Preparation method for and use of lactococcus lactis surface-modified with neoantigen peptide

Abstract

Provided are a preparation method for and a use of Lactococcus lactis surface-modified with a neoantigen peptide, relating to the field of biomedicine. The Lactococcus lactis comprises wild type or genetically engineered type. The specific modification strategy comprises: modifying a cell-penetrating peptide sequence in the N segment of a tumor neoantigen peptide, and then using the cell-penetrating peptide to insert into a bacterial surface; or using a biomineralization method to load the neoantigen peptide and connect it to a bacterial surface. It has been verified that both methods can stably bind the tumor neoantigen peptide to the surface of Lactococcus lactis. Animal experiments suggest that in situ lymph node injection of any neoantigen peptide-Lactococcus lactis complex is beneficial for presenting antigen peptides to initial T cells in the lymph nodes, stimulating the proliferation and differentiation of activated tumor-specific CD8+T cells, slowing tumor growth, and prolonging the survival time of tumor-bearing mice.

Description

Preparation method and application of Lactococcus lactis with surface modified new antigen peptide Technical Field

[0001] The present invention belongs to the field of biomedicine and pharmaceutical preparation technology, and specifically relates to a method for preparing and applying Lactococcus lactis with surface-modified neoantigen peptides. The present invention relates to two methods for preparing surface-modified neoantigen peptides of Lactococcus lactis. One method is to modify the cell-penetrating peptide sequence in the N segment of the tumor neoantigen peptide, and then use the cell-penetrating peptide to insert into the bacterial surface. The other method is to use a biomineralization method to load the neoantigen peptide and attach it to the bacterial surface. Background Art

[0002] The development and application of personalized neoantigen peptide tumor vaccines has become a hot topic in cancer treatment. They are beneficial for activating neoantigen-specific T cells, enabling precise tumor killing. However, naked peptide vaccines often face significant challenges, such as early enzymatic clearance in the body and inefficient phagocytosis and presentation by antigen-presenting cells (DCs).

[0003] Appropriate peptide vaccine carriers help new antigen peptide anti-tumor vaccines to work. Compared with other biomaterials such as Montanide ISA-5, mesoporous silica, nanoparticles and injectable hydrogels, probiotics are closely involved in human health. A series of preclinical experiments and clinical trials have confirmed the anti-tumor effects of some probiotics. Among the widespread applications of probiotics, lactic acid bacteria occupy a considerable proportion and have strong tumor regulation potential. Lactic acid bacteria are mainly divided into lactobacilli and lactococci (cocci can be further divided into pediococci, streptococci, and leuconostoc). Mature gene editing technologies such as the NICE system have made lactic acid bacteria often used as biological carriers for delivering active proteins.

[0004] The characteristics of bacteria themselves, such as the special lipid composition of the outer membrane, provide a theoretical basis for bacterial surface modification. In recent years, many studies have pointed out that the combination of bacteria and materials can greatly enhance the anti-tumor immune effect. Specific modification methods include: using chemical bonds to construct bacteria / nanomaterial complexes, so that nanomaterials bind to amino groups on the bacterial surface; using electrostatic interactions to construct bacteria / nanomaterial complexes; using specific biorecognition such as streptavidin-biotin interactions to construct bacteria / nanomaterial complexes; using antigen-antibody interactions to construct bacteria / nanomaterial complexes, etc.

[0005] Currently, tumor immunotherapy still faces many challenges. Therefore, there is an urgent need to develop new tumor immunotherapy and platforms to provide new ideas for clinical translation. The present invention provides a method for preparing Lactococcus lactis with surface-modified neoantigen peptides. The prepared Lactococcus lactis with surface-modified neoantigen peptides can be used as a tumor vaccine, which can induce tumor-specific immune responses by converting anti-bacterial immunity into anti-tumor immunity, significantly enhancing anti-tumor efficacy.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to address the deficiencies in the prior art and to provide a method for preparing and applying Lactococcus lactis with a surface modified neoantigen peptide.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] (1) The present invention provides a Lactococcus lactis with a surface modified neoantigen peptide, wherein the Lactococcus lactis comprises at least one tumor neoantigen peptide, wherein the tumor neoantigen peptide is non-genetically linked to the surface of the Lactococcus lactis, or the tumor neoantigen peptide is inserted into / through the cell wall of the Lactococcus lactis.

[0010] Furthermore, the length of the tumor neoantigen peptide is not limited, and is preferably a peptide consisting of 8-25 amino acids.

[0011] Furthermore, the tumor neoantigen peptides include tumor-specific antigens and tumor-associated antigens.

[0012] Furthermore, the tumor neoantigen peptide is an MHC-I restricted and / or MHC-II restricted antigen peptide.

[0013] Furthermore, the neotumor antigen peptide is a fusion peptide comprising multiple different antigens, which can trigger an immune response against one or more specific tumors.

[0014] Furthermore, the tumor neoantigen peptides are patient-recognized or patient-specific. The exact nature of the peptides can vary depending on the nature of the tumor to be treated. Specificity means that different anti-tumor, tumor-specific peptides will be used to treat individuals presenting different types of cancer, and even different anti-tumor, tumor-specific peptides can be used to treat individuals presenting the same type of cancer.

[0015] Furthermore, the Lactococcus lactis is wild-type Lactococcus lactis or genetically engineered Lactococcus lactis.

[0016] (2) The present invention also provides a method for constructing Lactococcus lactis with surface-modified neoantigen peptides, which is achieved by inserting tumor neoantigen peptides into the cell wall of Lactococcus lactis. The method specifically comprises: modifying the N-terminus of the tumor neoantigen peptide with a cell-penetrating peptide CPP sequence, and connecting the CPP sequence and the tumor neoantigen peptide sequence with a proteasome processing site; incubating the synthesized CPP-tumor neoantigen peptide with Lactococcus lactis in physiological saline at room temperature for about 2 hours to allow the Lactococcus lactis to bind to the CPP-tumor neoantigen peptide; and then centrifuging at 5000 rpm for 10 minutes to remove unbound free peptide in the supernatant, and the precipitate is Lactococcus lactis with the CPP-tumor neoantigen peptide inserted.

[0017] Furthermore, the cell-penetrating peptide CPP sequence is: GRKKRRQRRRPQ (SEQ ID NO: 1); the proteasome processing site sequence is: RWEKI (SEQ ID NO: 2). Cell-penetrating peptides (CPPs) are short peptides that promote cellular absorption / uptake of various molecular components (ranging from nanosized particles to small chemical molecules and large DNA fragments), and are bound to the peptide through chemical linkage via covalent bonds or through non-covalent interactions. The function of CPPs is to transport cargo into cells, a process that usually occurs through endocytosis. The CPP sequence used in the present invention contains a high relative abundance of positively charged amino acids (such as lysine or arginine), which can be firmly bound to the surface of Lactococcus lactis through the lipid bilayer and the attraction of positive and negative charges.

[0018] (3) The present invention also provides a method for constructing Lactococcus lactis with surface-modified neoantigen peptides, in which tumor neoantigen peptides are attached to the surface of Lactococcus lactis by a biomineralization method, specifically comprising: centrifuging and washing the Lactococcus lactis culture fluid, removing excess culture medium, and resuspending with ultrapure water to obtain a Lactococcus lactis solution; adding the Lactococcus lactis solution to a dimethylimidazole aqueous solution and stirring, then slowly pouring the neoantigen peptide solution (Ag) and a cationic solution into the above solution, stirring, and finally centrifuging and discarding the supernatant, and washing the precipitate with ultrapure water to obtain Lactis@MOF / Ag.

[0019] Furthermore, the cationic solution includes Zn 2+ 、Mn 2+ or Mg 2+ of aqueous solution.

[0020] Furthermore, the stirring method includes one or more of magnetic stirring, hand shaking, ultrasonic stirring, shaking table stirring, oscillator stirring, and static diffusion.

[0021] (IV) The present invention also provides the use of the above-mentioned Lactococcus lactis with surface modified neoantigen peptides in the preparation of tumor vaccines.

[0022] Furthermore, the injection methods of the Lactococcus lactis surface-modified with the neoantigen peptide include but are not limited to intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, and lymph node injection.

[0023] Furthermore, the tumor includes but is not limited to colon cancer, gastric cancer, breast cancer, liver cancer, lung cancer and cervical cancer tumors. Beneficial effects

[0024] (1) The present invention enables clinical use of probiotic lactic acid bacteria present in the human body by non-genetically linking or inserting / passing patient-recognized tumor-specific peptides into lactic acid bacteria. Simply by coating the lactic acid bacteria with a new set of tumor-specific peptides from the patient, the lactic acid bacteria can quickly respond to changes in the patient's tumor antigens presented on MHC-I.

[0025] (2) The lactic acid bacteria of the present invention are flexibly genetically modified, which is more conducive to their anti-tumor immune adjuvant effect. Moreover, the lactic acid bacteria used only need to undergo a strict quality control and approval stage once, thus saving time and money compared with the existing technology. Compared with the continuous construction of bacteria expressing different new antigens, which requires quality inspection each time, this platform is simpler, faster, more universal and more practical.

[0026] (3) The present invention provides a method for preparing Lactococcus lactis with a surface modified neoantigen peptide, which uses only water as a solvent without the need for an organic solvent, is environmentally friendly and safe, and the sample obtained by the synthesis method of the present invention has the advantage of a high peptide loading rate.

[0027] (4) After the wild-type or genetically engineered Lactococcus lactis of the present invention binds to the tumor neoantigen peptide, it increases the uptake and presentation of the neoantigen peptide by antigen-presenting cells such as dendritic cells (DC), stimulates the activation of DC cells, further triggers an immune response, and activates tumor-specific cytotoxic CD8 + The immune response of T cells achieves the effect of precise and lasting tumor killing with good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG1 is a flow cytometry test result of FOLactis combined with different amounts of CPP-OVA;

[0029] FIG2 is a statistical graph showing the flow cytometry results of FOLactis combined with different amounts of CPP-OVA;

[0030] FIG3 is a transmission electron microscopic diagram of FOLactis before and after binding to 100 μg of CPP-OVA;

[0031] FIG4 is a schematic diagram of confocal fluorescence microscopy of FOLactis combined with 100 μg of CPP-OVA;

[0032] FIG5 is a schematic diagram showing the results of high performance liquid chromatography (HPLC) of FOLactis combined with 100 μg, 200 μg, or 300 μg of CPP-OVA;

[0033] FIG6 is a schematic diagram of the mineralization method for connecting FOLactis and neoantigen peptides;

[0034] FIG7 is a schematic diagram showing the results of high performance liquid chromatography (HPLC) of FOLactis combined with 5 mg, 10 mg, and 20 mg of MAGE-A4 using the model antigen peptide MAGE-A4 as an example, ZnSO4·7H2O as a cation source, and a mineralization method;

[0035] FIG8 is a schematic diagram showing the results of confocal microscopy observation of BMDCs uptake of OVA, CPP-OVA, FOLactis+OVA, and OVA-FOLactis;

[0036] FIG9 is a statistical graph showing the flow cytometry results of MHC-I presentation and maturation of OVA peptide by BMDCs under different stimulation conditions;

[0037] FIG10 is a graph showing the tumor inhibition effects of different preparations after injection into the lymphatic region in a mouse melanoma B16F10-OVA model;

[0038] FIG11 is a graph showing the survival curves of mice after lymphatic injection of different preparations in a mouse melanoma B16F10-OVA model;

[0039] FIG12 is a graph showing the flow cytometry results for verifying the number of specific T cells in the draining lymph nodes and tumors of mice after injection of different preparations into the lymphatic regions in a mouse melanoma B16F10-OVA model;

[0040] FIG13 is a statistical graph showing the flow cytometry results of the number of specific T cells in the draining lymph nodes and tumors of mice after lymphatic regional injection of different preparations in the mouse melanoma B16F10-OVA model;

[0041] FIG14 is a diagram showing the HE immunohistochemistry results of important mouse organs after injection of different preparations into the lymphatic region in a mouse melanoma B16F10-OVA model. DETAILED DESCRIPTION

[0042] To make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the relevant drawings. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The implementation conditions not specified in the embodiments are generally those in routine experiments, and the reagents or instruments used without specifying the manufacturer are all conventional products that can be purchased commercially.

[0043] Example 1

[0044] The method of inserting the tumor neoantigen peptide into the cell wall of Lactococcus lactis achieves the surface modification of the tumor neoantigen peptide of Lactococcus lactis, specifically comprising:

[0045] (1) The N-terminus of the tumor neoantigen peptide (OVA) was modified with a cell-penetrating peptide CPP sequence, and a proteasome processing site was used to connect the CPP sequence and the tumor neoantigen peptide sequence to construct CPP-OVA. The sequence of CPP-OVA is GRKKRRQRRRPQRWEKISIINFEKL (SEQ ID NO: 3);

[0046] (2) 100-300 μg of synthesized CPP-OVA was mixed with 10 9 CFU FOLactis (recombinant Lactococcus lactis expressing Flt3L-OX40L fusion protein) was mixed and incubated in physiological saline at room temperature for about 2 hours to allow FOLactis to bind to CPP-OVA. The mixture was then centrifuged at 5000 rpm for 10 minutes to remove unbound free peptide in the supernatant. The precipitate was OVA-FOLactis.

[0047] Example 2

[0048] FOLactis was combined with Mage-A4 peptide via a biomineralization method to construct FOLactis@ZIF-8 / MA4, which includes:

[0049] (1) Synthesize the Mage-A4 peptide sequence AESLFREALSNKVDELAHFLLRKYRAKEL (SEQ ID NO: 4);

[0050] (2) 10 9CFU of FOLactis was added to 10 mL of 700 μmol / ml dimethylimidazole (MIM) aqueous solution and gently stirred for 10 minutes; 5 mg of Mage-A4 and 10 mL of 10 μmol / ml cationic solution (ZnSO4·7H2O solution) were slowly poured into the above solution and reacted for 20 minutes. The mixture was centrifuged at 5000 rpm for 10 minutes, the supernatant free peptide was discarded, and the precipitate was washed three times with ultrapure water to obtain FOLactis@ZIF-8 / MA4.

[0051] Example 3 HPLC method to detect the peptide ratio loaded by FOLactis

[0052] (1) Use HPLC to measure the standard curve of the model peptide.

[0053] (2) After FOLactis binds to the antigen peptide, centrifuge at 5000 rpm for 10 min, and measure the total amount of peptide in the supernatant by HPLC.

[0054] (3) Calculate the number of peptides bound to FOLactis. The amount of new antigen peptide loaded in the bacterial pellet = total amount of delivered peptide - supernatant peptide concentration × supernatant volume, and calculate the ratio.

[0055] FIG1 is a flow cytometry result diagram of FOLactis combined with different amounts of CPP-OVA, and FIG2 is a statistical diagram of the flow cytometry result diagram of FOLactis combined with different amounts of CPP-OVA.

[0056] Figure 3 is a schematic diagram of transmission electron microscopy of FOLactis before and after binding to 100 μg of CPP-OVA, Figure 4 is a schematic diagram of confocal fluorescence microscopy of FOLactis binding to 100 μg of CPP-OVA, and Figure 5 is a schematic diagram of the high performance liquid chromatography (HPLC) results of FOLactis binding to 100 μg, 200 μg, or 300 μg of CPP-OVA.

[0057] FIG7 is a graph showing the results of high performance liquid chromatography (HPLC) of FOLactis combined with 5 mg, 10 mg and 20 mg of Mage-A4.

[0058] As shown in Figures 1 to 5 and Figure 7 , the surface-modified neoantigenic peptides of Lactococcus lactis obtained by the two synthesis methods of the present invention both have the advantage of a high peptide loading rate.

[0059] Example 4 In vitro verification of the peptide presentation efficiency and activation efficiency of mouse bone marrow-derived cells (BMDCs)

[0060] (1) After C57BL / 6 mice were killed by cervical dislocation, they were soaked in alcohol and all femurs and tibias were surgically removed. Scissors and forceps were used to remove the muscle tissue around the bones as much as possible. The bones were moved to a clean bench and soaked in a sterile culture dish containing 70% alcohol for 2-5 minutes to disinfect and sterilize them. Then, they were washed twice with sterile NS (normal saline). The bones were moved to another new culture dish containing NS. The two ends of the bones were cut with scissors. Then, NS was extracted with a syringe. The needle was inserted into the bone marrow cavity from both ends of the bone. The bone marrow was repeatedly flushed into the culture dish until the bone turned completely white. The bone marrow suspension was collected and filtered with a 40μm filter to remove small fragments and muscle tissue. The filtrate was centrifuged at 1200rpm for 5 minutes and the supernatant was discarded. 2ml of red blood cell lysis buffer (about 3 times the volume) was added, the cells were resuspended, and incubated at room temperature for 3-5 minutes, up to 10 minutes. 10ml of NS was added to neutralize the effect of the lysis buffer, and then the filtrate was centrifuged at 1200rpm for 5 minutes and the supernatant was discarded. NS was washed once, and then the cells were resuspended in RPMI 1640 culture medium containing 10% FBS, so that mouse bone marrow cells BMDC were obtained.

[0061] (2) Adjust the above BMDC to 5*10 with 1640+10% FBS. 5 On day 0, cells were plated onto 24-well plates, with 1 ml of cells per well. RM GM-CSF 20 ng / ml and IL-4 10 ng / ml were added, and the cells were cultured in an incubator and sealed with adhesive tape to prevent liquid loss.

[0062] (3) On the second and fourth days, 3 / 4 of the medium was replaced with supplemented rm GM-CSF and rm IL-4.

[0063] (4) On the 6th day, gently pipette the culture medium to collect the suspended cells and loosely attached cells. Centrifuge at 1200 rpm for 5 min and discard the supernatant. Resuspend the cells in 1640 + 10% FBS and count them, then adjust the concentration to 1*10 6 cells / ml. rm GM-CSF (20 ng / ml) and IL-4 (10 ng / ml) were added and plated onto 6-well plates.

[0064] (5) On the 8th day, the suspended cells were collected and centrifuged at 1200 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in 1640 + 10% FBS to 1*10 6 Cells were plated in 24-well plates at 500 cells / ml. Eight groups were designed: NS, LSP, Lactis, FOLactis, OVA, CPP-OVA, FOLactis+OVA, and OVA-FOLactis. Three wells were plated in each group and incubated for the desired time. Cultures were kept in an incubator and sealed with adhesive tape to prevent loss of fluid.

[0065] (6) After incubation, select mouse CD11c-FITC, CD80-APC, and CD86-PE antibodies and incubate in the dark for 30 minutes. Add 1 ml of NS to each tube, centrifuge at 1200 rpm for 5 minutes, discard the supernatant, repeat the washing once, and analyze the results by flow cytometry.

[0066] FIG8 is a schematic diagram showing the results of confocal microscopy observation of BMDCs uptake of OVA, CPP-OVA, FOLactis+OVA, and OVA-FOLactis.

[0067] FIG9 is a statistical graph showing the flow cytometry results of MHC-I presentation and maturation of OVA peptide by BMDCs under different stimulation conditions (NS, LPS, Lactis, OVA, CPP-OVA, FOLactis+OVA, and OVA-FOLactis).

[0068] As shown in Figures 8 and 9, OVA-FOLactis can promote the MHC-I presentation of OVA peptide by BMDC cells and also significantly stimulate the activation of BMDC cells.

[0069] Example 5 In vivo verification of the tumor-suppressing effect of in situ injection of OVA-FOLactis into the lymphatic region

[0070] (1) Mouse experimental procedure: Experimental groups: saline control group (A), CPP-OVA group (B), FOLactis group (C), OVA-FOLactis group (D), 8 mice in each group, a total of 32 mice.

[0071] (2) Mouse tumor model: B16-OVA melanoma cells were resuspended to 5*10 6 / ml, and then each mouse was subcutaneously inoculated with 100ul B16-OVA, i.e. 5*10 5 cells, and the tumor can grow to 80-100 mm in size in about a week. 3 . Treatment started after 3 days.

[0072] (3) Mouse immunization: After induction and culture of lactic acid bacteria with Nisin, centrifuge at 12000g / min for 5min, discard the supernatant and collect the bacteria. Wash the precipitate twice with PBS buffer solution, incubate with CPP-OVA at room temperature for 2 hours, and discard the supernatant. Resuspend and adjust the concentration to 1×10 9 The above experimental and control groups were injected into the lymph node tumors on days 3, 5, 7, 9 and 11, for a total of 3 times, with 100 μL per mouse each time.

[0073] (4) Use a vernier caliper to record the tumor size 3-4 times a week. The tumor volume is length*width*width / 2. When the tumor volume of the mouse is greater than 1500mm3 The mice were euthanized at 4 ℃ and 1 ℃, and their weight was recorded using a scale to assess their health.

[0074] The tumor inhibition and survival results are shown in Figures 10 and 11 , indicating that injection of OVA-FOLactis into the lymphatic region can significantly inhibit tumor growth and prolong the survival time of mice.

[0075] Example 6 Flow cytometry detection of the number of specific T cells in mice

[0076] (1) Preparation of single cell suspension:

[0077] Lymph nodes: Three blood vessels converge under the skin of the mouse groin, where the inguinal lymph nodes are buried. Remove the lymph nodes and soak them in saline until ready for use. Gently grind the lymph nodes using the frosted surfaces of two glass slides against each other in a 6 cm diameter culture dish. Gently blow cells attached to the glass slides into the culture dish with saline. Filter small debris and fibrous tissue using a 40 μm filter. Centrifuge at 1200 rpm for 5 minutes and discard the supernatant. Resuspend the cells in saline at a concentration of 0.5-1×10 6 / ml, ready for flow staining.

[0078] Tumor: After the mouse tumor tissue is removed, it is soaked in physiological saline for later use. In a 6 cm diameter culture dish, the tumor tissue is cut into small pieces and placed in each well of a 24-well plate. Then, 1 ml of PRMI1640 culture medium containing 1 mg / ml collagenase IV is added and incubated at 37°C for 3-4 hours. After observing under a microscope that a large number of cells have decomposed from the tissue block, the culture medium is aspirated and the small fragments and fibrous tissue are filtered out with a 40 μm filter. Centrifuge at 1200 rpm for 5 minutes and discard the supernatant. Resuspend the cells with 1 ml of red blood cell lysis buffer and add 10 ml of physiological saline after 5-6 minutes to stop the lysis. Centrifuge at 1200 rpm for 5 minutes and discard the supernatant. Resuspend 1×10 cells in PRMI1640 culture medium containing 10% fetal bovine serum. 6 / ml, and let the cells adhere to the wall of 24-well plates overnight. The next day, gently shake to remove the supernatant, centrifuge at 1200 rpm for 5 minutes, and discard the supernatant. Resuspend the cells in saline at 0.5-1×10 6 / ml, ready for flow staining.

[0079] (2) Cell membrane surface antigen staining: After obtaining a single cell suspension sample (cell concentration: 0.5-1×10 6 The samples were incubated with each treatment group (NS, CPP-OVA, FOLactis, OVA-FOLactis) in the dark for 20-30 minutes, washed with 1 ml of PBS, centrifuged at 1200 rpm for 5 minutes, and the supernatant discarded. Vortex to mix thoroughly before loading onto the microcentrifuge.

[0080] Figure 12 shows flow cytometry results for the number of specific T cells in the draining lymph nodes and tumors of mice following regional lymph node injection of different formulations in the B16F10-OVA mouse melanoma model. Figure 13 shows flow cytometry results for the number of specific T cells in the draining lymph nodes and tumors of mice following regional lymph node injection of different formulations in the B16F10-OVA mouse melanoma model. Figure 12 demonstrates that OVA-FOLactis can promote the proliferation of specific T cells in mice.

[0081] Example 7 Paraffin Sections and HE Staining

[0082] The important organs (heart, liver, spleen, lung and kidney) of mice were subjected to paraffin sectioning and HE staining for safety testing.

[0083] (1) Preparation of paraffin sections: Dehydrate with ethanol gradient in a fully automatic tissue dehydrator, and then make the tissue transparent. Soak in wax overnight in a 60°C oven. Use ophthalmic forceps to take out the tissue block that has been soaked overnight, and place the tissue horizontally in an embedding box with the cross section facing up. Drop 60°C liquid paraffin into the embedding box to completely cover the tissue and avoid bubbles. After embedding, place the embedding box on a freezing table for rapid cooling and store at room temperature for later use. Before slicing, freeze the wax block in a 4°C refrigerator and trim it to the largest surface of the tissue. Cut the central part into sections with a thickness of 5μm, and place the tissue sections in 45°C warm water for rinsing.

[0084] (2) HE staining: Place the baked paraffin sections in an automatic tissue stainer for dewaxing and hydration, then place them in hematoxylin solution for staining for 5 minutes, rinse with running water for 3 minutes, differentiate with 1% hydrochloric acid alcohol for 30 seconds, rinse with running water for 1 minute, return to blue with dilute ammonia for tens of seconds, and rinse with running water for 1 minute. Place them in eosin solution for staining for 2 minutes, and rinse with running water for 3 minutes. After dehydration and transparency according to the steps, use neutral resin to seal the sections, air dry, and store at room temperature. The sections are observed and photographed under an optical microscope.

[0085] Figure 14 shows the HE immunohistochemistry results of key mouse organs after lymph node injection of different formulations in a mouse melanoma B16F10-OVA model. As shown in Figure 14, all formulations were safe and did not damage key mouse organs.

[0086] The above are only preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the scope of protection of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications without departing from the principle of the present invention should be regarded as the scope of protection of the present invention.

[0087] Sequence Listing

Claims

1. A Lactococcus lactis surface modified with a new antigen peptide, characterized in that: The Lactococcus lactis contains at least one tumor neoantigenic peptide, which is non-genetically linked to the surface of the Lactococcus lactis, or the tumor neoantigenic peptide is inserted into / through the cell wall of the Lactococcus lactis.

2. The surface-modified neoantigenic peptide Lactococcus lactis according to claim 1, characterized in that: The tumor neoantigen peptide is a peptide consisting of 8-25 amino acids.

3. The surface-modified neoantigenic peptide Lactococcus lactis according to claim 1, characterized in that: The tumor neoantigen peptide is an MHC-I restricted and / or MHC-II restricted antigen peptide.

4. The surface-modified neoantigenic peptide Lactococcus lactis according to claim 1, characterized in that: The tumor neoantigen peptide is a fusion peptide comprising multiple different antigens.

5. The surface-modified neoantigenic peptide Lactococcus lactis according to claim 1, characterized in that: The lactococcus lactis is wild-type lactococcus lactis or genetically engineered lactococcus lactis.

6. A method for constructing Lactococcus lactis with a surface modified new antigen peptide, characterized in that: The method of inserting the tumor neoantigen peptide into the cell wall of Lactococcus lactis realizes the surface modification of Lactococcus lactis with the tumor neoantigen peptide, which specifically includes: The N-terminus of the tumor neoantigen peptide is modified with a cell-penetrating peptide CPP sequence, and a proteasome processing site is used to connect the CPP sequence and the tumor neoantigen peptide sequence; The synthesized CPP-tumor neoantigen peptide is incubated with Lactococcus lactis in physiological saline at room temperature to allow the Lactococcus lactis to bind to the CPP-tumor neoantigen peptide; then the unbound free peptide in the supernatant is removed by centrifugation, and the precipitate is the Lactococcus lactis into which the CPP-tumor neoantigen peptide has been inserted.

7. The method for constructing Lactococcus lactis with surface modified new antigen peptides according to claim 6, characterized in that: The CPP sequence is shown in SEQ ID NO: 1; The proteasome processing site sequence is shown in SEQ ID NO:

2.

8. A method for constructing Lactococcus lactis with a surface modified new antigen peptide, characterized in that: The tumor neoantigen peptides were attached to the surface of Lactococcus lactis by biomineralization method, including: The Lactococcus lactis culture solution is centrifuged and washed to remove excess culture medium, and then resuspended in ultrapure water to obtain a Lactococcus lactis solution; The Lactococcus lactis solution was added to the dimethylimidazole aqueous solution and stirred, and then the new antigen peptide solution and the cationic solution were slowly poured into the above solution and stirred, and finally centrifuged and the supernatant was discarded, and the precipitate was washed with ultrapure water to obtain Lactis@MOF / Ag.

9. The method for constructing Lactococcus lactis with surface modified new antigen peptides according to claim 8, characterized in that: The cationic solution includes Zn 2+ , Mn 2+ or Mg 2+ of aqueous solution.

10. Use of Lactococcus lactis with surface modified neoantigenic peptides according to any one of claims 1 to 6 in the preparation of tumor vaccines.

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