Application of nano polypeptide vaccine for preventing colorectal cancer in treatment of colorectal cancer

Abstract

The invention relates to application of a nano-polypeptide vaccine for preventing colorectal cancer in treatment of colorectal cancer, in particular to application of the nano-polypeptide vaccine for preventing colorectal cancer in preparation of related products for treatment of colorectal cancer, the vaccine comprises an antigen, and the antigen contains a first polypeptide fragment and a second polypeptide fragment; the amino acid sequence of the first polypeptide fragment is VVGAVGVGK; and the amino acid sequence of the second polypeptide fragment is TPQDSRQVL. The new antigen polypeptide is selected as a tumor antigen, and the peptide vaccine has the advantages that the peptide vaccine can target multiple tumor-related antigens, the sequence is clear, the good modification potential is achieved, operability is high, preparation and quality control are easy, and the economic cost is relatively low. In addition, the nano polypeptide vaccine can be combined with a PD-1 inhibitor to treat colorectal cancer, so that a better treatment effect is achieved.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedicine technology, and in particular to the use of a nanopeptide vaccine for preventing colorectal cancer in the preparation of related products for treating colorectal cancer. Background Art

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors and has become one of the major diseases that seriously threaten human life and safety. Its incidence rate is increasing year by year. Traditional surgery, radiotherapy, chemotherapy and targeted therapy have various limitations. Surgical treatment may cause tumor metastasis. Radiotherapy and chemotherapy are prone to cause tolerance and recurrence of tumor cells, leading to poor prognosis. Targeted therapy has a higher response rate and survival rate than other therapies, but problems such as unsatisfactory drug development and high cost still exist. With the deepening of the understanding of the mechanism of tumor occurrence and development, therapeutic tumor vaccines, as a feasible option for immunotherapy, aim to induce the patient's immune system to produce a sustained tumor immune response to achieve the effect of treating and preventing tumor recurrence or metastasis. It can fill the gaps that are not currently available in conventional therapies or other immunotherapies. It has the characteristics of low toxicity, strong anti-tumor specificity, and the ability to induce immune memory in the body. It has a strong advantage in preventing tumor recurrence and metastasis.

[0003] Immunotherapy is one of the most successful therapies in the current field of cancer treatment. Among them, immune checkpoint inhibitor therapy (such as CTLA-4 monoclonal antibody, PD-1 / PD-L1 monoclonal antibody, etc.) has shined in the fields of melanoma, lung cancer, kidney cancer, squamous cell carcinoma and liver cancer. Many studies have shown that if cancer vaccines are combined with immune checkpoint inhibitors, the effect of "1+1>2" can be achieved. According to investigations and analyses, studies on "cancer vaccines" combined with immune checkpoint inhibitors show that this combination therapy can control tumors for a longer time than using immune checkpoint inhibitors alone. Therefore, the development of personalized therapeutic tumor vaccines combined with immune checkpoint inhibitors is very important and has practical significance, and has become a research hotspot and future direction in this field. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present disclosure provides an application of a nanopeptide vaccine for preventing colorectal cancer in the preparation of related products for treating colorectal cancer.

[0005] According to a first aspect of the present disclosure, there is provided a use of a nanopeptide vaccine for preventing colorectal cancer in the preparation of related products for treating colorectal cancer, wherein the vaccine comprises an antigen, wherein the antigen comprises a first polypeptide fragment and a second polypeptide fragment;

[0006] The amino acid sequence of the first polypeptide fragment is: VVGAVGVGK;

[0007] The amino acid sequence of the second polypeptide fragment is: TPQDSRQVL.

[0008] In one embodiment of the present disclosure, the antigen further includes a third polypeptide fragment, and the amino acid sequence of the third polypeptide fragment is: VVVGADGVGK.

[0009] In one embodiment of the present disclosure, the related products also include PD-1 inhibitors.

[0010] In one embodiment of the present disclosure, the administration mass ratio of the PD-1 inhibitor to the vaccine is 2:0.8-1.2.

[0011] In one embodiment of the present disclosure, the vaccine further comprises an adjuvant, and the adjuvant is Lycium barbarum polysaccharide.

[0012] In one embodiment of the present disclosure, the vaccine further comprises a carrier, and the carrier is a liposome.

[0013] In one embodiment of the present disclosure, the related product is used to enhance the anti-tumor immune response of colorectal cancer and inhibit the proliferation of colorectal cancer cells.

[0014] In one embodiment of the present disclosure, the related product is used to promote CD4 + T and CD8 + T cell proliferation.

[0015] In one embodiment of the present disclosure, the related product is used to promote CD8 + T cells secrete cytokines including at least TNF-α and IFN-γ.

[0016] In one embodiment of the present disclosure, the related product is an injectable preparation, an oral tablet or an oral capsule.

[0017] One beneficial effect of the present disclosure is that the present disclosure provides an application of a nanopeptide vaccine for preventing colorectal cancer in the preparation of related products for the treatment of colorectal cancer, which has broad prospects in the treatment of colorectal cancer. Specifically, personalized tumor vaccines prepared based on polypeptides can promote the regression of colorectal cancer, and these neoantigen peptides are not produced and expressed in normal cells, so they can more effectively stimulate the body's immune response. The present disclosure uses neoantigen polypeptides as tumor antigens, which has the advantage that peptide vaccines can target a variety of tumor-related antigens, have clear sequences, have good modification potential, are highly operable, are easy to prepare and quality control, and have relatively low economic costs.

[0018] In addition, nanopeptide vaccines can be combined with PD-1 inhibitors to treat colorectal cancer. As a "spot-type" anticancer drug, PD-1 inhibitors can be combined with "individualized" vaccines targeting new antigen peptides to control tumors for a longer period of time and achieve better therapeutic effects. Nanopeptide vaccines have good safety and have obvious therapeutic effects on the colorectum. They can significantly prolong the survival period of mice, inhibit tumor growth, enhance the anti-tumor immune response of colorectal cancer, and inhibit tumor cell proliferation.

[0019] Other features and advantages of the present disclosure will become apparent from the following detailed description of exemplary embodiments of the present disclosure with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

[0021] Figure 1 This is a diagram showing the results of the polypeptide immunogenicity identification in Example 1 of the present disclosure;

[0022] Figure 2 is a flow chart of the preparation of NPs nanoparticles in Example 2 of the present disclosure;

[0023] Figure 3 is a flow chart for preparing cationized LBP in Example 2 of the present disclosure;

[0024] Figure 4 is a schematic diagram of the preparation process of LBP-NPs nanovaccine in Example 2 of the present disclosure;

[0025] Figure 5 This is a schematic diagram of the mouse modeling process in Example 3 of the present disclosure;

[0026] Figure 6 This is a graph showing the results of a mouse experiment in Example 3 of the present disclosure;

[0027] Figure 7 This is a diagram of the spleen experiment results in Example 4 of the present disclosure;

[0028] Figure 8 is a graph showing the results of a mesenteric lymph node experiment in Example 4 of the present disclosure;

[0029] Fig. 9 This is a graph showing the results of a tumor experiment in Example 4 of the present disclosure;

[0030] Fig.10 This is a graph showing the results of the immunosuppressive cell experiment in Example 5 of the present disclosure;

[0031] Fig.11 This is a graph showing the results of an immunohistochemistry experiment in Example 6 of the present disclosure;

[0032] Fig.12 This is a diagram showing the results of a HE staining experiment in Example 7 of the present disclosure;

[0033] Fig.13 This is a schematic diagram of the mouse modeling process in Example 8 of the present disclosure;

[0034] Fig.14 This is a graph showing the results of a mouse experiment in Example 8 of the present disclosure;

[0035] Fig.15 This is a result diagram of the spleen experiment in Example 9 of the present disclosure;

[0036] Fig.16 is a graph showing the results of a mesenteric lymph node experiment in Example 9 of the present disclosure;

[0037] Fig.17 This is a graph showing the results of a tumor experiment in Example 9 of the present disclosure;

[0038] Fig.18 This is a graph showing the experimental results of M1 macrophages in Example 10 of the present disclosure;

[0039] Fig.19 This is a graph showing the results of the immunosuppressive cell experiment in Example 11 of the present disclosure. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present disclosure will now be described in detail with reference to the accompanying drawings. It should be noted that the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present disclosure unless otherwise specifically stated.

[0041] The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the present disclosure, its application, or uses.

[0042] Technologies, methods, and equipment known to ordinary technicians in the relevant art may not be discussed in detail, but where appropriate, the technologies, methods, and equipment should be considered as part of the specification.

[0043] It should be noted that like reference numerals and letters refer to similar items in the following figures, and therefore, once an item is defined in one figure, it need not be further discussed in subsequent figures.

[0044] In this document, “upper”, “lower”, “front”, “back”, “left”, “right”, etc. are only used to indicate the relative position relationship between related parts, rather than to limit the absolute positions of these related parts.

[0045] In this article, "first", "second", etc. are only used to distinguish each other, and do not indicate the importance and order, or the premise of each other's existence.

[0046] In this document, “equal”, “same”, etc. are not strictly limited in a mathematical and / or geometric sense, but also include errors that can be understood by those skilled in the art and are allowed in manufacturing or use.

[0047] The present disclosure provides an application of a nanopeptide vaccine for preventing colorectal cancer in the preparation of related products for treating colorectal cancer, wherein the vaccine comprises an antigen, wherein the antigen comprises a first polypeptide fragment and a second polypeptide fragment, wherein the amino acid sequence of the first polypeptide fragment is: VVGAVGVGK; and the amino acid sequence of the second polypeptide fragment is: TPQDSRQVL.

[0048] The first polypeptide fragment and the second polypeptide fragment are both new antigenic peptides associated with high-frequency mutations in colorectal cancer, among which the first polypeptide fragment is G12D (VVVGADGVGK) and the second polypeptide fragment is E545K (TPQDSRQVL). The vaccine contains G12D and E545K added in equal proportions. Both G12D and E545K can trigger T cell-mediated cytotoxic anti-tumor immune responses, so that the vaccine can promote the regression of colorectal cancer and play a role in treating colorectal cancer. In addition, G12D and E545K are not produced and expressed in normal cells, so they can more effectively stimulate the body's immune response.

[0049] The present disclosure has found through experimental research that the combination of G12D+E545K has strong immunogenicity, and the immunogenicity produced by the antigen combination is stronger than the immunogenicity produced by G12D and E545K acting alone. Based on this, the present disclosure uses G12D and E545K as antigens for a polypeptide vaccine, and verifies the effect of this vaccine in treating colorectal cancer through experiments.

[0050] In one embodiment of the present disclosure, the antigen also includes a third polypeptide fragment, and the amino acid sequence of the third polypeptide fragment is: VVVGADGVGK. The third polypeptide fragment is a new antigen peptide G12V (VVGAVGVGK) associated with high-frequency mutations in colorectal cancer. G12V can also trigger T cell-mediated cytotoxic anti-tumor immune responses so that the vaccine can promote the regression of colorectal cancer and play a role in treating colorectal cancer. And G12V will not be produced and expressed in normal cells, so it can more effectively stimulate the body's immune response.

[0051] The present disclosure has found through experimental research that the combination of G12D+G12V+E545K has strong immunogenicity, and the immunogenicity produced by the antigen combination is stronger than the immunogenicity produced by G12D, G12V and E545K acting alone. Based on this, the present disclosure uses G12D, G12V and E545K as antigens of a polypeptide vaccine, and verifies the effect of this vaccine in treating colorectal cancer through experiments.

[0052] In one embodiment of the present disclosure, the vaccine also includes an adjuvant. An adjuvant is a non-specific substance that is injected into the body together with an antigen or in advance, and can change the type of immune response and play an auxiliary role. As an immunopotentiator, adjuvants can not only non-specifically enhance the body's specific immune response to antigens, improve the body's inherent and adaptive immune response to antigens, but also save antigen dosage or reduce the number of vaccinations, thereby reducing vaccine costs. To some extent, adjuvants and vaccines are almost equally important. An ideal vaccine adjuvant should be easy to produce and use, have no toxic side effects, produce a strong immune enhancement effect in a small dose, and be metabolized by the body, and have a therapeutic effect.

[0053] The vaccine provided by the present disclosure has a small molecular weight and high purity, but its immunogenicity is relatively weak, so an adjuvant is needed to enhance its effect. As an immunopotentiator, adjuvant can not only non-specifically enhance the body's specific immune response to antigens, improve the body's inherent and adaptive immune response levels to antigens, but also save antigen dosage or reduce the number of vaccinations, thereby reducing vaccine costs.

[0054] In a specific embodiment of the present disclosure, the adjuvant is Lycium barbarum polysaccharide. Lycium barbarum polysaccharide (LBP), as the main active ingredient of Lycium barbarum, has multiple biological functions to improve human health and plays a vital role in the treatment of diseases. Previous studies have found that Lycium barbarum polysaccharide (LBPs) activates the immune system through the TLR4-NF-κB / MAPK-Blimp1 pathway and the TLR2-NF-κB / p38 pathway, promotes the maturation of lymphocytes and DCs, and thus exerts an anti-tumor effect.

[0055] Specifically, Lycium barbarum polysaccharide (LBP), as a new type of polysaccharide immune adjuvant, can promote the maturation of dendritic cells by activating the synergistic effect of the TLR4 / MGL pathway, thereby enhancing the activation of CD8+T cells and achieving a stronger anti-tumor effect. In order to further improve the targeting of nanopeptide vaccines to dendritic cells, the present invention uses LBP to modify the nanopeptide vaccine. The experimental results show that the vaccine modified with LBP can effectively enhance the killing of CD8+T cells against tumor cells.

[0056] The polypeptide vaccine provided in the present disclosure is a biological macromolecule, which can show good efficacy in the field of tumor treatment. How to maintain the activity of the vaccine and how to deliver it to tumor tissue to exert its efficacy are technical problems that need to be solved at present. To solve this problem, in one embodiment of the present disclosure, the vaccine also includes a carrier.

[0057] Specifically, the carrier is a liposome, and the present disclosure selects liposomes as carriers to deliver tumor-specific peptides. Liposomes are a nanocarrier system, a bilayer vesicle formed by self-assembly of a phospholipid bilayer in an aqueous medium, with high biocompatibility, diversification and high load capacity, and can encapsulate drugs in the bilayer. In recent years, in the development strategy of targeted drug delivery, liposomes, as an important way of drug delivery based on synthetic lipid membranes, have been widely welcomed in clinical applications.

[0058] In addition, liposomes are composed of components in naturally occurring biological membranes, and as drug carriers they are non-toxic and biodegradable. Based on their properties, including composition, size, and surface properties, liposome nanocarriers also have the ability to overcome biological barriers, such as skin and mucous membranes, and have the ability to control and release antigens, and can improve drug stability and targeting, reduce drug toxicity, and encapsulate drugs with liposomes to reduce renal excretion, prolong blood half-life, and ensure the high concentration of drugs required in plasma. The present disclosure uses liposomes to encapsulate colorectal cancer-specific neoantigen peptides to form drug-loaded liposomes.

[0059] The present disclosure provides a new vaccine that encapsulates colorectal cancer-specific antigen peptides with a liposome shell loaded with wolfberry polysaccharide, which provides a new idea for the treatment of colorectal cancer and has broad prospects. Specifically, personalized tumor vaccines prepared based on polypeptides can promote the regression of colorectal cancer, and these new antigen peptides will not be produced and expressed in normal cells, so they can more effectively stimulate the body's immune response. The present disclosure uses new antigen polypeptides as tumor antigens, which has the advantage that peptide vaccines can target a variety of tumor-related antigens, have clear sequences, have good modification potential, are highly operable, are easy to prepare and quality control, and have relatively low economic costs. Nanopeptide vaccines have good safety, have obvious therapeutic effects on the colorectum, can significantly prolong the survival period of mice, inhibit tumor growth, enhance the anti-tumor immune response of colorectal cancer, and inhibit tumor cell proliferation.

[0060] In one embodiment of the present disclosure, the related products also include PD-1 inhibitors. PD-1 (programmed death receptor 1) is an important immunosuppressive molecule that regulates the immune system and promotes self-tolerance by down-regulating the immune system's response to human cells and by inhibiting T-cell inflammatory activity. PD-1 can prevent autoimmune diseases, but it can also prevent the immune system from killing cancer cells. PD-1 inhibitors are a new class of drugs that block PD-1, activate the immune system to attack tumors, and are used to treat certain types of cancer.

[0061] In a specific embodiment of the present disclosure, the administration mass ratio of PD-1 inhibitors to vaccines is 2:0.8-1.2. PD-1 inhibitors are anticancer drugs currently used in clinical practice, and are effective against a variety of tumors. However, for most solid tumors, PD-1 inhibitors have the problem of low efficacy. In clinical practice, PD-1 inhibitors are only effective for about 20% of patients. Combining PD-1 inhibitors with other treatment methods can improve their efficacy. The nanopeptide vaccine disclosed in the present invention can be combined with PD-1 inhibitors to treat colorectal cancer. As a "spot-type" anticancer drug, PD-1 inhibitors can be combined with "personalized" vaccines targeted by new antigen peptides, thereby controlling tumors for a longer period of time and achieving better therapeutic effects.

[0062] In one embodiment of the present disclosure, the related product is used to enhance the anti-tumor immune response of colorectal cancer and inhibit the proliferation of colorectal cancer cells. + T and CD8 + T cell proliferation, and for promoting CD8 + T cells secrete cytokines, which include at least TNF-α and IFN-γ. The present invention detects CD4 T cells in the spleen, mesenteric lymph nodes and tumor tissues of colorectal cancer mice by flow cytometry. + T cells and CD8 + The number of T cells, as well as CD8 + The TNF-α and IFN-γ secreted by T cells confirmed that the aforementioned nanopeptide vaccine has the effect of enhancing the anti-tumor immune response of colorectal cancer.

[0063] The related products of the present disclosure may be pharmaceutical compounds, pharmaceutical compositions, etc. For example, the related products may be injection preparations, oral tablets or oral capsules. Specifically, the drug or pharmaceutical composition also includes a pharmaceutically acceptable excipient; a pharmaceutically acceptable excipient refers to any diluent, adjuvant and / or carrier that can be used in the pharmaceutical field. On the basis that the nanopeptide vaccine provided in the present disclosure can treat colorectal cancer, it is a conventional choice for those skilled in the art to reasonably select suitable diluents, adjuvants and / or carriers, which will not be described in detail here.

[0064] Example 1: Screening, synthesis and identification of colorectal cancer polypeptides

[0065] 1. Experimental Animals

[0066] SPF 6-week-old BALB / c female mice were purchased from Ningxia Medical University. All animal experiments were carried out in strict accordance with the operating procedures of the Experimental Animal Center of Ningxia Medical University and have been approved by the Ethics Committee of Ningxia Medical University.

[0067] The mice were randomly divided into 9 groups, 8 mice in each group. The 9 groups were negative control group (negative), positive control group (ConA), G12D, G12V, E545K, G12D+G12V, G12D+E545K, G12V+E545K and G12D+G12V+E545K groups.

[0068] 2. Experimental methods

[0069] Peptide screening and synthesis: Based on the top ten high-frequency mutation-related neoantigen peptides in existing colorectal cancer, the top three high-frequency mutation-related neoantigen peptides G12D (amino acid sequence: VVVGADGVGK), G12V (amino acid sequence: VVGAVGVGK), and E545K (amino acid sequence: TPQDSRQVL) were selected and synthesized as antigens for this experiment. The above three antigen peptides were synthesized by Shanghai Bioengineering Co., Ltd.

[0070] Immunization of mice: BALB / c mice were injected with peptides (50ug / mouse) intraperitoneally on day 0, day 7, and day 14, respectively. The negative control group was injected with 100uL PBS, and the positive control group was injected with 50ug ConA / mouse. The mice were killed on day 21, and the spleens of the mice were isolated aseptically.

[0071] Identification of peptide immunogenicity: The immunogenicity of the peptides was evaluated by detecting the amount of IFN-γ in the spleen of mice after intervention with the three antigenic peptides and their different combinations using the enzyme-linked immunosorbent spot (ELISPOT) assay. The specific steps are as follows:

[0072] ① Take out the ELISPOT 96-well plate and add 15 μL of 70% ethanol solution to each well under sterile conditions to pre-wet the PVDF membrane of the ELISPOT plate for 2 minutes.

[0073] ② Washing: After pre-wetting, add 200 μL sterile water to each well and wash 5 times for 3 minutes each time. After the last wash, gently dry the ELISPOT 96-well plate on sterile paper.

[0074] ③ Coating: Add 100 μL of primary antibody solution diluted with PBS to each well and incubate in a 4°C refrigerator overnight.

[0075] ④ Blocking: The next day, pour out the coating solution, then wash with PBS 3 times, soaking for 1 minute each time. After the last wash, place the ELISPOT 96-well plate on sterile absorbent paper and gently press to remove excess liquid. Then add 200 μL of blocking solution diluted with 1× PBS to each well and block at 37°C for 1 hour.

[0076] ⑤ Washing: After 1 hour, add 200 μL of sterile water to each well and wash 5 times, soaking for 1 minute each time. After discarding the liquid for the last time, gently dry it on sterile paper.

[0077] ⑥ Aseptically separate the spleen cells of the immunized mice, separate the mouse spleen lymphocytes with lymphocyte separation solution, add 1×104 mouse spleen lymphocytes to each well of the ELISPOT 96-well plate, and culture in an incubator at 37°C and 5% CO2 for 20 hours.

[0078] ⑦ Washing: After removing the cell culture medium, add 200 μL PBST solution to each well for five washes, soaking for 3 minutes each time to completely remove the cells. Then add 100 μL enzyme-labeled secondary antibody to each well and incubate for 1 hour at room temperature.

[0079] ⑧ Add detection antibody: After incubation for 1 hour, add 100 μL of pre-diluted biotin-labeled detection antibody to each well and incubate at 37°C for 1 hour.

[0080] ⑨ Washing: After incubation for 1 hour, add 200 μL of PBST solution to each well and wash 5 times, soaking for 5 minutes each time. After the last wash, dry the water on absorbent paper.

[0081] ⑩ Color development: Add 50 μL of TMB color development solution to each well in the dark. Let stand at room temperature for 15-45 minutes (all in the dark) until clear spots appear. Add 100 μL of deionized water to each well to terminate the color development reaction, store at room temperature in the dark, and read the plate using an ELISPOT plate reader after it is completely dry.

[0082] 2. Experimental results

[0083] Figure 1 This is the ELISPOT test result diagram. Figure 1 Figure A shows the spot diagram of IFN-γ detected by ELISPOT experiment. Figure 1 Figure B is the statistical result of the number of IFN-γ spots, and the experimental result is significant at p<0.001. The ELISPOT experimental results show that the immunogenicity of the combination of G12V+E545K is the strongest, and the immunogenicity of the combination of G12D+G12V+E545K is the second strongest.

[0084] Based on this experimental result, the present application selected G12V+E545K combination polypeptide and G12D+G12V+E545K combination polypeptide to prepare nano vaccines. Among them, the nano vaccine synthesized by G12V+E545K combination polypeptide is referred to as LBP-NPs1, and the nano vaccine synthesized by G12D+G12V+E545K combination polypeptide is referred to as LBP-NPs2.

[0085] Example 2: Preparation of LBP-NPs Nanovaccine

[0086] 1. Synthetic peptide fragments

[0087] The three antigen peptides G12D, G12V and E545K synthesized in Example 1 were used.

[0088] 15 mg of G12D and 15 mg of E545K were weighed, and 30 mg of the mixed polypeptides were dissolved in 10 mL of PBS for the preparation of the vaccine LBP-NPs1.

[0089] 10 mg of G12D, 10 mg of E545K and 10 mg of G12V were weighed, and 30 mg of the mixed polypeptides were dissolved in 10 mL of PBS for the preparation of vaccine LBP-NPs2.

[0090] 2. Preparation of liposome-peptide nanoparticles

[0091] like Figure 2 As shown, this embodiment adopts thin film dispersion-ultrasound method to prepare negatively charged liposome-peptide nanoparticles (NPs), and the specific steps are as follows:

[0092] Accurately weigh 60 mg of soybean lecithin and 30 mg of cholesterol in an eggplant-shaped bottle, and dissolve them in 30 mL of chloroform-methanol solvent (chloroform: methanol = 10: 20); ultrasonic dissolution in a 45°C water bath for 5 minutes to form an organic phase. Use a rotary evaporator to rotary evaporate, and evaporate under reduced pressure in a 45°C water bath for 1 hour to remove the organic solvent, so that the liposomes form a uniform film. Add 10 mL of aqueous phase (30 mg of polypeptide dissolved in 10 mL of PBS), shake and hydrate in a 40°C water bath for 2 hours to completely remove the film. Ultrasonicate in an ice water bath for 8 minutes (interval 3s, working 3s, power 55%) to a uniform system to obtain a liposome-polypeptide suspension. Pre-freeze the above mixed suspension in a -80°C refrigerator for 30 minutes, and quickly transfer the sample to a freeze dryer after pre-freezing, reduce the pressure to 0.2Mbar, and vacuum freeze-dry at -80°C for 24 hours to obtain NPs nanoparticles.

[0093] 3. Preparation of positively charged Lycium barbarum polysaccharide solution

[0094] like Figure 3 As shown, this embodiment adopts the reductive amination method to prepare positively charged wolfberry polysaccharides (LBP), and the specific steps are as follows:

[0095] 0.5g of Lycium barbarum polysaccharide was dissolved in 50mL of double distilled water (DDW), and then 0.716g of sodium periodate (the molar ratio of IO4- to Lycium barbarum polysaccharide was 1:1) was added. The mixture was quickly placed in a dark room and reacted with strong magnetic stirring at room temperature for 72 hours; the obtained dialdehyde polysaccharide and DDW were dialyzed in a dialysis bag with a relative molecular weight cutoff of 6kD-15kD for 48 hours. After freeze-drying, oxidized LBP was obtained, and 0.3g of oxidized LBP was accurately weighed, dissolved in 50mL of DDW and diluted to a certain concentration, thereby obtaining liquid A. 1.25 equimolar spermine (8.46mmol) was dissolved in 30mL of borate buffer (borate buffer 0.1M, pH=11) for more than 2 hours, dissolved and diluted to a certain concentration, thereby obtaining liquid B. Liquid B was slowly (by dropwise addition) added to liquid A under stirring at room temperature, and after the reaction liquid turned yellow, it was gently stirred at room temperature for 24 hours. Then, NaBH4 (0.3 g) was added to the mixture and reacted for 48 hours. Under the same conditions, NaBH4 (0.3 g) was added again and the above process was repeated for 24 hours; the dialysate was freeze-dried to obtain cationized LBP.

[0096] 4. Preparation of liposome-peptide nanoparticles

[0097] like Figure 4 As shown, in this example, LBP-NPs were prepared by electrostatic adsorption: the prepared negatively charged NPs nanoparticles were mixed with the cationic LBP solution in a ratio of 1:1, and slowly stirred overnight at room temperature to obtain LBP-NPs, which were then stored at 4°C. Among them, two mixed polypeptides were used to prepare LBP-NPs1 and LBP-NPs2, respectively.

[0098] Example 3: Mouse Experiment

[0099] 1. Experimental Animals

[0100] SPF 6- to 8-week-old BALB / c female mice were purchased from Ningxia Medical University. All animal experiments were conducted in strict compliance with the operating procedures of the Experimental Animal Center of Ningxia Medical University and have been approved by the Ethics Committee of Ningxia Medical University.

[0101] 2. Experimental methods

[0102] Forty-eight 6-8 week old female BALB / c mice were equally divided into 6 groups:

[0103] (1) PBS group: blank control group.

[0104] (2) LBP-NPs1 group: One week after modeling (tumor formation), immunotherapy with the LBP-NPs1 nanovaccine prepared in Example 2 was started, with a total of 3 subcutaneous inoculations, 100 μg each time, and an interval of 1 week between each inoculation (i.e., LBP-NPs1 was inoculated on the 7th, 14th, and 21st days).

[0105] (3) LBP-NPs2 group: One week after modeling (tumor formation), immunotherapy with the LBP-NPs2 nanovaccine prepared in Example 2 was started, with a total of 3 subcutaneous inoculations of 100 μg each time, with an interval of 1 week between each inoculation (i.e., LBP-NPs2 was inoculated on days 7, 14, and 21).

[0106] (4) αPD-1 group: One week after modeling (tumor formation), αPD-1 immunotherapy was started, with a total of six subcutaneous inoculations, 100 μg each time; the first three inoculations were separated by 1 week, and the next three inoculations were separated by 3 days (i.e., αPD-1 was inoculated on days 7, 14, 21, 24, 27, and 30).

[0107] (5) LBP-NPs1+αPD-1 group: One week after modeling (tumor formation), the LBP-NPs1 nanovaccine prepared in Example 2 and αPD-1 were inoculated for combined immunotherapy, with a total of 3 subcutaneous inoculations. Each time, 100 μg of vaccine and αPD-1 were inoculated, with an interval of 1 week between each inoculation (i.e., LBP-NPs1 and αPD-1 were inoculated on the 7th, 14th, and 21st days); after an interval of three days, αPD-1 was inoculated three times, with an interval of 3 days each time (i.e., αPD-1 was continued to be inoculated on the 24th, 27th, and 30th days).

[0108] (6) LBP-NPs2+αPD-1 group: One week after modeling (tumor formation), the LBP-NPs2 nanovaccine prepared in Example 2 and αPD-1 were inoculated for combined immunotherapy, with a total of three subcutaneous inoculations, 100 μg of vaccine and αPD-1 each time, with an interval of one week between each inoculation (i.e., LBP-NPs2 and αPD-1 were inoculated on the 7th, 14th, and 21st days); after an interval of three days, αPD-1 was continued to be inoculated three times, with an interval of three days each time (i.e., αPD-1 was continued to be inoculated on the 24th, 27th, and 30th days).

[0109] See also Figure 5 On day 0, a mouse subcutaneous colorectal cancer model was established: when CT-26 cells were grown to the logarithmic growth phase, they were digested with trypsin, washed three times by centrifugation, and resuspended in PBS to adjust the cell concentration to 1×10 7 pcs / mL in PBS and injected subcutaneously into the right scapula of mice (100 μL / mouse).

[0110] Starting from the injection of CT-26 cells, the length (L) and width (W) of colorectal cancer tumors were measured every 3 days, and the colorectal cancer tumor volume (V) was calculated using the formula V = 0.5 × L × W2. Five weeks after the injection of CT-26 cells (day 35), tumor-bearing mice were killed by cervical dislocation.

[0111] 3. Experimental results

[0112] Figure 6 Figure A in the figure shows the statistical results of mouse survival rate. As shown in the figure, as of the 35th day after the mice were inoculated with tumor cells, the survival rate of mice in the PBS group was 50%; compared with the PBS group, the survival rate of mice inoculated with nanovaccines LBP-NPs1, LBP-NPs2 or αPD-1 was improved. It can be seen that the nanovaccine of the present application can effectively improve the survival rate of mice.

[0113] In addition, compared with the use of nano vaccines alone or combined with αPD-1 treatment, the combined use of vaccines and αPD-1 treatment can significantly improve the survival rate of mice, among which the survival rate of mice in the LBP-NPs2+αPD-1 group reached 90%, and LBP-NPs2+αPD-1 treatment can increase the survival rate of mice to 100%. It can be seen that the combined use of nano vaccines and αPD-1 can more effectively improve the survival rate of mice and has an outstanding therapeutic effect.

[0114] Figure 6 Figure B shows the tumor volume detection results of mouse colorectal cancer. Figure 6 Figure C in the figure is a photo of mouse colorectal cancer tumor tissue on day 35. It can be seen from the figure that the colorectal cancer tumor volume of mice in the PBS group is significantly larger than the colorectal cancer tumor volume of mice inoculated with nanovaccines LBP-NPs1, LBP-NPs2 and αPD-1, and the results are statistically significant. It can be seen that inoculation with nanovaccines LBP-NPs1 and LBP-NPs2 can inhibit the growth of colorectal cancer tumors in mice.

[0115] In addition, compared with the use of nano-vaccine alone or combined with αPD-1 treatment, the colorectal cancer tumor volume of mice treated with the combination of vaccine and αPD-1 was significantly smaller, among which LBP-NPs2+αPD-1 treatment had the strongest inhibitory effect on mouse tumors. It can be seen that the combined use of nano-vaccine and αPD-1 can more effectively inhibit the growth of colorectal cancer tumors in mice and has an outstanding therapeutic effect.

[0116] Figure 6 Figure D shows the results of mouse weight test. It can be seen from the figure that LBP-NPs1, LBP-NPs2, and αPD-1 are safe when used alone or in combination, and there is no significant weight loss in mice after vaccination.

[0117] The above experiments show that vaccination with nanovaccines LBP-NPs1, LBP-NPs2, and αPD-1 can slow down the growth of colorectal cancer tumors and improve the survival rate of mice, and the therapeutic effect of using nanovaccines combined with αPD-1 is better; in addition, LBP-NPs1, LBP-NPs2, and αPD-1 are safe when used alone or in combination.

[0118] Example 4: Detection of T cells in mouse spleen, mesenteric lymph nodes and tumors by flow cytometry

[0119] Studies have shown that the replication potential and differentiation state of T cells are important regulators of anti-tumor activity. Therefore, promoting T cells is a potential way to improve the efficacy of immunotherapy. + T cells, called helper T cells, regulate immune responses by producing cytokines, which enhance or suppress inflammation. + T cells are called cytotoxic T cells. When CD8+ T cells recognize their antigens and are activated, they mainly produce inflammatory cytokines TNF-α and IFN-γ, which have anti-tumor and anti-viral microbial effects. In addition, CD8+ T cells can also kill pathogen-infected cells or malignant cells by secreting cell lytic molecules such as perforin and granzyme. + T and CD8 + T cells can generate anti-tumor immune responses, among which CD8 + T cells can directly kill tumor cells marked by tumor antigens, CD4 + T cells can help activate CD8 + T cells. In summary, detection of CD4 + T cells, CD8 + T cell count, and CD8 + The levels of TNF-α and IFN-γ secreted by T cells can evaluate the anti-tumor function of T cells.

[0120] The source of experimental animals and the modeling method are exactly the same as those in Example 3 and will not be repeated here.

[0121] 1. Experimental methods

[0122] The mice were raised until 5 weeks after CT-26 cell injection (day 35), and the tumor-bearing mice were killed by cervical dislocation. The spleen, mesenteric lymph nodes and colorectal cancer tumors of the mice were obtained by dissection.

[0123] Obtaining mononuclear cells: The spleen, mesenteric lymph nodes and tumors of mice were placed on a 200-mesh filter, respectively, and ground with a syringe. The ground suspension was filtered into a 15 mL centrifuge tube with a 200-mesh filter. The spleen and tumor cell suspensions were added to the mouse spleen mononuclear cell separation solution (Solarbio) and mouse tumor-infiltrating lymphocyte separation solution (Solarbio) at a ratio of 1:1, respectively. After centrifugation at 2000 rpm for 20 minutes, the buffy coat layer was aspirated and 5 times the volume of PBS was added. After thorough mixing, the mixture was centrifuged at 1500 r / min for 10 minutes. The supernatant was discarded and resuspended with 1 mL PBS to obtain a mononuclear cell suspension, which was counted and divided into tubes. The mesenteric lymph node suspension was directly resuspended with PBS to 5 mL, centrifuged at 1500 rpm for 10 minutes, the supernatant was discarded and resuspended with PBS to obtain a mononuclear cell suspension.

[0124] Flow staining mainly includes surface staining and intracellular staining. FITC-conjugated anti-CD8 antibody (BD Biosciences) and FITC-conjugated anti-CD4 antibody (BD Biosciences) are surface staining antibodies, and intracellular antibodies include PB450-conjugated anti-TNF-αantibody (BD Biosciences), PerCP-Cy TM 5.5-conjugated anti-IFN-γantibody (BD Biosciences). The surface antibody was incubated in the dark for 30 minutes. Before intracellular staining, the membrane was permeabilized for 25 minutes and then the intracellular antibody was added and incubated for 30 minutes. BD FACSCelesta TM Detection was performed by flow cytometry.

[0125] Among them, FITC-conjugated anti-CD8 antibody is used for surface staining of CD8+T cells; FITC-conjugated anti-CD4 antibody is used for surface staining of CD4+T cells; PB450-conjugatedanti-TNF-αantibody is used for staining TNF-α; PerCP-Cy TM 5.5-conjugated anti-IFN-γantibody was used to stain IFN-γ.

[0126] 2. Experimental results

[0127] 2.1 Spleen Experiment Results

[0128] See also Figure 7 , Figure 7 Figures A to D in the figure are the CD4 + T cells, CD8 + T cell count and CD8 + The experimental results of IFN-γ and TNF-α secreted by T cells.

[0129] like Figure 7 As shown in Figure A, compared with the PBS group, vaccine treatment alone and combined with αPD-1 treatment increased the number of CD4 + The number of T cells increased significantly, and CD4 + The increase in the number of T cells was more obvious. This indicates that LBP-NPs1 and LBP-NPs2 can promote the proliferation of CD4 + The number of T cells increased, and LBP-NPs1 and LBP-NPs2 could play a better promoting effect when combined with αPD-1.

[0130] like Figure 7 As shown in Figure B, compared with the PBS group, vaccine treatment alone and combined with αPD-1 treatment increased the number of CD8 + The number of T cells increased significantly, and CD8 + The increase in the number of T cells was more obvious. This indicates that LBP-NPs1 and LBP-NPs2 can promote the expression of CD8 + The number of T cells increased, and LBP-NPs1 and LBP-NPs2 could play a better promoting effect when combined with αPD-1.

[0131] like Figure 7 As shown in Figure C, compared with the PBS group, vaccine treatment alone and combined with αPD-1 treatment can significantly increase the IFN-γ content in the spleen of mice, and the increase in IFN-γ content after vaccine combined with αPD-1 treatment is more obvious. This shows that LBP-NPs1 and LBP-NPs2 can promote the expression of CD8 + T secretes IFN-γ, and LBP-NPs1 and LBP-NPs2 can have a better promoting effect when combined with αPD-1.

[0132] like Figure 7 As shown in Figure D, compared with the PBS group, the use of vaccine combined with αPD-1 treatment can significantly increase the content of TNF-α in the spleen of mice. This shows that LBP-NPs1 and LBP-NPs2 can promote the expression of CD8 in the spleen of colorectal cancer mice when combined with αPD-1. + T secretion of TNF-α can play a better promoting effect.

[0133] In summary, compared with the PBS group, the nanovaccine synthesized by the G12V+E545K combination peptide (LBP-NPs1) and the nanovaccine synthesized by the G12D+G12V+E545K combination peptide (LBP-NPs2) can promote the anti-tumor function of T cells in the spleen. Moreover, LBP-NPs1 and LBP-NPs2 can play a better promoting effect when combined with αPD-1, thereby enhancing the effect of treating colorectal cancer.

[0134] 2.2 Results of mesenteric lymph node experiments

[0135] See also Figure 8 , Figure 8 Figures A to D in the figure are the CD8 + T cells, CD4 + T cell count and CD8 + The experimental results of IFN-γ and TNF-α secreted by T cells.

[0136] like Figure 8 As shown in Figure A, compared with the PBS group, vaccine treatment alone and combined with αPD-1 treatment increased the number of CD8 + The number of T cells increased significantly, and CD8 + This indicates that LBP-NPs1 and LBP-NPs2 can promote the expression of CD8 T cells in the mesenteric lymph nodes of colorectal cancer mice. + The number of T cells increased, and LBP-NPs1 and LBP-NPs2 could play a better promoting effect when combined with αPD-1.

[0137] like Figure 8 As shown in Figure B, compared with the PBS group, vaccine treatment alone and combined with αPD-1 treatment increased the number of CD4 + The number of T cells increased significantly, and CD4 + The increase in the number of T cells was more obvious. This indicates that LBP-NPs1 and LBP-NPs2 can promote the expression of CD4 + The number of T cells increased, and LBP-NPs1 and LBP-NPs2 could play a better promoting effect when combined with αPD-1.

[0138] like Figure 8As shown in Figure C, compared with the PBS group, vaccine treatment alone and combined with αPD-1 treatment can significantly increase the content of IFN-γ in the mesenteric lymph nodes of mice, and the increase in IFN-γ content after vaccine combined with αPD-1 treatment is more obvious. This shows that LBP-NPs1 and LBP-NPs2 can promote the expression of CD8 + T secretes IFN-γ, and LBP-NPs1 and LBP-NPs2 can have a better promoting effect when combined with αPD-1.

[0139] like Figure 8 As shown in Figure D, compared with the PBS group, vaccine treatment alone and combined with αPD-1 treatment can significantly increase the content of TNF-α in the mesenteric lymph nodes of mice, and the increase in TNF-α content after vaccine combined with αPD-1 treatment is more obvious. This indicates that LBP-NPs1 and LBP-NPs2 can promote the expression of CD8 + T secretes TNF-α, and LBP-NPs1 and LBP-NPs2 can have a better promoting effect when combined with αPD-1.

[0140] In summary, compared with the PBS group, the nanovaccine synthesized by the G12V+E545K combination peptide (LBP-NPs1) and the nanovaccine synthesized by the G12D+G12V+E545K combination peptide (LBP-NPs2) can promote the anti-tumor function of T cells in mesenteric lymph nodes. Moreover, LBP-NPs1 and LBP-NPs2 can play a better promoting effect when combined with αPD-1, thereby enhancing the effect of treating colorectal cancer.

[0141] 2.3 Experimental results of colorectal cancer tumors

[0142] See also Fig. 9 , Fig. 9 Figures A to D in the figure are the CD4 + T cells, CD8 + T cell count and CD8 + The experimental results of IFN-γ and TNF-α secreted by T cells.

[0143] like Fig. 9 As shown in Figure A, compared with the PBS group, vaccine treatment alone and combined with αPD-1 treatment increased the number of CD4 + The number of T cells increased significantly, and CD4 + The increase in the number of T cells was more obvious. This indicates that LBP-NPs1 and LBP-NPs2 can promote the expression of CD4+ The number of T cells increased, and LBP-NPs1 and LBP-NPs2 could play a better promoting effect when combined with αPD-1.

[0144] like Fig. 9 As shown in Figure B, compared with the PBS group, vaccine treatment alone and combined with αPD-1 treatment increased the expression of CD8 + The number of T cells increased significantly, and CD8 + The increase in the number of T cells was more obvious. This indicates that LBP-NPs1 and LBP-NPs2 can promote the expression of CD8 + The number of T cells increased, and LBP-NPs1 and LBP-NPs2 could play a better promoting effect when combined with αPD-1.

[0145] like Fig. 9 As shown in Figure C, compared with the PBS group, vaccine treatment alone and combined with αPD-1 treatment can significantly increase the content of IFN-γ in colorectal cancer tumors of mice, and the increase in IFN-γ content after vaccine combined with αPD-1 treatment is more obvious. This shows that LBP-NPs1 and LBP-NPs2 can promote the expression of CD8 + T secretes IFN-γ, and LBP-NPs1 and LBP-NPs2 can have a better promoting effect when combined with αPD-1.

[0146] like Fig. 9 As shown in Figure D, compared with the PBS group, vaccine treatment alone and combined with αPD-1 treatment can significantly increase the content of TNF-α in colorectal cancer tumors of mice, and the increase in TNF-α content after vaccine combined with αPD-1 treatment is more obvious. This shows that LBP-NPs1 and LBP-NPs2 can promote the expression of CD8 + T secretes TNF-α, and LBP-NPs1 and LBP-NPs2 can have a better promoting effect when combined with αPD-1.

[0147] In summary, compared with the PBS group, the nanovaccine synthesized by the G12V+E545K combination peptide (LBP-NPs1) and the nanovaccine synthesized by the G12D+G12V+E545K combination peptide (LBP-NPs2) can promote the anti-tumor function of T cells in colorectal cancer tumors. Moreover, LBP-NPs1 and LBP-NPs2 can play a better promoting effect when combined with αPD-1, thereby enhancing the effect of treating colorectal cancer.

[0148] Example 5: Detection of immunosuppressive cells in mouse tumor tissues by flow cytometry

[0149] Among the various mechanisms of immune self-tolerance, the immunosuppression of endogenous CD4+Foxp3+Treg cells is essential. Treg cells can suppress abnormal antigen immune responses and also inhibit anti-tumor immune responses. The infiltration of a large number of Treg cells into tumor tissues is often associated with poor prognosis. More and more studies have shown that a large number of Treg cells infiltrate various types of tumors in humans and mice. Therefore, it is crucial to evaluate the heterogeneity of Foxp3+T cells in tumor tissues to evaluate their contribution to the anti-tumor immune response of various cancers.

[0150] Myeloid-derived suppressor cells (MDSCs) are involved in various aspects of disease immune regulation, including chronic inflammation, especially cancer, as well as infection, autoimmune diseases, trauma, tumors, etc. In most types of tumors, high infiltration of macrophages is closely associated with poor prognosis. Among them, M2 macrophages inhibit CD8 + T cell function hinders the efficacy of chemotherapy and radiotherapy, leading to tumor progression and poor prognosis. Macrophages can show remarkable plasticity within the tumor microenvironment (TME) and switch from one phenotype to another. Proinflammatory M1 macrophages in the TME may evolve into an M2-like phenotype after tumor progression, thereby playing a tumor-supporting role. The plasticity of macrophages highlights macrophage reprogramming as an attractive therapeutic strategy to inhibit tumor progression, allowing these cells to adjust their functions to meet the needs of anti-tumor defense. In summary, the number of tumor-infiltrating M1 macrophages, tumor-infiltrating M2 macrophages, Treg cells, and MDSCs cells in the tumor microenvironment can be detected to evaluate the anti-tumor function of each group of mice.

[0151] The source of experimental animals and the modeling method are exactly the same as those in Example 3 and will not be repeated here.

[0152] 1. Experimental methods

[0153] The mice were raised until 5 weeks (35th day) after CT-26 cell injection, and the tumor-bearing mice were killed by cervical dislocation, and the colorectal cancer tumor tissues of the mice were obtained by dissection.

[0154] Obtaining mononuclear cells: Place mouse tumor tissue on a 200-mesh filter, grind with a syringe, and filter the ground suspension with a 200-mesh filter into a 15 mL centrifuge tube. Add to mouse tumor-infiltrating lymphocyte separation solution (Solarbio) at a ratio of 1:1, centrifuge at 2000 rpm for 20 minutes, aspirate the buffy coat, add 5 times the volume of PBS, mix thoroughly, centrifuge at 1500 r / min for 10 minutes, discard the supernatant, and resuspend with 1 mL PBS to obtain a mononuclear cell suspension, count and divide into tubes.

[0155] Flow staining mainly includes surface staining and intracellular staining. FITC-conjugated anti-CD4 antibody (BD Biosciences), PB450-conjugated anti-CD86 antibody (BD Biosciences), APC-A750-conjugated anti-F4 / 80antibody (BD Biosciences), PE-conjugated anti-CD206antibody (BD Biosciences), APC-A00-conjugated anti-CD11b antibody (BD Biosciences) are surface staining antibodies. Intracellular antibodies include PerCP-Cy TM 5.5-conjugated anti-FoxP3 antibody (BD Biosciences). The surface antibody was incubated in the dark for 30 minutes. Before intracellular staining, the membrane was permeabilized for 25 minutes and then the intracellular antibody was added and incubated for 30 minutes. BD FACSCelesta TM Detection was performed by flow cytometry.

[0156] PB450-conjugated anti-CD86 antibody and APC-A750-conjugated anti-F4 / 80antibody were used to stain tumor-infiltrating M1 macrophages; PE-conjugated anti-CD206antibody and APC-A750-conjugated anti-F4 / 80antibody were used to stain tumor-infiltrating M2 macrophages; PerCP-Cy TM5.5-conjugated anti-FoxP3 antibody and FITC-conjugated anti-CD4 antibody were used to stain Treg cells in the tumor microenvironment; APC-A00-conjugated anti-CD11bantibody was used to stain MDSCs cells.

[0157] 2. Experimental results

[0158] See also Fig.10 , Fig.10 Figures A to B are the experimental results of the number of tumor-infiltrating M1 macrophages, tumor-infiltrating M2 macrophages, Treg cells in the tumor microenvironment, and MDSCs cells in the colorectal cancer tumor tissues of six groups of mice.

[0159] like Fig.10 As shown in Figure A, compared with the PBS group, vaccine treatment alone and combined with αPD-1 treatment can significantly increase the number of M1 macrophages in the spleen of mice, and the increase in the number of M1 macrophages after vaccine combined with αPD-1 treatment is more obvious. This shows that LBP-NPs1 and LBP-NPs2 can promote the increase in the number of M1 macrophages in the spleen of colorectal cancer mice, and LBP-NPs1 and LBP-NPs2 can play a better promoting effect when combined with αPD-1.

[0160] like Fig.10 As shown in Figure B, compared with the PBS group, vaccine treatment alone and combined with αPD-1 treatment can significantly reduce the number of M2 macrophages in the spleen of mice, and the reduction in the number of M2 macrophages after vaccine combined with αPD-1 treatment is more obvious. This shows that LBP-NPs1 and LBP-NPs2 can inhibit the number of M2 macrophages in the spleen of colorectal cancer mice, and LBP-NPs1 and LBP-NPs2 can have a stronger inhibitory effect when combined with αPD-1.

[0161] like Fig.10 As shown in Figure C, compared with the PBS group, vaccine treatment alone and combined with αPD-1 treatment can significantly reduce the number of Treg cells in the spleen of mice, and the reduction in the number of Treg cells after vaccine combined with αPD-1 treatment is more obvious. This shows that LBP-NPs1 and LBP-NPs2 can inhibit the number of Treg cells in the spleen of colorectal cancer mice, and LBP-NPs1 and LBP-NPs2 can have a stronger inhibitory effect when combined with αPD-1.

[0162] like Fig.10As shown in Figure D, compared with the PBS group, vaccine treatment alone and combined with αPD-1 treatment can significantly reduce the number of MDSCs cells in the spleen of mice, and the reduction in the number of MDSCs cells after vaccine combined with αPD-1 treatment is more obvious. This shows that LBP-NPs1 and LBP-NPs2 can inhibit the number of MDSCs cells in the spleen of colorectal cancer mice, and LBP-NPs1 and LBP-NPs2 can have a stronger inhibitory effect when combined with αPD-1.

[0163] Example 6: Immunohistochemistry experiment

[0164] The source of experimental animals and the modeling method are exactly the same as those in Example 3 and will not be repeated here.

[0165] 1. Experimental methods

[0166] Mice were raised until 5 weeks after CT-26 cell injection (day 35), and tumor-bearing mice were killed by cervical dislocation. Tumor tissues were obtained by dissection, and the expression of Ki67 and Caspase-3 in mouse tumor tissues was detected by immunohistochemistry. Ki67 is a protein present in the cell nucleus, and its gene is located on chromosome 10. Ki67 protein is present in all active stages of the cell cycle (G1, S, G2 and mitosis), but is not expressed in resting cells (G0). The expression of Ki67 protein is associated with the proliferation activity of malignant tumors and is used as a marker of tumor proliferation. The higher the expression of Ki67, the faster the tumor cells proliferate and the higher the malignancy of the tumor. Caspase-3 is a member of the caspase family and a key executor of apoptosis. The higher the expression of Caspase-3 in tumor cells, the more apoptosis of tumor cells and the better the prognosis. In this example, the expression of Ki67 and Caspase-3 in mouse tumor tissues was detected to determine the apoptosis of tumor cells in colorectal cancer tissues, and further determine the effects of nano-vaccine treatment alone, αPD-1 antibody treatment alone, and combined treatment on the progression of colorectal cancer.

[0167] The specific steps of immunohistochemistry are as follows:

[0168] 1.1 Fixation: The mouse tumor tissue was cleaned with PBS and fixed in 4% paraformaldehyde in a 4° refrigerator for 48 hours.

[0169] 1.2 Dehydration, embedding and sectioning: The fixed tumor tissue was placed in an embedding box and fully dehydrated with gradient ethanol from low to high. After passing through xylene, it was immersed in paraffin at 56°C for 2 hours. After the tissue was fixed, it was sectioned with a thickness of 3.5 μm.

[0170] 1.3 Dewaxing and hydrating: Place the slides in an oven at 60° for 2 hours before dewaxing and hydrating. The procedure is: 15 minutes in xylene I, 15 minutes in xylene II, 5 minutes in anhydrous ethanol I, 5 minutes in anhydrous ethanol II, 2 minutes in 95% ethanol, 2 minutes in 90% ethanol, 2 minutes in 80% ethanol, 2 minutes in 70% ethanol, and wash in tap water.

[0171] 1.4 Antigen repair: Place the tissue sections in a citric acid buffer (Seven Innovation (Beijing) Biotechnology Co., Ltd.) and repair them under high pressure for 15 minutes. After the air valve drops naturally, open the pressure cooker, take the sections out of the pressure cooker, rinse them in water for 3-5 minutes, and wash them three times with PBS, each time for 3 minutes.

[0172] 1.5 Inactivation of endogenous peroxidase: Place the slices in a humidified chamber, add endogenous peroxidase (Universal Two-Step Detection Kit, Cat.# PV-9000, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) to the sliced ​​tissue, incubate at room temperature for 10 min, and wash with PBS three times, 3 min each time.

[0173] 1.6 Blocking: Shake off excess water on the tissue slices, wipe off the water around the tissue with filter paper, and circle the tissue with an immunohistochemical pen. Then add goat serum (normal goat serum for blocking, Cat.#ZLI-9022, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) to cover the tissue, incubate in a 37℃ incubator for 30 minutes, and wash with PBS 3 times, 3 minutes each time.

[0174] 1.7 Incubate with primary antibody: shake off excess goat serum, directly add the prepared primary antibody and incubate at 37 degrees for 1.5 hours. Remove the wet box and wash with PBS three times, 5 minutes each time. Shake off excess water on the tissue sections, add reaction enhancement solution to cover the tissue, place it in the wet box at 37 degrees for 25 minutes, remove it and wash it with PBS three times, 3 minutes each time. The dilution ratios of Caspase-3 (ThermoFisher, Catalog #700182) and Ki-67 (ThermoFisher, Catalog #MA5-14520) are 1:200 and 1:100 respectively.

[0175] 1.8 Incubate with secondary antibody: Add appropriate amount of secondary antibody (Universal Two-step Detection Kit, Cat.#PV-9000, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) to completely cover the tissue, and incubate in a 37°C incubator for 30 minutes. Remove the wet box and wash with PBS three times, 5 minutes each time.

[0176] 1.9 DAB color development / re-staining: Add an appropriate amount of DAB color development solution (DAB color development kit (brown yellow), Cat.#AR1027-3, BOSTER) to the tissue to cover the tissue, observe the color development under a microscope, and stop the staining when the specific staining is strong and the background coloring is light. After rinsing the DAB color development solution on the slide with PBS, place the slice in tap water to stop the color development reaction and rinse with running water for 5 minutes. Then add hematoxylin (Hematoxylin Eosin (HE) staining kit, Cat.#G1120, Beijing Solebow Technology Co., Ltd.) to the slice, rinse with pure water after staining for 4 minutes, then add differentiation solution, rinse the differentiation solution on the slice with water after a few seconds, and then place it in tap water and rinse for 5-8 minutes to reverse blue.

[0177] 1.10 Dehydration, sealing and microscopic examination: Soak the slides in the following reagents for dehydration: 50% ethanol for 1 min, 75% ethanol for 1 min, 80% ethanol for 1 min, 85% ethanol for 1 min, 90% ethanol for 1 min, 95% ethanol for 1 min, xylene II for 2 min, and xylene I for 2 min. Take the tissue sections out of the last cylinder, seal them with neutral gum after drying, and dry them in a fume hood before microscopic examination.

[0178] 2. Experimental results

[0179] refer to Fig.11 The mouse tumor tissues were stained with Ki67 (cell proliferation marker) and Caspase3 (cell apoptosis marker). The results of immunohistochemical staining showed that the Ki67 expression level in the tumor tissue was significantly decreased after treatment with nanovaccine or αPD-1, but the Caspase-3 expression level was significantly increased, and the effect was most obvious in the combined treatment group. In other words, the use of nanovaccine combined with αPD-1 treatment can effectively promote the apoptosis of colorectal cancer cells and effectively inhibit the proliferation of colorectal cancer cells.

[0180] Example 7: HE staining experiment

[0181] Hematoxylin-eosin staining, referred to as HE staining. Among them, hematoxylin (H) is a basic dye that can dye the nucleus and ribosomes in the cell into blue-purple, and the structure stained by the basic dye is basophilic. Eosin (Eosin, E) is an acidic dye that can dye the cytoplasm into red or light red, and the structure stained by the acidic dye is eosinophilic. This example uses HE staining experiments to observe the morphological characteristics of the heart, liver, spleen, lung, and kidney of three groups of mice, such as tissue structure, tissue type, and cell hierarchy.

[0182] The source of experimental animals and the modeling method are exactly the same as those in Example 3 and will not be repeated here.

[0183] 1. Experimental methods

[0184] The mice were raised until 5 weeks (35 days) after CT-26 cell injection, and the tumor-bearing mice were killed by cervical dislocation. The heart, liver, spleen, lung, and kidney tissues of the mice in the PBS group, LBP-NPs1 group, LBP-NPs2 group, αPD-1 group, LBP-NPs1+αPD-1 group, and LBP-NPs2+αPD-1 group were obtained by dissection.

[0185] 1.1 Fixation: The heart, liver, spleen, lung, and kidney tissues of mice were cleaned with PBS, and then fixed in 4% paraformaldehyde in a 4°C refrigerator for 48 hours to prevent autolysis of cells or decomposition of bacteria after death, thereby maintaining the original morphological structure of the cells.

[0186] 1.2 Dehydration, embedding and sectioning: The fixed tumor tissue was placed in an embedding box and fully dehydrated with gradient ethanol from low to high. After passing through xylene, it was immersed in paraffin at 56°C for 2 hours. After the tissue was fixed, it was sectioned with a thickness of 3.5 μm.

[0187] 1.3 Dewaxing and hydrating: Place the slides in an oven at 60° for 2 hours before dewaxing and hydrating. The procedure is: 15 minutes in xylene I, 15 minutes in xylene II, 5 minutes in anhydrous ethanol I, 5 minutes in anhydrous ethanol II, 2 minutes in 95% ethanol, 2 minutes in 90% ethanol, 2 minutes in 80% ethanol, 2 minutes in 70% ethanol, and wash in tap water.

[0188] 1.4 Staining: Stain the tissue sections with hematoxylin and eosin. Specifically, dry the water on the slide, add hematoxylin staining solution, stain for 5 minutes, rinse off the hematoxylin staining solution with running water, and observe the degree of staining under a microscope. Differentiate with 1% hydrochloric acid alcohol for 1 to 3 seconds, wash with water, soak in tap water to turn blue for 5 to 10 minutes, and observe the color under a microscope. If the color is not dark enough, directly add hematoxylin staining solution to stain for 1 minute, then differentiate again and wash immediately with water; if it is too dark, increase the differentiation time appropriately. After that, add 1 drop of 0.5% eosin staining solution, stain for 10 to 15 seconds, wash with distilled water, and observe under a microscope.

[0189] 1.5 Dehydration, sealing and microscopic examination: Soak the slides in the following reagents for dehydration: 50% ethanol for 1 min, 75% ethanol for 1 min, 80% ethanol for 1 min, 85% ethanol for 1 min, 90% ethanol for 1 min, 95% ethanol for 1 min, xylene II for 2 min, and xylene I for 2 min. Take the tissue sections out of the last cylinder, seal them with neutral gum after drying, and dry them in a fume hood before microscopic examination.

[0190] 2. Experimental results

[0191] See also Fig.12 , Fig.12 The results of histological changes in the heart, liver, spleen, lung, and kidney of six groups of mice. The HE staining results show that the use of LBP-NPs nanovaccine alone, αPD-1 treatment alone, and combined treatment did not produce any significant changes in the morphology of the main organs of mice, heart, liver, spleen, lung, and kidney. Therefore, the use of LBP-NPs nanovaccine to treat colorectal cancer has a certain degree of safety and will not produce toxic side effects.

[0192] According to the experimental results of Examples 3 to 7, the two LBP-NPs nanovaccines prepared in Example 2 have good safety, have obvious therapeutic effects on the colorectum, and can play a stronger therapeutic effect when combined with αPD-1. Specifically, vaccine treatment alone and combined with αPD-1 treatment can significantly prolong the survival period of mice, inhibit tumor growth, improve the anti-tumor immune response of colorectal cancer, promote the apoptosis of colorectal cancer cells, and inhibit tumor cell proliferation. In tumor tissues and peripheral immune organs, the levels of CD8+T and its secretion of TNF-α and IFN-γ are significantly increased, and the increase in the number of CD4+T cells better promotes the proliferation of cytotoxic T lymphocytes. After treatment, Treg and MDSCs in the immune microenvironment are reversed, and macrophages are polarized, changing from M1 phenotype to M2. The above changes are closely related to the better prognosis of patients with colorectal cancer, indicating the potential role of vaccines in the anti-colorectal cancer effect.

[0193] Example 8: Mouse Experiment (Prevention + Treatment)

[0194] 1. Experimental Animals

[0195] SPF 6- to 8-week-old BALB / c female mice were purchased from Ningxia Medical University. All animal experiments were conducted in strict compliance with the operating procedures of the Experimental Animal Center of Ningxia Medical University and have been approved by the Ethics Committee of Ningxia Medical University.

[0196] 2. Experimental methods

[0197] Twenty-four 6-8 week old female BALB / c mice were equally divided into three groups:

[0198] (1) PBS group: blank control group.

[0199] (2) NPs1@NPs1+αPD-1 group: Before modeling, mice were subcutaneously vaccinated with 100 μg of the LBP-NPs1 nanovaccine prepared in Example 2 on days -21, -14, and -7 for prevention; a subcutaneous colorectal cancer model was established on day 0, and one week after modeling (tumor formation), the mice were vaccinated with LBP-NPs1 nanovaccine and αPD-1 for combined immunotherapy, for a total of 3 subcutaneous vaccinations, 100 μg of the vaccine and αPD-1 each time, with an interval of 1 week between each vaccination (i.e., LBP-NPs1 and αPD-1 were vaccinated on days 7, 14, and 21); after an interval of three days, αPD-1 was continued to be vaccinated three times, with an interval of 3 days each time (i.e., αPD-1 was continued to be vaccinated on days 24, 27, and 30).

[0200] (3) NPs2@NPs2+αPD-1 group: Before modeling, mice were subcutaneously vaccinated with 100 μg of the LBP-NPs2 nanovaccine prepared in Example 2 on days -21, -14, and -7 for prevention; a subcutaneous colorectal cancer model was established on day 0, and one week after modeling (tumor formation), the mice were vaccinated with LBP-NPs2 nanovaccine and αPD-1 for combined immunotherapy, for a total of three subcutaneous vaccinations, with 100 μg of vaccine and αPD-1 each time, with an interval of one week between each vaccination (i.e., LBP-NPs2 and αPD-1 were vaccinated on days 7, 14, and 21); after an interval of three days, αPD-1 was continued to be vaccinated three times, with an interval of 3 days each time (i.e., αPD-1 was continued to be vaccinated on days 24, 27, and 30).

[0201] See also Fig.13 On day 0, a mouse subcutaneous colorectal cancer model was established: when CT-26 cells were grown to the logarithmic growth phase, they were digested with trypsin, washed three times by centrifugation, and resuspended in PBS to adjust the cell concentration to 1×10 7 pcs / mL in PBS and injected subcutaneously into the right scapula of mice (100 μL / mouse).

[0202] Starting from the injection of CT-26 cells, the length (L) and width (W) of colorectal cancer tumors were measured every 3 days, and the colorectal cancer tumor volume (V) was calculated using the formula V = 0.5 × L × W2. Five weeks after the injection of CT-26 cells (day 35), tumor-bearing mice were killed by cervical dislocation.

[0203] 3. Experimental results

[0204] Fig.14Figure A in the figure shows the statistical results of mouse survival rate. As shown in the figure, as of the 35th day after the mice were inoculated with tumor cells, the survival rate of mice in the PBS group was 50%. Compared with the PBS group, the survival rate of the two groups of mice that were prevented by LBP-NPs1 and LBP-NPs2 and then received the combined treatment of nanovaccines and αPD-1 was significantly increased to 100%. It can be seen that preventive vaccination with nanovaccines and combined treatment with nanovaccines and αPD-1 after tumor formation can more effectively improve the survival rate of mice.

[0205] Fig.14 Figure B shows the tumor volume detection results of mouse colorectal cancer. Figure 6 Figure C is a photo of mouse colorectal cancer tumor tissue on day 35. It can be seen from the figure that the colorectal cancer tumor volume of mice in the PBS group was significantly larger than the colorectal cancer tumor volume of the two groups of mice that were prevented by LBP-NPs1 and LBP-NPs2 and then received the combined treatment of nanovaccines and αPD-1. The results were statistically significant. It can be seen that preventive vaccination with nanovaccines and combined treatment with nanovaccines and αPD-1 after tumor formation can inhibit the growth of colorectal cancer tumors in mice.

[0206] Fig.14 Figure D shows the results of mouse weight test. It can be seen from the figure that compared with the PBS group, there was no significant weight loss in the mice in the NPs1@NPs1+αPD-1 group and the NPs2@NPs2+αPD-1 group. It can be seen that it is safe to receive LBP-NPs1, LBP-NPs2 for prevention and combined treatment with nanovaccine and αPD-1.

[0207] The above experiments show that preventive vaccination with nanovaccines LBP-NPs1 and LBP-NPs2 and combined treatment with nanovaccines and αPD-1 after tumor formation can slow the growth of colorectal cancer tumors, improve the survival rate of mice, and is safe for mice.

[0208] Example 9: Detection of T cells in mouse spleen, mesenteric lymph nodes and tumors by cytometry (prevention + treatment)

[0209] The source of experimental animals and the modeling method are exactly the same as those in Example 8 and will not be repeated here.

[0210] 1. Experimental methods

[0211] The mice were raised until 5 weeks after CT-26 cell injection (day 35), and the tumor-bearing mice were killed by cervical dislocation. The spleen, mesenteric lymph nodes and colorectal cancer tumors of the mice were obtained by dissection.

[0212] Obtaining mononuclear cells: The spleen, mesenteric lymph nodes and tumors of mice were placed on a 200-mesh filter, respectively, and ground with a syringe. The ground suspension was filtered into a 15 mL centrifuge tube with a 200-mesh filter. The spleen and tumor cell suspensions were added to the mouse spleen mononuclear cell separation solution (Solarbio) and mouse tumor-infiltrating lymphocyte separation solution (Solarbio) at a ratio of 1:1, respectively. After centrifugation at 2000 rpm for 20 minutes, the buffy coat layer was aspirated and 5 times the volume of PBS was added. After thorough mixing, the mixture was centrifuged at 1500 r / min for 10 minutes. The supernatant was discarded and resuspended with 1 mL PBS to obtain a mononuclear cell suspension, which was counted and divided into tubes. The mesenteric lymph node suspension was directly resuspended with PBS to 5 mL, centrifuged at 1500 rpm for 10 minutes, the supernatant was discarded and resuspended with PBS to obtain a mononuclear cell suspension.

[0213] Flow staining mainly includes surface staining and intracellular staining. FITC-conjugated anti-CD8 antibody (BD Biosciences) and FITC-conjugated anti-CD4 antibody (BD Biosciences) are surface staining antibodies, and intracellular antibodies include PB450-conjugated anti-TNF-αantibody (BD Biosciences), PerCP-Cy TM 5.5-conjugated anti-IFN-γantibody (BD Biosciences). The surface antibody was incubated in the dark for 30 minutes. Before intracellular staining, the membrane was permeabilized for 25 minutes and then the intracellular antibody was added and incubated for 30 minutes. BD FACSCelesta TM Detection was performed by flow cytometry.

[0214] Among them, FITC-conjugated anti-CD8 antibody is used for surface staining of CD8+T cells; FITC-conjugated anti-CD4 antibody is used for surface staining of CD4+T cells; PB450-conjugatedanti-TNF-αantibody is used for staining TNF-α; PerCP-Cy TM 5.5-conjugated anti-IFN-γantibody was used to stain IFN-γ.

[0215] 2. Experimental results

[0216] 2.1 Spleen Experiment Results

[0217] See also Fig.15 , Fig.15 Figures A, B, C, and D in the figure are the CD4 + T cells, CD8 + T cell count and CD8 + The experimental results of IFN-γ and TNF-α secreted by T cells.

[0218] like Fig.15 As shown in Figure A, compared with the PBS group, after preventive treatment with LBP-NPs1 and LBP-NPs2 nanovaccines, combined with αPD-1 treatment, the expression of CD4 + Increased number of T cells.

[0219] like Fig.15 As shown in Figure B, compared with the PBS group, after preventive treatment with LBP-NPs1 and LBP-NPs2 nanovaccines, combined with αPD-1 treatment, the expression of CD8 + Increased number of T cells.

[0220] like Fig.15 As shown in Figure C, compared with the PBS group, after preventive treatment with LBP-NPs1 and LBP-NPs2 nanovaccines, combined with αPD-1 treatment, the expression of CD8 + T cells secrete IFN-γ.

[0221] like Fig.15 As shown in Figure D, compared with the PBS group, after preventive treatment with LBP-NPs1 and LBP-NPs2 nanovaccines, combined with αPD-1 treatment, the expression of CD8 + T cells secrete TNF-α.

[0222] In summary, compared with the PBS group, the nanovaccine synthesized by the G12V+E545K combination peptide (LBP-NPs1) and the nanovaccine synthesized by the G12D+G12V+E545K combination peptide (LBP-NPs2) can promote the anti-tumor function of T cells in the spleen. In addition, the strategy of first using nanovaccines for prevention and then using the combination of nanovaccines and αPD-1 for treatment can effectively promote the proliferation of antigen-specific T lymphocytes, thereby building a strong immune response in the body and enhancing the effect of treating colorectal cancer.

[0223] 2.2 Results of mesenteric lymph node experiments

[0224] See also Fig.16 , Fig.16Figures A, B, C, and D in the figure are the CD4 + T cells, CD8 + T cell count and CD8 + The experimental results of IFN-γ and TNF-α secreted by T cells.

[0225] like Fig.16 As shown in Figure A, compared with the PBS group, the preventive treatment with LBP-NPs1 and LBP-NPs2 nanovaccines, combined with αPD-1 treatment, can promote the expression of CD4 + Increased number of T cells.

[0226] like Fig.16 As shown in Figure B, compared with the PBS group, after preventive treatment with LBP-NPs1 and LBP-NPs2 nanovaccines, combined with αPD-1 treatment, the expression of CD8 + Increased number of T cells.

[0227] like Fig.16 As shown in Figure C, compared with the PBS group, after preventive treatment with LBP-NPs1 and LBP-NPs2 nanovaccines, combined with αPD-1 treatment, the expression of CD8 + T cells secrete IFN-γ.

[0228] like Fig.16 As shown in Figure D, compared with the PBS group, the preventive treatment with LBP-NPs1 and LBP-NPs2 nanovaccines, combined with αPD-1 treatment, can promote the expression of CD8 + T cells secrete TNF-α.

[0229] In summary, compared with the PBS group, the nanovaccine synthesized by the G12V+E545K combination peptide (LBP-NPs1) and the nanovaccine synthesized by the G12D+G12V+E545K combination peptide (LBP-NPs2) can promote the anti-tumor function of T cells in mesenteric lymph nodes. In addition, the strategy of first using nanovaccine for prevention and then using the combination of nanovaccine and αPD-1 for treatment can effectively promote the proliferation of antigen-specific T lymphocytes, thereby building a strong immune response in the body and enhancing the effect of treating colorectal cancer.

[0230] 2.3 Experimental results of colorectal cancer tumors

[0231] See also Fig.17 , Fig.17Figures A, B, C, and D in the figure are the CD4 + T cells, CD8 + T cell count and CD8 + The experimental results of IFN-γ and TNF-α secreted by T cells.

[0232] like Fig.17 As shown in Figure A, compared with the PBS group, the preventive treatment with LBP-NPs1 and LBP-NPs2 nanovaccines, combined with αPD-1 treatment, can promote the expression of CD4 + Increased number of T cells.

[0233] like Fig.17 As shown in Figure B, compared with the PBS group, the preventive treatment with LBP-NPs1 and LBP-NPs2 nanovaccines, combined with αPD-1 treatment, can promote the expression of CD8 + Increased number of T cells.

[0234] like Fig.17 As shown in Figure C, compared with the PBS group, the preventive treatment with LBP-NPs1 and LBP-NPs2 nanovaccines, combined with αPD-1 treatment, can promote the expression of CD8 + T cells secrete IFN-γ.

[0235] like Fig.17 As shown in Figure D, compared with the PBS group, the preventive treatment with LBP-NPs1 and LBP-NPs2 nanovaccines, combined with αPD-1 treatment, can promote the expression of CD8 + T cells secrete TNF-α.

[0236] In summary, compared with the PBS group, the nanovaccine synthesized by the G12V+E545K combination peptide (LBP-NPs1) and the nanovaccine synthesized by the G12D+G12V+E545K combination peptide (LBP-NPs2) can promote the anti-tumor function of T cells in colorectal cancer tumors. In addition, the strategy of first using nanovaccines for prevention and then using the combination of nanovaccines and αPD-1 for treatment can effectively promote the proliferation of antigen-specific T lymphocytes, thereby building a strong immune response in the body and enhancing the effect of treating colorectal cancer.

[0237] Example 10: Post-prophylactic NPs combined with αPD-1 therapy exerts anti-tumor effects in vivo by increasing tumor-infiltrating M1 macrophages

[0238] The source of experimental animals and the modeling method are exactly the same as those in Example 8 and will not be repeated here.

[0239] 1. Experimental methods

[0240] The mice were raised until 5 weeks after CT-26 cell injection (day 35), and the tumor-bearing mice were killed by cervical dislocation, and the tumor tissues were obtained by dissection.

[0241] Obtaining mononuclear cells: Place mouse tumor tissue on a 200-mesh filter and grind it with a syringe. Filter the ground suspension with a 200-mesh filter into a 15 mL centrifuge tube. The cells were added to the mouse tumor infiltrating lymphocyte separation medium (Solarbio) at a ratio of 1:1. After centrifugation at 2000 rpm for 20 minutes, the buffy coat layer was aspirated and 5 times the volume of PBS was added. After thorough mixing, the cells were centrifuged at 1500 r / min for 10 minutes. The supernatant was discarded and resuspended with 1 mL of PBS to obtain a mononuclear cell suspension. After counting, the cells were divided into tubes and resuspended with 100 mL of PBS containing 2% FBS. Subsequently, 0.35 uL of surface-stained antibodies of PB450-conjugated anti-CD86antibody (BD Biosciences) and APC-A750-conjugated anti-F4 / 80antibody (BD Biosciences) were added respectively. After gently blowing and mixing, the cells were incubated in a 4°C incubator away from light for 30 minutes. 350 mL of PBS solution containing 2% FBS was added, the cells were gently mixed, and the cells were incubated at 4°C at 1500 r·min. -1 Centrifuge for 5 minutes, discard the supernatant, add 300 mL of PBS solution containing 2% FBS to resuspend the cells, gently pipette to mix, and then test on the machine.

[0242] 2. Experimental results

[0243] Fig.18 The figure shows the experimental results of the number of tumor-infiltrating M1 macrophages in colorectal cancer tumor tissues of three groups of mice. Fig.18 Compared with the PBS group, the mouse groups of NPs1@NPs1+αPD-1 and NPs2@NPs2+αPD-1 showed a significant increase in the number of M1 macrophages in the tumor. Among them, the NPs1@NPs1+αPD-1 group performed more prominently in promoting the activation and function of M1 macrophages. The experimental results show that the potential of LBP-NPs1 and LBP-NPs2 nanovaccines combined with αPD-1 treatment in enhancing the activity of M1 macrophages in the tumor microenvironment provides a new strategy for tumor immunotherapy.

[0244] Example 11: Post-prophylactic NPs combined with αPD-1 therapy exerts anti-tumor effects in vivo by reducing immunosuppressive cells

[0245] The source of experimental animals and the modeling method are exactly the same as those in Example 8 and will not be repeated here.

[0246] 1. Experimental methods

[0247] The mice were raised until 5 weeks after CT-26 cell injection (day 35), and the tumor-bearing mice were killed by cervical dislocation, and the tumor tissues were obtained by dissection.

[0248] According to the above steps, the mononuclear cell suspension of tumor tissue was obtained, counted and divided into tubes, and the cells were resuspended in 100 mL of PBS containing 2% FBS, and then FITC-conjugated anti-CD4 antibody (BD Biosciences), APC-A750-conjugated anti-F4 / 80antibody (BD Biosciences), PE-conjugated anti-CD206antibody (BD Biosciences), APC-A00-conjugated anti-CD11b antibody (BD Biosciences) were added as surface staining antibodies. Intracellular antibodies included PerCP-Cy TM 5.5-conjugated anti-FoxP3 antibody (BD Biosciences). The surface antibody was incubated in the dark for 30 minutes. Before intracellular staining, the membrane was permeabilized for 25 minutes and then the intracellular antibody was added and incubated for 30 minutes. BD FACSCelesta TM Detection was performed by flow cytometry.

[0249] 2. Experimental results

[0250] See also Fig.19 , Fig.19 Figures A, B, C, and D are the experimental results of the number of tumor-infiltrating M2 macrophages, Treg cells, and MDSCs cells in the colorectal cancer tumor tissues of the three groups of mice.

[0251] like Fig.19 As shown in Figure A, the number of M2 macrophages in colorectal cancer tumors of the two groups of mice that were prevented by LBP-NPs1 and LBP-NPs2 nanovaccines and subsequently treated with αPD-1 was lower than that of the PBS group. This shows that LBP-NPs1 and LBP-NPs2 can inhibit the number of M2 macrophages in colorectal cancer tumors of colorectal cancer mice.

[0252] like Fig.19As shown in Figure B, the number of Treg cells in colorectal cancer tumors of the two groups of mice that were prevented by LBP-NPs1 and LBP-NPs2 nanovaccines and then treated with αPD-1 was lower than that of the PBS group. This shows that LBP-NPs1 and LBP-NPs2 can inhibit the number of Treg cells in colorectal cancer tumors of colorectal cancer mice.

[0253] like Fig.19 As shown in Figure C, the number of MDSCs cells in colorectal cancer tumors of the two groups of mice that were prevented by LBP-NPs1 and LBP-NPs2 nanovaccines and then treated with αPD-1 was lower than that of the PBS group. It can be seen that LBP-NPs1 and LBP-NPs2 can inhibit the number of MDSCs cells in colorectal cancer tumors of colorectal cancer mice.

[0254] In summary, compared with the control group treated with PBS alone, both groups of mice (NPs1@NPs1+αPD-1 group and NPs2@NPs2+αPD-1 group) that underwent vaccine prevention and subsequent combined αPD-1 treatment showed a significant reduction in the number of immunosuppressive cells in the tumor microenvironment, including Treg cells, MDSC cells, and M2 macrophages. This result shows that the vaccine combined with αPD-1 treatment strategy can effectively regulate the tumor microenvironment and reduce the number of immunosuppressive cells, thus providing a new perspective for anti-tumor treatment.

[0255] The embodiments of the present disclosure have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The choice of terms used herein is intended to best explain the principles of the embodiments, practical applications, or technical improvements in the marketplace, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein. The scope of the present disclosure is defined by the appended claims.

Claims

1. Use of a nanopeptide vaccine for preventing colorectal cancer in the preparation of related products for treating colorectal cancer, characterized in that: The vaccine comprises an antigen, wherein the antigen comprises a first polypeptide fragment and a second polypeptide fragment; The amino acid sequence of the first polypeptide fragment is: VVGAVGVGK; The amino acid sequence of the second polypeptide fragment is: TPQDSRQVL.

2. The use according to claim 1, characterized in that: The antigen also includes a third polypeptide fragment, and the amino acid sequence of the third polypeptide fragment is: VVVGADGVGK.

3. The use according to claim 1 or 2, characterized in that: The related products also include PD-1 inhibitors.

4. The use according to claim 3, characterized in that: The administration mass ratio of the PD-1 inhibitor to the vaccine is 2:0.8-1.

2.

5. The use according to any one of claims 1 or 2, characterized in that: The vaccine also includes an adjuvant, which is Lycium barbarum polysaccharide.

6. The use according to any one of claims 1 or 2, characterized in that: The vaccine further comprises a carrier, which is a liposome.

7. The use according to any one of claims 1 or 2, characterized in that: The related products are used to enhance the anti-tumor immune response of colorectal cancer and inhibit the proliferation of colorectal cancer cells.

8. The use according to claim 7, characterized in that: The related products are used to promote CD4 + T and CD8 + T cell proliferation.

9. The use according to claim 7, characterized in that: The related products are used to promote CD8 + T cells secrete cytokines including at least TNF-α and IFN-γ.

10. The use according to claim 1, characterized in that: The related products are injectable preparations, oral tablets or oral capsules.

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