 96-well plate. The absorbance of the supernatant was measured using full-function enzyme calibration at 570 nm to evaluate heme lysis. The results in FIG. 9a showed that the color of the cell supernatant in the MCN-NP incubation group was similar to that of the PBS control group. Furthermore, the measurement of the absorbance of the supernatant at 570 nm revealed that heme lysis, associated with the fragmentation of the red blood cell membrane, was hardly detected in the supernatant for the administration of the maximum amount of MCN-NPs (FIG. 9b). The constructed antigen delivery system of the present disclosure was also shown to have good biological safety, which provided important assurance for the subsequent use of the nanosystem in tumor antigen immunotherapy.

[0100] [Example 9] Evaluation of MCN-NP uptake by macrophages RAW264.7 Tumor antigen uptake by immune cells was a key step in activating the immune system. In this disclosure, flow cytometry and laser confocal microscopy (Confocal) were used to evaluate MCN-NP uptake by immune cells. First, RAW264.7 cells were treated with green fluorescent tagged OVA peptide (FAM group), CpG-OVA covalent conjugate, and MCN-NP (1 μM as OVA), respectively. The uptake of MCN-NP into RAW264.7 cells was analyzed after 4 hours by flow cytometry and laser confocal microscopy. (1) For flow analysis: The cells treated for 4 hours were washed three times with PBS, then digested with 0.25% trypsin for 1 min, and 10% RPMI1640 was added to neutralize the trypsin. Centrifugation was performed at 1000 rpm and 4°C, and the supernatant was removed. The cells were washed with PBS according to resuspension-centrifugation-resuspension, and on-machine detection was performed. The flow results in Figure 10a showed that the MCN-NP experimental group had the strongest fluorescence shift compared with the OVA group, confirming that the M-CP nanocomposite significantly improved the cell membrane permeation ability of the OVA peptide. (2) For confocal experiments: Cells were spread on cell climbing pieces 12 hours before, the climbing pieces were carefully washed with PBS after administration treatment, and then the fixed cells were treated with 4% paraformaldehyde for 15 minutes. Residual formaldehyde was washed twice with PBS. Finally, the pieces were sealed with a sealing tablet containing DAPI. After drying in the dark at room temperature, the intensity and localization of green fluorescence in RAW264.7 cells of different experimental groups were observed using a 488 nm laser. The confocal results in Figure 10b also showed that the cells in the MCN-NP-treated group had the strongest green fluorescence of FAM, which was consistent with the flow detection results in Figure 10a. In addition, M-CP significantly increased the uptake of OVA antigen by macrophages.

[0101] [Example 10] Evaluation of MCN-NP uptake by antigen-presenting cells DC2.4 As important antigen-presenting cells, the uptake of antigen by DC cells directly determines the ability of DC cells to induce T cell activation. The present disclosure further evaluated the uptake of MCN-NPs by the stably culturable antigen-presenting cell line DC2.4. Flow cytometry and laser confocal microscopy were also used to analyze the membrane penetration and localization of MCN-NPs after internalization. The flow-through procedure was the same as in Example 9. For confocal experiments, paraformaldehyde fixation significantly reduced DC cells, in which the degree of internalization of MCN-NPs was analyzed using live cell imaging technology. The procedure is as follows: DC2.4 cells were seeded in an 8-well plate for live cell imaging 12 hours before administration, and the culture supernatant was removed 4 hours after administration, and a mixed solution of PBS containing Hoechst 33342 (nuclear blue fluorescent dye for live cells) and Lysotracker (lysosomal red fluorescent dye for live cells) was added, and the supernatant was discarded after incubation at 37 °C for 30 minutes. The cells were carefully washed three times with PBS, and then 10% RPMI1640 medium was added to make it detectable on-machine. The flow results in Figure 11a and Figure 11b showed that in the DC2.4 cell line, the MCN-NPs-treated group had the largest fluorescence shift. Similarly, the confocal data (Figure 11c) showed the strongest green fluorescence in DC2.4 cells in the MCN-NPs group. The above data again demonstrated that M-CP was effective in helping the penetration of antigen peptides.

[0102] [Example 11] Evaluation of MCN-NP uptake by mouse bone marrow-derived BMDCs The mature cell lines RAW264.7 and DC2.4 could first demonstrate the internalization of the nanocomposite by immune cells at the cellular level, and whether MCN-NPs had the same effect on DC cells in vivo required further experimental validation. To further evaluate the in vivo immune cell internalization of MCN-NPs, bone marrow DC cells from C57BL / 6 mice were extracted according to a well-established published method (see BMDC extraction and culture procedure, available from Abcam, Inc.: https: / / www.abcam.cn / proteins / bmdc-isolation-protocol) and tested for MCN-NPs internalization.

[0103] The BMDC culture procedure is as follows: C57BL / 6 mice aged 2 to 4 weeks were used and sacrificed using CO2. The femurs and tibiae of the mice were harvested under sterile conditions. Both ends of the bone joint were cut using scissors and the bone marrow was washed with serum-free RPMI medium until the bone cavity turned white. After centrifugation at 1500 rpm for 5 min, the supernatant was removed and the cells were resuspended in red blood cell lysis solution for 3 min, after which lysis was completed in RPMI1640 medium supplemented with 5 mL of serum to contain 10% serum. After centrifugation at 1500 rpm for 5 min, the supernatant was removed and then washed twice with RPMI1640. The cells were resuspended using 10% RPMI1640 containing 20 ng / mL GM-CSF and 10 ng / mL IL-4 factors. After counting, the cells were diluted to 1 × 10 6 Cells / mL were seeded into 6-well plates at a density of 10 ...

[0104] The results in Figure 12 showed that BMDCs had higher uptake of MCN-NPs compared with OVA peptide alone at 4 hours after administration. With increasing administration time, the green fluorescence intensity of BMDCs first increased and then decreased, and significant green fluorescence was observed in the cells of the MCN-NP-treated group even after 48 hours (Figure 13). This result was consistent with the data of DC2.4, and again fully demonstrated that the peptide-based nanocomposite prepared according to the present disclosure can significantly improve the stability, membrane permeability, and intracellular controlled release effect of tumor antigen peptides.

[0105] [Example 12] Evaluation of the immune activation ability of MCN-NPs The secretion of tumor-associated factors and interleukins from immune cells stimulated by drugs may reflect the activity of the immune system to some extent. In this disclosure, the ability of MCN-NPs to stimulate the activation of the immune system was evaluated by the secretion of tumor-associated factors TNF-α and interleukins IL-6, IL12p40 by RAW264.7 cells after administration of drugs using an enzyme-linked immunosorbent assay (ELISA) kit. RAW264.7 cells were treated with CpG, OVA, CpG-OVA, and MCN-NPs (at a concentration of 1 μM on an OVA basis) for 48 hours, respectively. The cell supernatant was collected. Subsequently, the suspended cells were removed by centrifugation at 1000 g for 20 minutes. 100 μL of the cell supernatant was added to an enzyme-linked immunoassay plate. The secretion of each cytokine by RAW264.7 cells was analyzed according to the instructions of the ELISA kit. The contents of tumor-associated factors TNF-α (FIG. 14a), interleukin IL-6 (FIG. 14b), and IL-12p40 (FIG. 14c) in the cell culture supernatant were calculated by drawing a standard curve from the ELISA results of the standard samples in the kit. The results in FIG. 14 showed that the MCN-NP nanocomposite significantly increased the secretion amount of each associated factor compared to the administration of OVA or CpG alone, demonstrating its potential tumor immunotherapy ability.

[0106] [Example 13] Evaluation of the ability of MCN-NPs to activate the in vivo immune system The experimental results at the cellular level preliminarily confirmed that the nanoparticles constructed according to the present disclosure had potential antitumor immune effects. To further evaluate the efficacy of the vaccine, the activation of the immune system stimulated by MCN-NP was evaluated at the animal level. To measure the enrichment of the nanocomposite in lymph nodes, Cy5.5 was fluorescently modified at the 5' end of CpG, and then MCN-NP-Cy5.5 was prepared according to Example 6. 24 hours after subcutaneous administration in the tail, the mice were treated with CO2, and mouse lymph nodes were obtained and subjected to in vitro tissue fluorescence imaging analysis. The results in Figure 15a showed that the lymph nodes in MCN-NP were significantly increased compared with the control group and CpG group. Further image analysis revealed that the fluorescence intensity of the lymph nodes in the MCN-NP group was 3.77 times higher than that of the lymph nodes in the CpG group (Figure 15b). It was shown that the nanocomposite effectively entered the lymph nodes under the action of the draining lymph nodes.

[0107] To further evaluate whether the nanocomposite that entered the lymph nodes was effective in activating relevant immune cells, the MCN-NP nanocomposite (for the amount of 15 nmol / OVA) was administered every 7 days according to the time points in Figure 16a, and the mixture of M-CP and CpG (for the amount of 15 nmol / CpG) was added once at 7-day intervals. Seven days after the last administration of the MCN-NP nanocomposite, lymph node stimulation was analyzed again, revealing that the inguinal lymph nodes of the MCN-NP group were the largest and had the highest cell count (Figures 16b, 16c, and 16d). The lymph nodes were then ground into single cells in a 70 μM disposable nylon filter using a syringe plunger, 1 mL of PBS was added to resuspend the single cells, and centrifuged at 500 g for 5 minutes at room temperature, after which the supernatant was removed. 1 mL of PBS was added to resuspend again, 50 μL of cell resuspension was taken, and the cell count was counted by flow cytometry. 2×10 7The number of cells / tube was divided into three parallel groups. After centrifugation, the cells were resuspended in 100 μL cell flow buffer and 1.0 μL CD16 / CD32 antibody was added. After 10 min incubation at room temperature, CD11c-APC (for labeling DC cells), CD19-PE (for labeling B cells), and F4 / 80-APC (for labeling macrophages) were added respectively and incubated at room temperature for 20 min. 1 mL PBS was added. Then, after 5 min centrifugation at room temperature, the supernatant was removed. PBS was used to wash again. The cells were resuspended in 500 μL cell flow buffer. The fluorescently labeled cells were evaluated on the machine. The results in Figure 17 showed that DC cells (Figure 17a) and B cells (Figure 17b) were significantly activated for proliferation by MCN-NPs in lymph nodes. The results in Fig. 17c showed that the proportion of macrophages was not significantly increased in the MCN-NPs group, which may be due to the fact that macrophages are associated with innate immunity, and the levels of cells associated with innate immunity were returned to equilibrium 7 days after administration.

[0108] After that, the changes of T cells in the blood of the mice were continuously detected, and the specific procedure was as follows: fresh mouse blood was obtained by orbital blood sampling, and sodium heparin was added to prevent coagulation. Then, the blood was divided into 50 μL, and lysed by adding 1 mL of 1× red blood cell lysis solution for 10 minutes, and centrifuged at 500 g for 5 minutes at room temperature, after which the supernatant was removed. The cells were resuspended with 1 mL of cell flow buffer, centrifuged, and washed again. Then, the cells were resuspended in 100 μL of cell flow buffer. Labeling of T cells in the blood was performed according to the lymph node staining procedure. CD3e-efluor450, CD4-APC, and CD8-FITC were added simultaneously to the above samples to detect CD4 in the blood. + and CD8 +The T cell population was labeled. The simultaneous addition of CD3e-efluor450, CD8-FITC, and MHC-OVA-APC was performed to label OVA-specific T cells in the CD8+ cell population in the blood. The results in FIG. 18 showed that the total population of T cells in the blood of mice (FIG. 18a), the percentage of CD8+ T cells (FIG. 18b), and the percentage of OVA-specific killer T cells (FIG. 18c) were increased 7 days after the end of immunization. Furthermore, the ability of MCN-NPs to induce T cells to differentiate into CD8+ killer T cells was improved compared to the physical co-administration of CpG and OVA, and the percentage of OVA-specific CD8+ T cells was significantly increased (FIG. 18d). The nanocomposite constructed according to the present disclosure was shown to significantly activate the cellular immune system of mice.

[0109] [Example 14] MCN-NPs for tumor prevention Furthermore, the present disclosure established two tumor prevention models to evaluate the prevention of tumor colonization and growth using nanocomposites. The specific model settings were as follows: Tumor prevention model: A control group (0% 1640), an OVA group (15 nmol / mouse), a CpG+OVA group (CpG 15 nmol / mouse, OVA 15 nmol / mouse), a CpG-OVA covalent conjugate (15 nmol / mouse), MCN-NP (CpG-OVA 15 nmol / mouse, M-CP 150 nmol / mouse), and an M-CP group (150 nmol / mouse) were set up. They were administered three times every 7 days according to the protocol in Figure 19a. The MCN-NP group was supplemented once with a mixture of M-CP and CpG (15 nmol / mouse, M-CP 150 nmol / mouse, calculated as the amount of CpG) at 2-day intervals after each administration. Ten days after the end of the last immunization, 1 × 10 6 B16-OV cells / mouse were inoculated subcutaneously. After 6 days, tumor size was measured and body weight change was counted. Tumors reached a tumor statistical endpoint of 1500 mm 3 The experiment was terminated when the number of plants reached 100. Life cycle statistics were then performed.

[0110] The results in Figures 19b to 19d showed that the tumors in mice in the nanocomposite group were significantly inhibited with an inhibition rate of 100%, and the life cycle of mice in this group was significantly increased (Figure 19e). No significant changes in body weight were detected throughout the study (Figure 20), indicating that the nanocomposite was biologically safe and played a positive role in preventing tumor formation. Lung metastasis model: Furthermore, a lung metastasis model was chosen to evaluate the prevention of melanoma lung colonization using the nanocomposite (Figure 21a). The dose was 5 x 10 per mouse. 5 The administration was similar to that in the tumor model, except that B16-OVA cells were injected via the tail vein 7 days after the end of the last administration. Mice were dissected on the 20th day after injection, and the lungs of the mice were removed and washed with Fekete's elution solution (70 mL alcohol, 10 mL formaldehyde, 5 mL glacial acetic acid, 15 mL water). After decolorization, the lung tumor nodules were counted. Finally, all tissues were sent to Univ company for tissue sectioning and H&E staining analysis. The results in Figure 21b and Figure 21c showed that the MCN-NP group had the lowest number of black nodules in the lungs. H&E staining analysis in the lung tissue revealed that the control group could detect significant lung tissue demonstration and melanoma cell infection. Similarly, lung cells infected with melanoma cells were also evident in the OVA, CpG+OVA, and CpG-OVA covalent conjugate groups. The absence of this phenomenon in the MCN-NP nanocomposite group indicated that the nanocomposite was effective in preventing lung metastasis of melanoma. No significant pathological and physiological structural changes were detected in H&E staining of vital tissues other than the lung compared with the control group (Figure 22), which also predicted that the nanocomposite constructed in this study had good safety.

[0111] [Example 15] MCN-NPs for the treatment of tumors The present disclosure then constructed a tumor treatment model to evaluate the efficacy of the nanocomposite in treating melanoma. According to the time points in Figure 23a, 1 × 10 per mouse was administered. 6 B16-OVA cells were subcutaneously inoculated on day 0. Six days later, tumor size was measured, the weight change of the mice was measured, and the first administration was performed. After that, a total of three administrations were performed every 7 days. The MCN-NP group was supplemented once with a mixture of M-CP and CpG (15 nmol / g, M-CP 150 nmol / g, calculated as the amount of CpG) at an interval of 2 days after each administration. The increase in tumor volume in the mice was continuously measured during the period. 3 The tumor growth to 100 mg / kg / day was the statistical end point of the tumor. Subsequently, a life cycle analysis was performed. The results in Fig. 23b and Fig. 23e showed that the tumor growth was slowest in the MCN-NP group, and the tumors of three mice were eliminated 27 days after inoculation, achieving 75% tumor treatment. The life cycle in this group of mice was also significantly increased (Fig. 23c), again demonstrating that the nanoparticle vaccine constructed according to the present disclosure had a significant tumor treatment effect. Similarly, no significant decrease in body weight was detected in the mice throughout the experiment, indicating the safety of the nanocomposite (Fig. 23d).

[0112] [Example 16] MCN-NPs for the treatment of other tumors To verify the suitability and general applicability of the delivery system in this disclosure, the model antigen peptide OVA was replaced with the tumor antigen peptide Adpgk (Ac-ELASMTNMELMSS, SEQ ID NO4) of mouse colon cancer MC-38. The covalent conjugate of CpG-Adpgk was prepared according to the protocols in Examples 1 to 4. The covalent conjugate was mixed with M-CP peptide to obtain a nanocomposite containing the novel antigen peptide of Adpgk (MCN-NP (Adpgk) The present disclosure further established an antitumor model of MC-38 as follows: 1 × 10 per mouse 6MC-38 cells were subcutaneously inoculated on day 0. After 7 days, tumor size was measured, body weight change was measured, and the first administration was performed. After that, administration was performed three times every 7 days. Control group (0% 1640), Adpgk group (15 nmol / mouse), CpG+Adpgk group (CpG 15 nmol / mouse, Adpgk 15 nmol / mouse), MCN-NP (Adpgk) The mice were provided with the CpG-Adpgk group (15 nmol / mouse, M-CP group (150 nmol / mouse) and the M-CP group (150 nmol / mouse). The increase in tumor volume in the mice was continuously measured during the period. Statistical data was recorded when the tumor reached 1000 mm 3 The data included tumors from each mouse group until they grew to 100%. For data statistical analysis, the tumors of each mouse group were obtained on the 34th day after tumor seeding. From the MC-38 tumor growth curves in Fig. 24a (statistical data) and Fig. 24d (individual data), it can be seen that the tumor volume increased most slowly in the MCN-NP-treated group, followed by the M-CP-treated group, which was consistent with the results in Fig. 23b. This indicated that the nanoparticle vaccine constructed according to the present disclosure had a significant and tumor-specific tumor therapeutic effect. As can be more intuitively revealed from the tumor weight and comparison diagrams in Figs. 24b, 24c, and 24e, the MCN-NP-treated group showed a slower tumor volume increase, followed by the M-CP-treated group. (Adpgk) The tumors of three mice in the treatment group disappeared on day 34, and the tumor treatment rate reached 60%, again demonstrating the efficacy and general applicability of the nanoparticle vaccine constructed in this disclosure. Similarly, no significant loss in mouse body weight was detected throughout the experiment (Figure 25), indicating the safety of the nanocomposite.

[0113] In this disclosure, relational terms such as first and second are used only to distinguish one entity or operation from another, and do not necessarily require or imply any actual relationship or ordering between such entities or operations.

[0114] From the foregoing, it will be appreciated that, although specific embodiments of the present disclosure have been described herein for purposes of illustration, various modifications or improvements may be made by those skilled in the art without departing from the spirit and scope of the present disclosure, which changes or modifications are intended to be included within the scope of the claims appended hereto.

Claims

1. 1. A composition comprising: a closed loop peptide comprising at least two methionines and at least two sulfonium centers; and A composition comprising a conjugate formed by a negatively charged immune adjuvant and a peptide.

2. The negatively charged immune adjuvants include nucleic acid immune adjuvants, activators of immune pathways, and adjuvants that have the effect of blocking immune suppressive pathways, preferably CpG to silence immune suppressive pathways or activate immune pathways. １８２６ 2. The composition of claim 1, wherein the nucleic acid is selected from the group consisting of poly(I:C), poly(I:C), and siRNA / shRNA.

3. The composition of claim 1 or claim 2, wherein the negatively charged immunoadjuvant comprises at least 20 nucleotides.

4. The composition according to any one of claims 1 to 3, wherein the negatively charged immunoadjuvant comprises the sequence of SEQ ID NO6.

5. The peptide is an antigenic peptide, preferably an antigenic peptide of melanoma B16-OVA (OVA ２５７－２６４ The composition according to any one of claims 1 to 4, wherein the antigen peptide is a neo-antigen peptide (Adpgk:Ac-ELASMTNMELMSS) of MC-38 tumor cells.

6. The composition of any one of claims 1 to 5, wherein the peptide comprises at least 8 amino acids.

7. The composition of any one of claims 1 to 6, wherein the peptide comprises the sequence of SEQ ID NO2, the sequence of SEQ ID NO3, or the sequence of SEQ ID NO4.

8. The composition of any one of claims 1 to 7, wherein the closed loop peptide comprises at least 9 amino acids.

9. The composition of any one of claims 1 to 8, wherein the closed loop peptide comprises the sequence of SEQ ID NO1.

10. The composition of claim 1 , wherein the conjugate is a reversible covalent conjugate.

11. 11. The composition of claim 1, wherein the composition has a particle size of from 50 nm to 200 nm.

12. A process for preparing the composition of claim 1, comprising the steps of: dissolving a linear peptide containing at least two methionines in a weakly acidic polar solution and adding a closed-loop reagent to obtain a closed-loop peptide; mixing the immune adjuvant with the sulfonium peptide under basic conditions to obtain a conjugate by addition reaction; and mixing said conjugate with said closed loop peptide to obtain said composition; A process comprising:

13. 13. The process according to claim 12, wherein the immune adjuvant is a nucleic acid adjuvant, preferably a sulfhydryl-terminated immune adjuvant, more preferably a nucleic acid immune adjuvant comprising the sequence of SEQ ID NO6.

14. 14. The process according to claim 12 or 13, wherein the sulfonium peptide is selected from the group consisting of alkynyl-terminated sulfonium peptides and vinyl-terminated sulfonium peptides, preferably selected from the group consisting of a peptide comprising the sequence of SEQ ID NO2, a peptide comprising the sequence of SEQ ID NO3, and a peptide comprising the sequence of SEQ ID NO4.

15. 15. The process according to any one of claims 12 to 14, wherein the molar ratio of the immune adjuvant to the sulfonium peptide is from 1:2 to 1:

10.

16. 16. The process according to any one of claims 12 to 15, wherein the molar ratio of the immune adjuvant to the closed-loop peptide is from 1:50 to 1:

400.

17. 17. The process according to any one of claims 12 to 16, wherein the conjugate self-assembles with the closed peptide to obtain a composition.

18. 18. The process according to any one of claims 12 to 17, wherein the immune adjuvant is mixed with the sulfonium peptide under basic conditions at 4°C to 28°C to obtain the conjugate by addition reaction.

19. 19. A process according to any one of claims 12 to 18, wherein the linear peptide containing at least two methionines comprises the sequence of SEQ ID NO1.

20. 12. A method of treating or preventing a tumor comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a composition according to any one of claims 1 to 11.

21. 21. The method of claim 20, wherein the tumor is selected from the group consisting of melanoma and colon cancer.

22. 22. The method of claim 20 or claim 21, wherein the subject is a mammal, preferably a human.

23. 23. The method of any one of claims 20 to 22, further comprising administering to the subject another agent for preventing or treating a tumor.

24. The method of claim 23, wherein the agent is selected from the group consisting of a chemotherapeutic agent, a biological agent, or any combination thereof, preferably a small molecule chemotherapeutic agent, a peptide agent, an antibody agent, or any combination thereof, more preferably paclitaxel, a PD-1 monoclonal antibody, a PD-L1 monoclonal antibody, a biological agent with targeted therapeutic effect, or any combination thereof.

25. A method for activating immunity comprising administering to a subject in need thereof an effective amount of a composition according to any one of claims 1 to 11.

26. The method of claim 25, wherein the immunity is tumor immunity.

27. The method of claim 25 or claim 26, wherein the immunity is cellular immunity.

28. A nanoparticle, a closed loop peptide comprising at least two methionines and at least two sulfonium centers; and Conjugates formed by negatively charged immune adjuvants and peptides Including, wherein the closed loop peptide self-assembles with the conjugate to form a nanoparticle.

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