Universal hydrogel vaccine based on tumor neoantigen peptide as well as preparation method and application of universal hydrogel vaccine

Abstract

The invention relates to a universal hydrogel vaccine based on tumor neoantigen peptide as well as a preparation method and application of the universal hydrogel vaccine. The hydrogel vaccine comprises tumor neoantigen peptide hydrogel and an adjuvant loaded on the tumor neoantigen peptide hydrogel, the tumor neoantigen peptide-peptidyl assembly sequence conjugate is assembled in a solvent to form a nanofiber solution, and the nanofiber solution is triggered to form hydrogel under physiological conditions; the conjugate is obtained by coupling a tumor neoantigen peptide and a peptidyl assembly sequence through a covalent bond; the adjuvant is at least any one of a cell factor, an STING agonist, a Toll-like receptor agonist and an immune cell. The vaccine disclosed by the invention has universality, aiming at different tumor neoantigen peptides, self-assembly of a conjugate in a solvent is realized by regulating and controlling the quantity of valine or phenylalanine in a peptidyl assembly sequence to form a nanofiber solution, and the vaccine adjuvant is loaded through physical action to obtain a nanofiber solution containing the vaccine adjuvant; therefore, personalized cancer immunotherapy is realized.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a universal hydrogel vaccine based on tumor neoantigen peptides, and a preparation method and application thereof. Background Art

[0002] The development of next-generation sequencing and bioinformatics tools has promoted the rapid identification and prediction of tumor neoantigen peptides, bringing great hope for personalized tumor immunotherapy. However, vaccines based on tumor neoantigen peptides face rapid degradation and clearance by the body after subcutaneous and intramuscular injection. At the same time, problems such as size effect, charge effect, and solubility also hinder the migration of vaccines to lymph nodes, resulting in insufficient utilization of neoantigen peptides, limited activation of antigen-presenting dendritic cells, and short-lived anti-tumor immune responses. Therefore, the development of efficient and safe personalized tumor vaccines remains a huge challenge.

[0003] At present, hydrogel-based scaffold materials have attracted widespread attention in the field of vaccine delivery. They have the characteristics of high delivery efficiency, easy storage and transportation at room temperature, and long sustained-release drug efficacy. Hydrogels can deliver vaccines to specific sites, increase drug concentration and bioavailability, control and continuously release drugs to effector sites, and minimize off-target toxic side effects. In addition, their porous structure also provides space for the retention of immune cells and the interaction between cells and vaccine components, thereby inducing a strong anti-tumor immune response. Therefore, the construction of vaccines based on hydrogel scaffold materials is expected to improve the controllable and long-term release of vaccine components, change the spatiotemporal distribution of antigens and adjuvants in lymph nodes, and thus improve the anti-tumor effect.

[0004] It is worth noting that directly linking tumor neoantigen peptides to designed peptide-based assembly sequences is expected to convert tumor neoantigen peptides into effective hydrogel vaccines, but there is currently little research in this area. Summary of the invention

[0005] The purpose of the present invention is to overcome the problems of low immune activation efficiency, long development cycle and high cost of existing vaccines, and to provide a universal hydrogel vaccine based on tumor neoantigen peptides and its preparation method and application.

[0006] Expanding the application scope of the above hydrogel scaffold material platform and developing highly versatile and rapidly scalable hydrogel vaccines for different tumors are of great significance for future clinical applications and personalized treatment. Therefore, according to the physicochemical properties of tumor neoantigen peptides, screening and optimizing the number of key amino acids in the peptide assembly sequence is conducive to promoting the assembly of tumor neoantigen peptide-peptide assembly sequence conjugates and subsequent hydrogel formation, so as to construct hydrogel vaccines based on tumor neoantigen peptides and realize rapid preparation and immediate application of vaccines.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] One of the technical solutions of the present invention provides a universal hydrogel vaccine based on tumor neoantigen peptides, comprising a tumor neoantigen peptide hydrogel and an adjuvant loaded on the tumor neoantigen peptide hydrogel;

[0009] The tumor neoantigen peptide hydrogel is assembled from tumor neoantigen peptide-peptide assembly sequence conjugates in a solvent to form a nanofiber solution, which is converted from a solution state to a hydrogel state under physiological conditions;

[0010] The tumor neoantigen peptide-peptide assembly sequence conjugate is obtained by covalently coupling a tumor neoantigen peptide and a peptide assembly sequence; the tumor neoantigen peptide serves as a tumor antigen; and the peptide assembly sequence contains valine V or phenylalanine F with assembly and folding properties.

[0011] The universal hydrogel vaccine based on tumor neoantigen peptides directly uses neoantigen peptides as hydrogel building units, has the advantages of easy adjustment of drug content and reduced drug burst effect. Compared with the technical solutions in the prior art that directly use self-assembled polypeptide hydrogels to encapsulate tumor-derived exosomes, RBD proteins, S proteins, S1 proteins, S2 proteins, N proteins, E proteins or M proteins and other antigens, it has obvious advantages in technical effects.

[0012] Furthermore, the sources of the tumor neoantigen peptides include but are not limited to: mutated peptides obtained from genetic changes in the genome of any one or more tumors of breast cancer, melanoma, colon cancer, glioma, gastric cancer, lung cancer, pancreatic cancer, prostate cancer, bladder cancer, endocrine tumors, adenomas, blastomas, desmoid tumors, sarcomas, lymphomas, germ cell tumors, rectal cancer, hepatocellular carcinoma, leukemia, Wilms' tumor, gastric cancer, esophageal cancer, cervical cancer, and head and neck cancer; the tumor neoantigen peptides include but are not limited to: SIINFEKLC, ASMTNMELMC, LCPGNKYEM, TYVPANASL, CYTWNQMNL, SYGVLLWEIF, SVYDFFVWL ,LMLGEFLKL,SGLEQLESIINFEKL,RFVPDGNRI,GWVKPIIIGH,RVLELFRAAQLANDDVVLQIMELC,GIPVHLELASMTNMELMSSIVHQQVF,REGVELCPGNKYEMRRHGT THSLVIHD, ALLEIASCL, KVPRNQDWL, MTYENMDIL, VDWENVSPELNSTDQ, PSKPSFQEFVDWENVSPELNSTDQPFL, KPIIIGHHAYGDQYRA, FMTYWHLLNAFTVTVPKDL.

[0013] Furthermore, the peptidyl assembly sequence is any one of the following: C(V) n GRGD, C(V) n GRGDR, C(F) n GRGD, C(F) n GRGDR regulates the gel properties by adjusting the number of valine V or phenylalanine F (n=any integer from 0 to 5, and can be 0).

[0014] Among them, A is alanine, D is aspartic acid, E is glutamic acid, F is phenylalanine, G is glycine, H is histidine, I is isoleucine, K is lysine, L is leucine, N is asparagine, P is proline, Q is glutamine, R is arginine, S is serine, T is threonine, V is valine, W is tryptophan, and Y is tyrosine; that is, SIINFEKLC represents a polypeptide having an amino acid sequence of SIINFEKLC, ASMTNMELMC represents a polypeptide having an amino acid sequence of ASMTNMELMC, and the same applies to the others.

[0015] The hydrogel vaccine based on tumor neoantigen peptides provided in the present application can be customized for different types of tumors, and can be designed by optimizing the number of key amino acids in the peptide assembly sequence, designing the corresponding tumor neoantigen peptide-peptide assembly sequence conjugates and subsequently producing a hydrogel vaccine based on tumor neoantigen peptides for personalized cancer immunotherapy.

[0016] Furthermore, the adjuvant is selected from one or a combination of cytokines, STING agonists, Toll-like receptor agonists, and immune cells.

[0017] Furthermore, the cytokines include but are not limited to: IL-2, IL-12, IL-6, IL-15, IL-21, IL-22, IFN-α, IFN-β, IFN-γ, TNF-β, GM-CSF, CXCL-9, CXCL-10, CXCL-11, CCL27, and CCL28.

[0018] Furthermore, the STING agonists include but are not limited to: c-di-GMP, c-di-AMP, 3',3'-cGAMP, 2',3'-cGAMP, MSA-2, ADU-S100, MK-1454, SR-717lithium, MAVU-140, MK-1454, IMSA-101, GSK3745417, SB11285, BMS-986301, and HG381.

[0019] Furthermore, the Toll-like receptor agonist includes but is not limited to: PAM3CSK4, PAM2CSK4, arabinomannolipid, lipoteichoic acid, GPI membrane anchor, yeast polysaccharide, Poly (I: C), monophospholipid A, imiquimod, imidazoquinoline, pterin, pyrimidine, pyridine pyrimidine, pyrrolopyrimidine, benzimidazole, CpG 1826, ODN 1018, ODN 2006, ODN 2216, ODN2395, ODN 1668.

[0020] Furthermore, the immune cells include but are not limited to natural killer cells, lymphokine-activated killer cells, dendritic cells, cytokine-induced killer cells, cytotoxic T lymphocytes, T cell receptor engineered T cells, chimeric antigen receptor T cells, chimeric antigen receptor macrophages, and chimeric antigen receptor natural killer cells.

[0021] The second technical solution of the present invention provides a method for preparing a universal hydrogel vaccine based on tumor neoantigen peptides, and the steps are as follows:

[0022] S1, synthetic tumor neoantigen peptides;

[0023] S2, coupling the tumor neoantigen peptide and the peptidyl assembly sequence to prepare a tumor neoantigen peptide-peptidyl assembly sequence conjugate;

[0024] S3, dissolving the tumor neoantigen peptide-peptide assembly sequence conjugate in a solvent, and self-assembling to obtain a nanofiber solution;

[0025] S4. Adding an adjuvant to the nanofiber solution obtained in step S3 and incubating the mixture. The resulting nanofiber solution containing the adjuvant is injected into the body to form a hydrogel vaccine based on tumor neoantigen peptides.

[0026] In some specific embodiments of the present invention, the tumor neoantigen peptide is selected from the antigen peptide SIINFEKLC of B16-OVA melanoma cells, and the peptide base assembly sequence is C(V) n GRGD, n=0, 1, 2; the tumor neoantigen peptide-peptide assembly sequence conjugate SIINFEKLC-ssC (V) n GRGD is conjugated via a disulfide bond.

[0027] In some specific embodiments of the present invention, the tumor neoantigen peptide is selected from the Adpgk antigen peptide ASMTNMELMC of MC-38 colon cancer cells; the peptidyl assembly sequence is CVGRGD; and the tumor neoantigen peptide-peptidyl assembly sequence conjugate ASMTNMELMC-ss-CVGRGD is coupled via a disulfide bond.

[0028] In some specific embodiments of the present invention, the tumor neoantigen peptide is selected from the M27 antigen peptide LCPGNKYEMC of B16-F10 melanoma cells; the peptidyl assembly sequence is CVVVGRGD; and the tumor neoantigen peptide-peptidyl assembly sequence conjugate LCPGNKYEMC-ss-CVVVGRGD is coupled via a disulfide bond.

[0029] In some specific embodiments of the present invention, the adjuvant is a combination of a cytokine and a STING agonist;

[0030] In some more preferred embodiments of the present invention, the cytokine is GM-CSF, and when the amount of the tumor neoantigen peptide-peptide assembly sequence conjugate is 1 to 100 mg, the mass of the GM-CSF is 0.01 to 50 mg.

[0031] In some more preferred embodiments of the present invention, the STING agonist is ADU-S100 (CDA), and when the amount of the tumor neoantigen peptide-peptide assembly sequence conjugate is 1 to 100 mg, the mass of the CDA is 0.01 to 50 mg.

[0032] The third technical solution of the present invention provides an application of a universal hydrogel vaccine based on tumor neoantigen peptides. The universal hydrogel vaccine based on tumor neoantigen peptides is used to prepare drugs for treating / inhibiting cancer.

[0033] The drug for treating / suppressing cancer is used to activate antigen-specific immune cells to achieve personalized cancer immunotherapy; the universal hydrogel vaccine based on tumor neoantigen peptides is used to deliver antigens and adjuvants and control their long-term release.

[0034] Furthermore, the cancer includes, but is not limited to, breast cancer, melanoma, colon cancer, glioma, gastric cancer, lung cancer, pancreatic cancer, prostate cancer, bladder cancer, endocrine tumors, adenoma, blastoma, desmoid tumor, sarcoma, lymphoma, germ cell tumor, rectal cancer, hepatocellular carcinoma, leukemia, Wilms' tumor, gastric cancer, esophageal cancer, cervical cancer, and head and neck cancer.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) The universal hydrogel vaccine based on tumor neoantigen peptide prepared by the present invention is easy to prepare and has universality. It can be customized for different types of tumors. By optimizing the number of key amino acids in the peptide assembly sequence, the tumor neoantigen peptide-peptide assembly sequence conjugate is converted into a tumor neoantigen peptide hydrogel, thereby providing a theoretical basis for the development and broad-spectrum application of vaccines.

[0037] (2) The present invention provides a universal hydrogel vaccine based on tumor neoantigen peptides, which does not require any additional carrier, avoids the inherent potential toxicity of the carrier itself, and also provides the possibility of multi-component synergistic effects.

[0038] (3) The universal hydrogel vaccine based on tumor neoantigen peptides prepared by the present invention can activate antigen-specific immune responses and increase the infiltration of anti-tumor immune cells in the tumor microenvironment, thereby effectively inhibiting tumor growth, postoperative recurrence and metastasis;

[0039] (4) The present invention provides a universal hydrogel vaccine based on tumor neoantigen peptides, which has good safety and high stability. It can be used as a local delivery platform for the sustained delivery of antigens and adjuvants, thereby achieving controllable and long-term delivery of antigens and adjuvants after a single immunization, inducing the production of anti-tumor immune responses to effectively inhibit tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Transmission electron microscopy images of the self-assembly of the tumor neoantigen peptide-peptide assembly sequence conjugates (A) SIINFEKLC-ss-CVGRGD, (B) SIINFEKLC-ss-CVVGRGD, and (C) SIINFEKLC-ss-CGRGD of Example 1 of the present invention;

[0041] Figure 2 The rheological characterization diagrams of the tumor neoantigen peptide-peptide assembly sequence conjugate (A) SIINFEKLC-ss-CVGRGD and (B) SIINFEKLC-ss-CVVGRGD self-assembly according to Example 1 of the present invention;

[0042] Figure 3 This is a characterization diagram of the "solution-gel" transition of the tumor neoantigen peptide-peptide assembly sequence conjugate SIINFEKLC-ss-CVVGRGD self-assembly of Example 2 of the present invention;

[0043] Figure 4 This is a Zeta potential diagram of the tumor neoantigen peptide-peptide assembly sequence conjugate SIINFEKLC-ss-CVVGRGD self-assembly and the adjuvant loaded thereafter according to Example 2 of the present invention;

[0044] Figure 5 This is a photo of the tumor neoantigen peptide hydrogel formed in vivo in Example 3 of the present invention;

[0045] Figure 6 This is a graph showing the release curves of CDA and GM-CSF in the hydrogel vaccine of the tumor neoantigen peptide of Example 4 of the present invention;

[0046] Figure 7The hydrogel vaccine of the tumor neoantigen peptide of Example 5 of the present invention promotes CD8 + Characterization of T cell tumor infiltration;

[0047] Figure 8 This is a diagram showing the effect of the hydrogel vaccine of the tumor neoantigen peptide of Example 5 of the present invention on inhibiting the growth of B16-OVA melanoma cells;

[0048] Fig. 9 This is a diagram showing the effect of the hydrogel vaccine of the tumor neoantigen peptide of Example 6 of the present invention on inhibiting the postoperative recurrence of MC-38 colon cancer cells;

[0049] Fig.10 This is a diagram showing the effect of the hydrogel vaccine of the tumor neoantigen peptide of Example 7 of the present invention on inhibiting B16-F10 melanoma lung metastasis. DETAILED DESCRIPTION

[0050] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, several variations and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0051] In the following examples, the required materials are as follows: amino acids were purchased from AAPPTEC, CDA and GM-CSF were purchased from InvivoGen. Other materials not specified can be purchased by those skilled in the art through commercial channels.

[0052] In the following examples, the concentration of PBS buffer is 0.01M or 0.1M, and the pH is 7.2-7.4; other raw materials or processing techniques, unless otherwise specified, are conventional commercially available raw materials or conventional processing techniques in the art.

[0053] Example 1: Synthesis of Tumor Neoantigen Peptide-Peptide Assembly Sequence Conjugates

[0054] The tumor neoantigen peptide SIINFEKLC used in this example has a peptide assembly sequence of C(V) n GRGD, n is 0, 1, 2, the specific experimental steps are as follows:

[0055] S1. The tumor neoantigen peptide SIINFEKLC was added dropwise to the 2,2'-disulfide dipyridine solution at a molar ratio of 1:2. After stirring for overnight reaction, the mixture was washed with ether several times until the supernatant was clear and free of yellow. SIINFEKLC-ss-Pyr was obtained after drying.

[0056] S2. Dissolve 117.35 mg of the above SIINFEKLC-s-s-Pyr and 171.11 mg of the peptide-based assembly sequence C(V) n GRGD in DMSO, react under N2 condition for 48 hours, then purify the sample by high performance liquid chromatography, and obtain the tumor neoantigen peptide-peptide-based assembly sequence conjugate SIINFEKLC-s-s-C(V) n GRGD after freeze-drying.

[0057] Dissolve the tumor neoantigen peptide-peptide-based assembly sequence conjugate SIINFEKLC-s-s-C(V) n GRGD prepared in step S2 of this example in deionized water at a concentration of 5 mM, let it stand at room temperature and self-assemble to obtain a nanofiber solution. This nanofiber solution can load adjuvants CDA and GM-CSF through electrostatic interaction to obtain a nanofiber solution containing vaccine adjuvants. After adding 0.1 M PBS (used to simulate the in vivo physiological environment) to this nanofiber solution, a hydrogel vaccine of tumor neoantigen peptide can be formed.

[0058] Figure 1 This is the self-assembly structure of the tumor neoantigen peptide-peptide-based assembly sequence conjugate prepared in this example. Transmission electron microscopy shows that: SIINFEKLC- S-S -CVGRGD( Figure 1 A, n = 1) and SIINFEKLC- S-S -CVVGRGD( Figure 1 B, n = 2) form nanofiber structures in aqueous solution and have good assembly performance; however, when n = 0, SIINFEKLC- S-S -CGRGD( Figure 1 C) has poor assembly performance in aqueous solution, resulting in the fracture of the formed fiber structure and lack of cross-linking.

[0059] Figure 2 This is the rheological property of the self-assembly of the tumor neoantigen peptide-peptide-based assembly sequence conjugate prepared in this example. In the figure, G′ represents the storage modulus of the hydrogel, which is an important index to measure its mechanical properties. A hydrogel with a high storage modulus means that it can maintain good shape stability under force and is not prone to plastic deformation. This property makes hydrogels widely used in the biomedical field. In the figure, G″ represents the loss modulus of the hydrogel, which is also an important index to measure its mechanical properties. With a high loss modulus, it can consume less energy under force and maintain good resilience and deformation ability. Generally, when G′ < G″, the material is in a liquid state, and when G′ > G″, the material is in a solid state.

[0060] The results show that SIINFEKLC- S-S-CVGRGD( Figure 2 A,n=1) and SIINFEKLC- S-S -CVVGRGD( Figure 2 B, n = 2) the storage modulus (G') of the self-assembly increased, and a crossover point (G'>G") appeared, indicating that the self-assembly of the two tumor neoantigen peptide-peptide assembly sequence conjugates quickly transformed from a liquid state to a solid state after adding 0.1M PBS. Moreover, when n = 2, SIINFEKLC- S-S The storage modulus (G') of the -CVVGRGD self-assembly increased rapidly, and a hydrogel was formed within a few seconds. However, when n = 1, it took about 1.5 minutes for the intersection to appear after adding 0.1M PBS, and the speed of hydrogel formation was slightly slower. S-S -CVVGRGD (ie n=2) is taken as an example for study.

[0061] Example 2: Preparation of hydrogel vaccine of tumor neoantigen peptide

[0062] S1. The tumor neoantigen peptide-peptide assembly sequence conjugate SIINFEKLC-ss-CVVGRGD prepared in Example 1 was dissolved in deionized water at a concentration of 5 mM, and self-assembled to obtain a nanofiber solution after standing at room temperature. After subcutaneous injection, the tumor neoantigen peptide hydrogel was formed in vivo.

[0063] S2. Then add adjuvants CDA and GM-CSF (CDA 0.02 mg, GM-CSF 0.002 mg) to 0.1 mL of the above nanofiber solution. After vortex stirring, incubate the mixed solution at 37°C for 30 minutes. The resulting nanofiber solution containing the adjuvant forms a hydrogel vaccine of tumor neoantigen peptides in vivo after subcutaneous injection.

[0064] The adjuvant-containing nanofiber solution prepared in this example has the rheological properties of the "solution-gel" transition as described in Example 1, and a hydrogel vaccine of tumor neoantigen peptides can be obtained by adding 0.1 M PBS.

[0065] In this example, 0.1 M PBS was used to simulate the physiological environment in vivo, and the inversion method was used to test the "solution-gel" transition characteristics. Figure 3 The "solution-gel" transition of the self-assembly provided in this embodiment was determined by an inversion method to have an instant "solution-gel" transition characteristic.

[0066] Zetasizer (Malvern Zetasizer Nano ZS) was used to measure the change in zeta potential after CDA and GM-CSF were added to the nanofiber solution. Figure 4 This is the Zeta potential diagram of Example 2 of the present invention. After loading the adjuvant, the decrease in Zeta potential indicates that the nanofiber solution adsorbs the adjuvant through electrostatic action, that is, a nanofiber solution containing the vaccine adjuvant is obtained.

[0067] Example 3: Formation of tumor neoantigen peptide hydrogel in vivo

[0068] The nanofiber solution prepared in Example 1 was subcutaneously inoculated into mice. After 15 minutes, the mice were dissected to observe the formation of tumor neoantigen peptide hydrogel in vivo and take pictures.

[0069] from Figure 5 It can be seen that the self-assembly has a "solution-gel" transition property in the body. It is injected in a solution state and triggers the formation of tumor neoantigen peptide hydrogel under physiological conditions, which then slowly releases and exerts its effect.

[0070] Example 4: Hydrogel vaccine long-acting release adjuvant for tumor neoantigen peptides

[0071] This example uses the hydrogel vaccine of tumor neoantigen peptide prepared in Example 2 to study the performance of in vitro release adjuvant. The in vitro release study was carried out at 37°C in a 0.01M PBS release medium supplemented with 10% FBS. The cumulative release of CDA was analyzed using a high performance liquid chromatograph. The cumulative release of GM-CSF was detected by an enzyme-linked immunosorbent assay kit. The above-mentioned high performance liquid chromatography detection method and enzyme-linked immunosorbent assay kit detection method are both routine operations in the field, and the kit detection is carried out according to the instructions in the kit; the conditions for the high performance liquid chromatography analysis are: wavelength A = 220nm, acetonitrile and water are selected as solvents, and the elution time is 20min.

[0072] Figure 6 is the in vitro release curve of CDA and GM-CSF, and the calculation formula in the figure is:

[0073]

[0074] Among them, C n is the concentration at the nth sampling point, V0 is the volume of the release medium, V is the sampling volume each time, and m is the total mass of the drug.

[0075] The results showed that the hydrogel vaccine of the tumor neoantigen peptide can slowly release CDA and GM-CSF. Within 20 days of the experiment, the release of CDA and GM-CSF was about 99% and 81.5%, respectively. Therefore, the hydrogel vaccine of the tumor neoantigen peptide can be used as a drug reservoir to effectively control the slow and sustained release of adjuvants.

[0076] Example 5: Hydrogel vaccine of tumor neoantigen peptide promotes antigen-specific CD8 + T cells infiltrate tumors and effectively inhibit the growth of tumor cells

[0077] In this example, B16-OVA melanoma cells and C57BL / 6 mice were used to establish a subcutaneous melanoma model: 5×10 5 B16-OVA cells were subcutaneously injected into the right axilla of female C57BL / 6 mice to establish a subcutaneous melanoma model.

[0078] The hydrogel vaccine of tumor neoantigen peptide was prepared according to the method described in Example 2 and set aside.

[0079] On the 8th day after the model was established, the mice were randomly divided into 6 groups (6 mice in each of the normal saline group, blank hydrogel group, and aPD1 group; 8 mice in each of the free vaccine group, hydrogel vaccine group, and hydrogel vaccine + aPD1 group), and the corresponding therapeutic drugs were subcutaneously injected at the contralateral side of the tumor (see Table 1 for details, 100 μg SIINFEKL, 20 μg CDA, 2 μg GM-CSF, and 100 μg aPD1 each time per mouse).

[0080] Table 1 Injections of different groups of mouse models in Example 5

[0081] Group Therapeutic drugs injected into each mouse Saline group Normal saline Blank gel group Tumor neoantigen peptide hydrogel aPD1 group (aPD1) aPD1 Bolus vaccine A mixture of SIINFEKL, CDA, and GM-CSF Gel vaccine group Hydrogel vaccine of neoantigen peptides for tumor Gel vaccine+aPD1 group Tumor neoantigen peptide hydrogel vaccine, aPD1

[0082] (1) Hydrogel vaccines of tumor neoantigen peptides promote antigen-specific CD8 + T cell infiltration in tumors

[0083] Anti-PD1 antibody (i.e., aPD1, only the (aPD1) group and the gel vaccine + aPD1 group (Gel vaccine + aPD1) need to be injected intraperitoneally on days 9, 12, 15, and 18. The role of the injected antibody is to block the PD-1 / PD-L1 pathway and enhance the killing ability of T cells. Tumor size and body weight were monitored every two days. After 7 days of treatment, the mouse tumor tissue was taken, digested into single cells, T cells were labeled with antibodies, and antigen-specific CD8 + T cell infiltration in tumor tissue.

[0084] Figure 7 Antigen-specific CD8+ Infiltration effect of T cells in tumors, %SIINFEKL in the figure + of CD8 + T cells can be directly analyzed from the FLOWJO software used after detection by a flow cytometer. *** indicates 0.0001 < p < 0.001. The infiltration effect of the hydrogel vaccine group was significantly higher than that of the free vaccine group, indicating that the same components had better effects in the hydrogel state, and this effect was more obvious after adding aPD1. The above results show that the hydrogel vaccine of tumor neoantigen peptides can effectively promote the infiltration of antigen-specific CD8 + T cells in the tumor microenvironment.

[0085] (2) The hydrogel vaccine of tumor neoantigen peptides effectively inhibits the growth of B16-OVA melanoma cells

[0086] After receiving drug injection treatment, the tumor volume of the mice was recorded, and the tumor volume curve was plotted to evaluate the in vivo therapeutic effect of the hydrogel vaccine of this tumor neoantigen peptide.

[0087] Figure 8 For the effect of the hydrogel vaccine of tumor neoantigen peptides in treating tumor-bearing mice, in the figure, ** indicates 0.001 < p < 0.01, indicating that the hydrogel vaccine of this tumor neoantigen peptide can effectively inhibit the growth of B16-OVA, which confirms the effective therapeutic ability of the hydrogel vaccine of this tumor neoantigen peptide against B16-OVA melanoma cells.

[0088] Example 6 The hydrogel vaccine of tumor neoantigen peptides effectively inhibits the postoperative recurrence of MC-38 colon cancer

[0089] In this example, the tumor neoantigen peptide-peptide assembly sequence conjugate ASMTNMELMC-s-s-CVGRGD was prepared by selecting the Adpgk antigen peptide ASMTNMELMC and the peptide assembly sequence CVGRGD; then the tumor neoantigen peptide-peptide assembly sequence conjugate was dissolved in deionized water, and after standing at room temperature, it self-assembled to obtain a nanofiber solution. Then, the adjuvants CDA and GM-CSF were added to the above solution. After vortex stirring, the mixed solution was incubated at 37°C for 30 minutes to obtain a nanofiber solution containing vaccine adjuvants, which was subcutaneously injected into the body to obtain the hydrogel vaccine of tumor neoantigen peptides.

[0090] An MC-38 colon cancer postoperative mouse model was established using MC-38 colon cancer cells and C57BL / 6 mice: On the first day, 5×10 5MC-38 cells. On the 10th day after the model was established, all visible tumor tissues were surgically removed, and the wounds were sutured with tissue glue and wound clips. The mice were randomly divided into 6 groups (normal saline, aPD1 and Bolus vaccine groups, 5 mice in each group; hydrogel vaccine group, 7 mice in each group; hydrogel vaccine + aPD1 group, 8 mice in each group), and the corresponding therapeutic drugs were subcutaneously injected into the contralateral part (see Table 2 for details, 100μg ASMTNMELM, 20μg CDA, 2μg GM-CSF per mouse, 100μg aPD1 each time), the tumor volume of the mice was recorded, and the tumor volume curve was drawn to evaluate the in vivo therapeutic effect of the hydrogel vaccine.

[0091] Table 2 Injections of different groups of mouse models in Example 6

[0092]

[0093] Fig. 9 The results show that the hydrogel vaccine of tumor neoantigen peptide can effectively inhibit the postoperative recurrence of MC-38 colon cancer, which confirms the effective therapeutic ability of the hydrogel vaccine of tumor neoantigen peptide on the postoperative recurrence of MC-38 colon cancer.

[0094] Example 7 Hydrogel vaccine of tumor neoantigen peptide effectively inhibits lung metastasis of B16-F10 melanoma

[0095] In this example, the M27 antigen peptide LCPGNKYEM and the peptide assembly sequence CVVVGRGD were selected to prepare the tumor neoantigen peptide-peptide assembly sequence conjugate LCPGNKYEMC-ss-CVVVGRGD; the tumor neoantigen peptide-peptide assembly sequence conjugate LCPGNKYEMC-ss-CVVVGRGD was then dissolved in deionized water, and the nanofiber solution was self-assembled after standing at room temperature, and then the adjuvants CDA and GM-CSF were added to the above solution. After vortex stirring, the mixed solution was incubated at 37° C. for 30 minutes to obtain a nanofiber solution containing a vaccine adjuvant, which was subcutaneously injected into the body to obtain a hydrogel vaccine of the tumor neoantigen peptide.

[0096] Melanoma mouse model was established using B16-F10 cells and C57BL / 6 mice: On day 0, female C57BL / 6 mice were injected with 2.5×10 5B16-F10 cells. On day 1, tumor-bearing mice were randomly assigned to 5 groups (n = 5 mice in each group) and injected subcutaneously on the right side of the mice with saline, aPD1, Bolus vaccine, hydrogel vaccine or hydrogel vaccine + aPD1 (see Table 3 for details, 100 μg LCPGNKYEM, 20 μg CDA, 2 μg GM-CSF per mouse, 100 μg anti-PD1 each time). Anti-PD1 (i.e., aPD1) was injected intraperitoneally on days 2, 5, 8, and 11. On day 30, the lungs of euthanized mice were resected and the number of lung metastases was counted.

[0097] Table 3 Injections of different groups of mouse models in Example 7

[0098]

[0099]

[0100] Fig.10 The results show that the hydrogel vaccine of tumor neoantigen peptide can effectively inhibit the occurrence of B16-F10 melanoma lung metastasis, which confirms the effective therapeutic ability of the hydrogel vaccine of tumor neoantigen peptide on B16-F10 melanoma lung metastasis in mice.

[0101] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A universal hydrogel vaccine based on tumor neoantigen peptides, characterized in that: It comprises a tumor neoantigen peptide hydrogel and an adjuvant loaded on the tumor neoantigen peptide hydrogel; the tumor neoantigen peptide hydrogel is self-assembled by a tumor neoantigen peptide-peptide assembly sequence conjugate in a solvent to form a nanofiber solution, which is converted from a solution state to a hydrogel state under physiological conditions; The tumor neoantigen peptide-peptide assembly sequence conjugate is obtained by covalently coupling a tumor neoantigen peptide and a peptide assembly sequence; the tumor neoantigen peptide serves as a tumor antigen; and the peptide assembly sequence contains valine V or phenylalanine F with assembly and folding properties.

2. A universal hydrogel vaccine based on tumor neoantigen peptides according to claim 1, characterized in that: The sources of the tumor neoantigen peptides include, but are not limited to, mutant peptides obtained from genetic changes in the genome of any one or more tumors of breast cancer, melanoma, colon cancer, glioma, gastric cancer, lung cancer, pancreatic cancer, prostate cancer, bladder cancer, endocrine tumors, adenomas, blastomas, desmoid tumors, sarcomas, lymphomas, germ cell tumors, rectal cancer, hepatocellular carcinoma, leukemia, Wilms tumor, gastric cancer, esophageal cancer, cervical cancer, and head and neck cancer; The tumor neoantigen peptide sequences include but are not limited to: TYVPANASL, SIINFEKLC, ASMTNMELMC, LCPGNKYEM, CYTWNQMNL, SYGVLLWEIF, SVYDFFVWL, LMLGEFLKL, SGLEQLESIINFEKL, RFVPDGNRI, GWVKPIIIGH, RVLELFRAAQLANDDVVLQIMELC, GIPVHLELASMTNMELMSSIVHQQVF, REGVELCPGNKYEMRRHGTTHSLVIHD, ALLEIASCL, KVPRNQDWL, MTYENMDIL, VDWENVSPELNSTDQ, PSKPSFQEFVDWENVSPELNSTDQPFL, KPIIIGHHAYGDQYRA, FMTYWHLLNAFTVTVPKDL.

3. A universal hydrogel vaccine based on tumor neoantigen peptides according to claim 1, characterized in that: The peptidyl assembly sequence is any of the following: C(V) n GRGD, C(V) n GRGDR, C(F) n GRGD, C(F) n GRGDR, the gel properties are regulated by adjusting the number of valine V or phenylalanine F, wherein n=any integer from 0 to 5, and can be 0.

4. A universal hydrogel vaccine based on tumor neoantigen peptides according to claim 1, characterized in that: The adjuvant is selected from one or a combination of cytokines, STING agonists, Toll-like receptor agonists, and immune cells.

5. A universal hydrogel vaccine based on tumor neoantigen peptides according to claim 4, characterized in that: The cytokines include but are not limited to: IL-2, IL-12, IL-6, IL-15, IL-21, IL-22, IFN-α, IFN-β, IFN-γ, TNF-β, GM-CSF, CXCL-9, CXCL-10, CXCL-11, CCL27, CCL28; The STING agonists include but are not limited to: c-di-GMP, c-di-AMP, 3',3'-cGAMP, 2',3'-cGAMP, MSA-2, ADU-S100, MK-1454, SR-717lithium, MAVU-140, MK-1454, IMSA-101, GSK3745417, SB11285, BMS-986301, and HG381.

6. A universal hydrogel vaccine based on tumor neoantigen peptides according to claim 4, characterized in that: The Toll-like receptor agonists include but are not limited to: PAM3CSK4, PAM2CSK4, arabinomannolipid, lipoteichoic acid, GPI membrane anchor, yeast polysaccharide, Poly (I: C), monophospholipid A, imiquimod, imidazoquinoline, pterin, pyrimidine, pyridine pyrimidine, pyrrolopyrimidine, benzimidazole, CpG 1826, ODN 1018, ODN 2006, ODN 2216, ODN 2395, ODN 1668; The immune cells include, but are not limited to, natural killer cells, lymphokine-activated killer cells, dendritic cells, cytokine-induced killer cells, cytotoxic T lymphocytes, T cell receptor engineered T cells, chimeric antigen receptor T cells, chimeric antigen receptor macrophages, and chimeric antigen receptor natural killer cells.

7. A method for preparing a universal hydrogel vaccine based on tumor neoantigen peptides according to any one of claims 1 to 6, characterized in that: The specific steps are as follows: S1, synthetic tumor neoantigen peptides; S2, coupling the tumor neoantigen peptide and the peptidyl assembly sequence to prepare a tumor neoantigen peptide-peptidyl assembly sequence conjugate; S3, dissolving the tumor neoantigen peptide-peptide assembly sequence conjugate in a solvent, and self-assembling to obtain a nanofiber solution; S4. Adding an adjuvant to the nanofiber solution obtained in step S3 and incubating the mixture. The resulting nanofiber solution containing the adjuvant is injected into the body to form a universal hydrogel vaccine based on neoantigen peptides of tumors.

8. The use of a universal hydrogel vaccine based on tumor neoantigen peptides according to any one of claims 1 to 6, characterized in that: The universal hydrogel vaccine based on tumor neoantigen peptides is used to prepare drugs for treating / inhibiting cancer.

9. The use of a universal hydrogel vaccine based on tumor neoantigen peptides according to claim 8, characterized in that: The drug for treating / suppressing cancer is used to activate antigen-specific immune cells to achieve personalized cancer immunotherapy.

10. The use of a universal hydrogel vaccine based on tumor neoantigen peptides according to claim 8, characterized in that: The cancers include, but are not limited to, breast cancer, melanoma, colon cancer, glioma, gastric cancer, lung cancer, pancreatic cancer, prostate cancer, bladder cancer, endocrine tumors, adenoma, blastoma, desmoid tumor, sarcoma, lymphoma, germ cell tumor, rectal cancer, hepatocellular carcinoma, leukemia, Wilms tumor, gastric cancer, esophageal cancer, cervical cancer, and head and neck cancer.

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