Surface-drug-loaded multifunctional engineering probiotics with antigen capturing capacity, preparation method and application of surface-drug-loaded multifunctional engineering probiotics

Abstract

The invention discloses a surface drug-loaded multifunctional engineering probiotic with antigen capture capability, a preparation method and application, the strain takes non-pathogenic probiotic as an original strain, after the engineering probiotic expressing protein stimulating immune cell development or differentiation is constructed, the surface of the engineering probiotic is modified with maleimide functional groups and drug-loaded nano-particles, and the surface drug-loaded multifunctional engineering probiotic with antigen capture capability is obtained. Therefore, the compound is a multifunctional bacterium with antigen capture, drug delivery and anti-tumor effects at the same time, and the effect of effectively promoting antigen tumors and far-end tumors through multifunction synergism is achieved. Meanwhile, the drug-loaded nanoparticles loaded on the surface of the engineering probiotics can form a slow-release carrier, so that the onset time of the drug is prolonged, and the bioavailability of the drug is improved; the ICD effect is induced, and the conventional cytotoxic effect is converted into the immune activation effect, so that the systemic toxicity caused by the application of large-dose chemotherapeutic drugs is avoided, and the immune activation function of bacteria is retained. The engineering probiotic preparation process is simple and efficient, the whole process is synthesized in a water phase, and the safety is high.

Description

Technical Field

[0001] The present invention relates to tumor immunotherapy, and in particular to a surface-drug-loaded multifunctional engineered probiotic with antigen-capturing capability, a preparation method and applications thereof. Background Art

[0002] As an important branch of tumor immunotherapy, bacterial therapy has the core advantage of pathogen-associated molecular patterns (PAMPs) inherent in bacteria. These patterns can effectively trigger systemic anti-tumor immune responses by activating pattern recognition receptors (PRRs) on the surface of immune cells. Studies have shown that engineered bacteria that express proteins that promote immune cell differentiation can effectively inhibit tumor growth in mice. However, the complex immunosuppressive microenvironment and physical barriers of solid tumors severely restrict the therapeutic effect of a single engineered bacterium, especially for large tumors, which are difficult to completely eliminate.

[0003] In recent years, the mechanism of chemotherapy-induced immunogenic cell death (ICD) has provided new insights into combination therapy: chemotherapeutic drugs such as oxaliplatin can reshape the tumor immune microenvironment and promote dendritic cell maturation and T cell infiltration by triggering the exposure of calreticulin (CRT) and the release of damage-associated molecular patterns (DAMPs) such as high-mobility group protein B1 (HMGB1) and adenosine triphosphate (ATP). However, clinical data show that the ICD effect induced by chemotherapy alone has bottlenecks such as low antigen presentation efficiency and short duration of immune response, which is closely related to insufficient capture of antigen-presenting cells (APCs) due to rapid degradation of tumor antigens. The mechanism is that highly expressed matrix metalloproteinases (MMPs) and nucleases in the tumor microenvironment can degrade more than 80% of tumor-associated antigens (TAAs) within 6-12 hours, while it takes at least 24 hours for APCs to complete the antigen uptake, processing, and presentation process, resulting in a delay in the initiation of the immune response.

[0004] To address this critical spatiotemporal mismatch, recent studies have shown that maleimide (MAL) groups can covalently bind to the thiol groups of tumor antigens, a process known as Michael addition reaction, to form a stable complex that effectively prolongs the residence time of tumor antigens. This antigen fixation strategy not only circumvents the antigen dilution effect caused by systemic administration, but also effectively improves the efficiency of antigen uptake by immune cells. However, multifunctional engineered bacteria with the ability to simultaneously activate immune cells, deliver chemotherapy drugs, and capture tumor antigens remain to be developed. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a new type of multifunctional engineered probiotics with antigen capture ability, drug loading ability, and anti-tumor ability and a preparation method thereof; the second purpose is to provide applications of the multifunctional engineered probiotics.

[0006] Technical solution: The multifunctional engineered probiotic with antigen capture ability and drug loading on the surface according to the present invention is characterized in that the engineered probiotic expresses a protein that stimulates the development or differentiation of immune cells, and is surface-modified with maleimide functional groups and drug-loaded nanoparticles;

[0007] Among them, the protein that stimulates the development or differentiation of immune cells is any one or more of FIt3L, OX40L, IL-2, GM-CSF, RANKL, or a fusion protein of multiple proteins; the drug-loaded nanoparticles are any one of metal-organic frameworks loaded with drugs, gold nanoparticles, and mesoporous silica.

[0008] Preferably, the original strain of the engineered probiotic is any non-pathogenic strain of Lactococcus, Lactobacillus, Streptococcus, Pediococcus, or Leuconostoc.

[0009] Preferably, the protein that stimulates the development and differentiation of immune cells is the FIt3L-PVGLIG-OX40L fusion protein.

[0010] Preferably, the drug-loaded nanoparticles are any one of magnesium-based metal frameworks or zeolitic imidazolate frameworks.

[0011] Preferably, the drug loaded on the drug-loaded nanoparticles is any one of platinum drugs, DNA alkylating agents, antimetabolic drugs, antitumor antibiotics, plant alkaloids, and topoisomerase inhibitors.

[0012] Preferably, the drug loaded on the drug-loaded nanoparticles is any one of cisplatin, carboplatin, nedaplatin, oxaliplatin, and lobaplatin.

[0013] The preparation method of the multifunctional engineered probiotic with antigen capture ability and drug loading on the surface according to the present invention includes:

[0014] (1) Construct an engineered probiotic that expresses a protein that stimulates the development or differentiation of immune cells;

[0015] (2) In the engineered probiotic solution obtained in step 1, add a maleimide-functionalized amphiphilic polymer conjugate solution, mix and incubate, centrifuge and resuspend to obtain an engineered probiotic solution surface-modified with maleimide functional groups;

[0016] (3) In the engineered probiotic solution obtained in step 2, add an imidazole ligand solution, stir and mix and incubate, centrifuge and resuspend;

[0017] (4) In the engineered probiotic solution obtained in step 3, add a chemotherapeutic drug, mix evenly and then add a metal salt, stir and mix and incubate, centrifuge and discard the supernatant to obtain a multifunctional engineered probiotic with antigen capture ability and drug loading on the surface.

[0018] Preferably, in step 1, the concentration of the engineered probiotic expressing the protein stimulating the development or differentiation of immune cells is 0.5 - 1.5×10 9 CFU / mL;

[0019] Preferably, in step 2, the maleimide-functionalized amphiphilic polymer conjugate is any one of DSPE-PEG-MAL, DSPC-PEG-MAL, DSPG-PEG-MAL, DOPE-PEG-MAL, with a concentration of 10 - 30 mg / mL, the solvent is dimethyl sulfoxide or absolute ethanol, the addition amount is 2 - 8% of the volume of the bacterial liquid, and the concentration of the engineered probiotic liquid with surface-modified maleimide functional groups obtained by resuspension is 0.5 - 1.5×10 10 CFU / mL;

[0020] Preferably, in step 3, the imidazole ligand is 2-methylimidazole, with a concentration of 60 - 80 mM, the addition amount is 50 - 150 times the volume of the bacterial liquid, and after centrifugation and discarding the supernatant, it is resuspended with physiological saline at 40 - 60 times the initial volume of the bacterial liquid;

[0021] Preferably, in step 4, the chemotherapeutic drug is any one of cisplatin, carboplatin, nedaplatin, oxaliplatin, lobaplatin, with a final concentration of 0.5 - 1.5 μg / μL, and the metal salt is any one of magnesium sulfate, magnesium chloride, zinc nitrate, zinc acetate, cobalt chloride, with a final concentration of 0.5 - 1.5 M.

[0022] Application of the multifunctional engineered probiotic with antigen capture ability and surface drug loading in the preparation of anti-tumor drugs.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0024] 1. This engineered probiotic simultaneously has the functions of antigen capture, drug delivery, and tumor treatment, and can effectively improve the effects on primary tumors and distal tumors;

[0025] 2. The drug-loaded nanoparticles loaded on the surface of this engineered probiotic can form a sustained-release drug carrier, prolong the drug onset time, and improve the drug bioavailability;

[0026] 3. This engineered probiotic can induce the ICD effect of tumor cells, transform the conventional cytotoxic effect into an immune activation effect, avoid the systemic toxicity of high-dose chemotherapy drugs, and retain the immune activation function of bacteria;

[0027] 4. The preparation process of this engineered probiotic is simple and efficient, the whole process is synthesized in an aqueous phase, and it has high safety. Description of the Drawings

[0028] Figure 1Schematic diagram for the preparation of the multifunctional engineered probiotic FOLactis-MAL-MOF / O with antigen capture ability and drug loading on the surface;

[0029] Figure 2 Characterization results of FOLactis-MAL. Among them, a is the Fourier transform infrared spectroscopy (FTIR) spectrum of FOLactis before and after MAL modification, b is the Zeta potential diagram of FOLactis-MAL and FOLactis-MAL+peptide, and c is the particle size diagram of FOLactis-MAL and FOLactis-MAL+peptide;

[0030] Figure 3 OXA release curve of FOLactis-MAL-MOF / O;

[0031] Figure 4 Antigen capture function results of FOLactis-MAL-MOF / O. Among them, a is the capture efficiency and average fluorescence intensity diagram of cy5-peptide detected by flow cytometry, and b is the capture function result diagram of CT26 polypeptide mixture of FOLactis-MAL-MOF / O detected by BCA;

[0032] Figure 5 Confocal imaging diagram of FOLactis-MAL-MOF / O capturing tumor antigens;

[0033] Figure 6 Internalization results of DC cells with FOLactis-MAL-MOF / O. Among them, the left figure is the detection result diagram by flow cytometry, and the right figure is the confocal imaging diagram;

[0034] Figure 7 Function verification diagram of FOLactis-MAL-MOF / O promoting DC cell activation;

[0035] Figure 8 Evaluation diagram of in vivo anti-tumor effect and abscopal effect of FOLactis-MAL-MOF / O. Among them, a is the change diagram of the primary tumor volume of mice after treatment, b is the change diagram of the distal tumor volume of mice after treatment, c is the statistical chart of the survival period of mice after treatment, and d is the change diagram of the body weight of mice after treatment. Detailed implementation manners

[0036] The technical solutions of the present invention will be further described below.

[0037] Example 1: Preparation and characterization of the multifunctional engineered probiotic FOLactis-MAL-MOF / O with antigen capture ability and drug loading on the surface

[0038] 1. Preparation of FOLactis-MAL-MOF / O

[0039] (1) According to the method described in Zhu J, Ke Y, Liu Q, et al. Engineered Lactococcus lactis secreting Flt3L and OX40 ligand for in situ vaccination-based cancer immunotherapy. Nat Commun. 2022;13(1):7466., using Lactococcus lactis as the starting strain, an engineered probiotic FOLactis was constructed. Among them, the protein stimulating the development and differentiation of immune cells is the FIt3L-PVGLIG-OX40L fusion protein, and its amino acid sequence is shown in SEQ ID No: 1;

[0040] (2) Prepare a solution of distearoyl phosphatidylethanolamine-methoxypolyethylene glycol-maleimide (DSPE-PEG-MAL) with a concentration of 20 mg / mL, and the solvent is dimethyl sulfoxide (DMSO);

[0041] (3) Add 50 μL of the solution obtained in step (2) to 1 mL of the FOLactis bacterial solution obtained in step (1) with a concentration of 1.0×10 10 CFU / mL. After mixing and incubating at 25 °C for 16 h, centrifuge at 5000 rpm for 10 min. Discard the supernatant and resuspend to obtain an engineered probiotic solution with maleimide functional groups modified on the surface;

[0042] (4) Prepare a 2-methylimidazole-saline solution with a concentration of 70 mM;

[0043] (5) Add 10 mL of the solution obtained in step (4) to 100 μL of the engineered probiotic solution obtained in step (3) with a concentration of 1.0×10 10 CFU / mL. After stirring and mixing at 25 °C for 10 min, centrifuge at 5000 rpm for 10 min. Discard the supernatant and resuspend with 5 mL of saline;

[0044] (6) Add oxaliplatin with a final concentration of 1 μg / μL to the engineered probiotic solution obtained in step (5), mix evenly, then add magnesium sulfate with a final concentration of 1 M. After stirring and mixing at 25 °C for 20 min, centrifuge at 5000 rpm for 10 min. Discard the supernatant to obtain a surface drug-loaded multifunctional engineered probiotic with antigen capture ability.

[0045] The preparation process of FOLactis-MAL-MOF / O is as Figure 1 shown.

[0046] 2. Characterization of FOLactis-MAL-MOF / O

[0047] FOLactis-MAL-MOF / O was characterized by Fourier transform infrared spectroscopy and dynamic light scattering nanoparticle size analyzer.

[0048] The results are as Figure 2 shown, where Figure 2 a is the Fourier transform infrared spectrogram of FOLactis before and after MAL modification. It can be seen that compared with unmodified FOLactis, in the FT-IR spectrogram of FOLactis-MAL, the antisymmetric stretching vibration peak of methyl, the antisymmetric stretching vibration peak of methylene and the symmetric stretching vibration peak of methylene appear at 2957.09 cm -1 , 2918.21 cm -1 and 2850.60 cm -1 respectively. The in-plane rocking vibration peak of CH2 appears at 717.75 cm -1 , all of which are the infrared spectral characteristics of DSPE-PEG-MAL, indicating the successful modification of maleimide groups.

[0049] The Zeta potential changes of FOLactis-MAL and the mixture of FOLactis-MAL + CT26 polypeptide (FOLactis-MAL + peptide) are as Figure 2 shown in b. After FOLactis was modified with maleimide groups, the surface potential increased from -10.5 mV to -8.83 mV. After incubation with CT26 neoantigen peptide, the surface potential further increased to -7.43 mV; Figure 2 c is the particle size change of FOLactis-MAL and FOLactis-MAL + peptide. It can be seen that after FOLactis was modified with maleimide groups, the particle size increased from 1573 nm to 1762 nm. After incubation with CT26 neoantigen peptide, the particle size further increased to 1993 nm. The changes in Zeta potential and particle size prove the successful modification of MAL and its ability to effectively capture polypeptides.

[0050] Among them, the preparation method of CT26 polypeptide mixture is as follows:

[0051] (1) CT26 mouse colon cancer cells were cultured in a cell culture dish for 48 hours, rinsed 3 times with PBS, and then cultured in serum-free medium containing 10 μg / mL OXA for another 48 hours;

[0052] (2) The supernatant was centrifuged at 13000 rpm for 20 minutes at 4 °C, and the supernatant was collected to obtain the polypeptide mixture. The protein concentration was determined by BCA assay, which is the CT26 polypeptide mixture.

[0053] 3. Release of OXA Loaded in FOLactis-MAL-MOF / O

[0054] (1) Prepare FOLactis-MAL-MOF / O according to the aforementioned method;

[0055] (2) Resuspend with normal saline, take 1×10 9 bacteria, place at room temperature, and centrifuge at 3 h, 6 h, 12 h, 24 h, 36 h, and 48 h respectively, then take the supernatant;

[0056] (3) Detect the concentration of OXA in the supernatant by HPLC method: on a C18 reverse-phase column (4.6 mm×250 mm), the injection volume is 10 μL, the mobile phase is acetonitrile: water = 75:25 (v / v), the flow rate is 1 mL / min for isocratic elution, the detection wavelength is 240 nm, calculate the release curve of OXA, and the results are as Figure 3 shown.

[0057] Example 2: Protein Antigen Capture Ability of FOLactis-MAL-MOF / O

[0058] 1. Cy5-labeled Peptide (Cy5-peptide) as Protein Antigen

[0059] (1) Take 100 μL of FOLactis or FOLactis-MAL-MOF / O with a concentration of 1×10 8 CFU / mL prepared in Example 1 and mix with 100 μL of Cy5-peptide with a concentration of 2 mg / mL. Use FOLactis or FOLactis-MAL-MOF / O without adding Cy5-peptide as a control. Make up all samples to 600 μL with PBS solution, incubate at room temperature for 1 day, centrifuge at 5000 rpm for 10 minutes, discard the supernatant, and obtain FOLactis or FOLactis-MAL-MOF / O that captures Cy5-peptide;

[0060] (4) Analyze the change in fluorescence intensity by flow cytometry.

[0061] 2. CT26 Polypeptide Mixture (CT26 cell lysate) as Protein Antigen

[0062] (1) Culture CT26 mouse colon cancer cells in a cell culture dish for 48 hours, rinse 3 times with PBS, and add serum-free medium containing 10 μg / mL OXA and culture for another 48 hours;

[0063] (2) Centrifuge the supernatant at 13,000 rpm for 20 minutes at 4°C, collect the supernatant to obtain a polypeptide mixture, and determine the protein concentration by the BCA assay. Dilute it to obtain a 2 mg / mL CT26 cell lysate;

[0064] (3) Take 100 μL each of FOLactis or FOLactis-MAL-MOF / O at a concentration of 1×10 9 CFU / mL and mix with 200 μL of CT26 neoantigen peptide at a concentration of 2 mg / mL. Use FOLactis or FOLactis-MAL-MOF / O without adding CT26 neoantigen peptide as a control. Make up the control samples to 200 μL with PBS solution and incubate at room temperature for 1 day;

[0065] (4) After incubation, wash 3 times with PBS, centrifuge at 5000 rpm for 10 min, and resuspend in 200 μL of PBS. Determine the mass of the captured protein by the BCA assay.

[0066] 3. Fluorescence Observation of Cy5-Labeled Peptide (Cy5-peptide) as a Protein Antigen

[0067] (1) Mix DIO dye at a concentration of 10 mM with FOLactis at a volume ratio of 1:100, let it stand at 30°C for 30 min, then centrifuge at 5000 rpm for 10 min, repeat the washing 3 times, and discard the supernatant to obtain DIO + -FOLactis;

[0068] (2) Under a light-shielded environment, use DIO + -FOLactis and prepare DIO + -FOLactis-MAL and DIO + -FOLactis-MAL-MOF / O according to the procedure described in Example 1;

[0069] (3) Take 100 μL each of DIO 8 -FOLactis or DIO + -FOLactis-MAL or DIO + -FOLactis-MAL-MOF / O at a concentration of 1×10 + CFU / mL and mix with 100 μL of Cy5-peptide at a concentration of 2 mg / mL. Make up all samples to 600 μL with PBS solution, incubate together at room temperature for 1 day, and centrifuge at 5000 rpm for 10 minutes to obtain DIO + -FOLactis or DIO + -FOLactis-MAL or DIO+ -FOLactis-MAL-MOF / O;

[0070] (4) Observe the fluorescence by confocal microscopy.

[0071] The results are as Figure 4 , where Figure 4 The flow cytometry detection of the capture efficiency and average fluorescence intensity of cy5-peptide shown in a both reflect that FOLactis-MAL-MOF / O has better protein antigen capture ability than FOLactis; Figure 4 The BCA results shown in b verify this. From Figure 5 the confocal microscopy results shown, it can be seen that the yellow fluorescence intensity in the Merge result of DIO + -FOLactis-MAL-MOF / O is higher than that in the DIO + -FOLactis group, that is, FOLactis-MAL-MOF / O has better protein antigen capture ability.

[0072] Example 3: In vitro immunomodulatory effect of FOLactis-MAL-MOF / O

[0073] 1. Internalization of FOLactis-MAL-MOF / O by DC cells

[0074] 1.1 Detection of the internalization of FOLactis-MAL-MOF / O by DC cells by flow cytometry

[0075] (1) After preparing CT26 cell lysate according to the steps described in Example 2, add Cy5 dye with a concentration of 10 mM to the CT26 cell lysate mixture at a volume ratio of 1:60 to prepare Cy5-cell lysate;

[0076] (2) Prepare FOLactis-MAL-ZIF / O according to the steps described in Example 1. Take 100 μL of FOLactis, FOLactis-MAL or FOLactis-MAL-ZIF / O with a concentration of 1×10 9 CFU / mL and mix with 200 μL of Cy5-cell lysate with a concentration of 2 mg / mL, incubate at room temperature for 1 day, centrifuge and discard the supernatant to obtain FOLactis-MAL-ZIF / O that captures Cy5-cell lysate; that is, Cy5-FOLactis-MAL-ZIF / O;

[0077] (3) Mix the FOLactis-MAL-ZIF / O that captured Cy5-cell lysate obtained in step 2 with mouse BMDC cells at a cell number ratio of 50:1, co-incubate at 37 °C for 2 h, then discard the supernatant to remove free Cy5-FOLactis-MAL-ZIF / O; detect the proportion of Cy5+ BMDC (i.e., BMDC that captured Cy5-FOLactis-MAL-ZIF / O) using a flow cytometer.

[0078] 1.2. Observation of the internalization of FOLactis-MAL-MOF / O by DC cells under a confocal microscope

[0079] (1) Mix the DIO dye at a concentration of 10 mM with FOLactis at a volume ratio of 1:100, let it stand at 30 °C for 30 min, then centrifuge at 5000 rpm for 10 min, repeat the washing 3 times, and discard the supernatant to obtain DIO + -FOLactis;

[0080] (2) Under a light-shielded environment, use DIO + -FOLactis to prepare DIO + -FOLactis-MAL-MOF / O through the process described in Example 1;

[0081] (3) After preparing the CT26 polypeptide mixture (CT26 cell lysate) according to the steps described in Example 2, mix DIO + -FOLactis-MAL-MOF / O with CT26 cell lysate, incubate at room temperature for 1 day, then centrifuge at 5000 rpm for 10 min to obtain DIO + -FOLactis-MAL-MOF / O that captured CT26 cell lysate;

[0082] (4) Mix the DIO + -FOLactis-MAL-MOF / O that captured CT26 cell lysate obtained in step 3 with mouse BMDC cells at a cell number ratio of 50:1, co-incubate at 37 °C for 2 h, then discard the supernatant to remove free DIO + -FOLactis-MAL-MOF / O;

[0083] (5) Add the DIL dye at a concentration of 10 mM to BMDC cells that internalized DIO + -FOLactis-MAL-MOF / O at a volume ratio of 1:100, incubate at 37 °C for 5 min, wash 3 times with physiological saline, and take pictures using a confocal microscope.

[0084] The results of flow cytometry are as Figure 6 shown in the left figure. Compared with the control group, more bacteria capturing Cy5-cell lysate were taken up by BMDCs in the FO-MAL and FO-MAL-MOF / O groups, that is, the proportion of Cy5 + BMDCs was higher. The fluorescence results are as Figure 6 shown in the right figure. The red color represents the BMDC cell membrane, and the green color represents the bacteria capturing cell lysate. Compared with the control group, more bacteria were taken up by BMDCs in the cytoplasm of the FO-MAL-MOF / O+cell lysate group.

[0085] 2. Immunostimulatory effect of FOLactis-MAL-MOF / O on DC cells

[0086] (1) Prepare CT26 cell lysate according to the steps described in Example 2, and use FOLactis or FOLactis-MAL or FOLactis-MAL-MOF / O to capture antigen proteins;

[0087] (2) Mouse BMDC cells were seeded into 96-well plates at a density of 2×10 6 cells / well and cultured for 48 h;

[0088] (3) Dilute the drugs using blank 1640 medium, including: a. physiological saline (control); b. CT26 cell lysate; c. FOLactis; d. FOLactis+CT26 cell lysate; e. FOLactis-MAL+CT26 cell lysate, f. FOLactis-MAL-MOF / O+CT26 cell lysate, where the final concentration of FOLactis in groups c-f is 2×10 8 cfu / mL;

[0089] (4) Add to each well at a number ratio of DC to FOLactis of 1:10, with 3 replicate wells in each group. After co-incubation at 37 °C for 24 h, the activation level of DC cells was detected by flow cytometry.

[0090] The results are as Figure 7 shown. DC cells can endocytose a large amount of FO-MAL-MOF / O in a short time. After the captured CT26 cell lysate is phagocytosed, it further stimulates DC activation. Compared with other groups, the activation effect of the FO-MAL-MOF / O+CT26 cell lysate group on DCs is significantly increased, verifying that the mode of combining antigen-capturing bacteria with chemotherapeutic drugs has a stronger immunostimulatory effect.

[0091] Example 4: Evaluation of the in vivo anti-tumor effect and abscopal effect of FOLactis-MAL-MOF / O

[0092] (1) Four- to five-week-old BALB / c mice were selected, and 2×10 6 CT26 cells were subcutaneously injected into the left lower abdomen of each mouse as the primary tumor. Three days later, the same dose of CT26 cells was subcutaneously injected into the right lower abdomen of the mouse as the distal tumor. When the primary tumor grew to 80±10 mm 3 , the mice were randomly divided into 4 groups, with 5 mice in each group;

[0093] (2) Intratumoral injections were performed on the 0th, 3rd, 10th, and 13th days after tumor formation. The 4 groups of mice were injected with: a. Normal saline (NS); b. 5×10 8 CFU of FOLactis; c. FOLactis-MOF / O containing 90 μg OXA and 5×10 8 CFU; d. FOLactis-MAL-MOF / O containing 90 μg OXA and 5×10 8 CFU. The tumor volume was measured every 2 days, and the calculation formula was 0.5×length×width 2 .

[0094] As Figure 8 shown in a, FOLactis-MAL-MOF / O could significantly inhibit the growth of the directly treated primary tumor, and the tumor volume was less than 300 mm 3 . As Figure 8 shown in b, FOLactis-MAL-MOF / O could also mobilize the systemic immune response to inhibit the untreated distal tumor. Compared with the FOLactis-MOF / O group, the FOLactis-MAL-MOF / O group had a stronger anti-tumor effect. At the same time, Figure 8 c showed that the administration of FOLactis-MAL-MOF / O could effectively improve the survival of mice and had no adverse effect on body weight ( Figure 8 d).

[0095] In summary, it is proved that the surface drug-loaded multifunctional engineered probiotic with antigen capture ability described in the present invention can kill tumor cells by releasing chemotherapeutic drugs, capture the released tumor antigens, prolong the retention time of tumor antigens, increase the antigen presentation efficiency, enhance the anti-tumor immune response, and improve the treatment effect.

Claims

1. A multifunctional engineered probiotic with antigen capture ability and surface drug loading, characterized in that, The engineered probiotics express proteins that stimulate the development or differentiation of immune cells, and are surface-modified with maleimide functional groups and drug-loaded nanoparticles; Among them, the protein that stimulates the development or differentiation of immune cells is any one or more of FIt3L, OX40L, IL-2, GM-CSF, RANKL, or a fusion protein of multiple proteins; the drug-loaded nanoparticles are any one of metal-organic frameworks loaded with drugs, gold nanoparticles, and mesoporous silica.

2. The multifunctional engineered probiotic with drug-loaded surface having antigen capture ability according to claim 1, wherein The original strain of the engineered probiotics is any one of non-pathogenic strains of the genus Lactococcus, Lactobacillus, Streptococcus, Pediococcus, and Leuconostoc.

3. The multifunctional engineered probiotic with drug-loaded surface having antigen capture ability according to claim 1, wherein The protein that stimulates the development and differentiation of immune cells is the FIt3L-PVGLIG-OX40L fusion protein.

4. The multifunctional engineered probiotic with drug-loaded surface having antigen capture ability according to claim 1, wherein The drug-loaded nanoparticles are any one of magnesium-based metal frameworks or zeolitic imidazolate frameworks.

5. The multifunctional engineered probiotic with drug-loaded surface having antigen capture ability according to claim 1, characterized in that, The drug loaded on the drug-loaded nanoparticles is any one of platinum drugs, DNA alkylating agents, antimetabolic drugs, antitumor antibiotics, plant alkaloids, and topoisomerase inhibitors.

6. The multifunctional engineered probiotic with drug-loaded surface having antigen capture ability according to claim 1, characterized in that, The drug loaded on the drug-loaded nanoparticles is any one of cisplatin, carboplatin, nedaplatin, oxaliplatin, and lobaplatin.

7. A method for preparing a multifunctional engineered probiotic with drug-loaded surface and antigen capture ability, characterized in that, Including: (1) Construct engineered probiotics that express proteins that stimulate the development or differentiation of immune cells; (2) In the engineered probiotic solution obtained in step 1, add a maleimide-functionalized amphiphilic polymer conjugate solution, mix and incubate, centrifuge and resuspend to obtain an engineered probiotic solution surface-modified with maleimide functional groups; (3) In the engineered probiotic solution obtained in step 2, add an imidazole ligand solution, stir, mix and incubate, centrifuge and resuspend; (4) In the engineered probiotic solution obtained in step 3, add a chemotherapeutic drug, mix evenly, add a metal salt, stir, mix and incubate, centrifuge and discard the supernatant to obtain a surface drug-loaded multifunctional engineered probiotic with antigen capture ability.

8. The preparation method of the multifunctional engineered probiotic with antigen capture ability according to claim 7, characterized in that, In step 2, the maleimide-functionalized amphiphilic polymer conjugate is any one of DSPE-PEG-MAL, DSPC-PEG-MAL, DSPG-PEG-MAL, and DOPE-PEG-MAL.

9. The preparation method of the multifunctional engineered probiotic with drug-loaded surface having antigen capture ability according to claim 7, characterized in that, In step 6, the chemotherapeutic drug is any one of cisplatin, carboplatin, nedaplatin, oxaliplatin, and lobaplatin; the metal salt is any one of magnesium sulfate, magnesium chloride, zinc nitrate, zinc acetate, and cobalt chloride.

10. Use of the surface drug-loaded multifunctional engineered probiotic with antigen capture ability according to claim 1 in the preparation of antitumor drugs.

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