An engineered bacteria and non-natural sugar composition for treating solid tumors and its application

Abstract

This invention discloses a composition for treating solid tumors. The composition comprises engineered bacteria and one or more azide-based non-natural sugars. The azide-based non-natural sugars are used to label tumor antigens, and the engineered bacteria are used to specifically bind to and deliver the labeled tumor antigens, activating dendritic cells. Application of this composition significantly improves the presentation efficiency of tumor antigens by dendritic cells, thereby enhancing the immune response and demonstrating significant therapeutic efficacy in the treatment of solid tumors.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology for the treatment of solid tumors, and specifically to a method for preparing and applying a composition for treating solid tumors. Background Technology

[0002] Cancer is one of the most serious public health problems of the 21st century, and its diversity, heterogeneity, and complexity pose enormous challenges to scientific research and medical practice. In 2022, nearly 20 million new cancer cases were diagnosed globally, with approximately 9.7 million deaths. The situation is expected to worsen, with new cancer cases projected to reach 35 million by 2050, highlighting the urgency of addressing this crisis. Utilizing the immune system to attack cancer has become an effective cancer treatment strategy. Antigens released by tumors after radiation therapy can serve as in situ vaccines, presented via dendritic cells (DCs) to trigger an adaptive immune response. Therefore, developing effective in situ vaccine strategies to enhance the immune activation level of tumor autoantigens for the treatment of solid tumors holds promise.

[0003] Live bacteria are considered immunostimulants that can synergistically treat tumors with radiotherapy. Bacterial pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), CpG DNA, and flagellin, can be recognized by Toll-like receptors (TLRs) on various immune cells, including neutrophils, natural killer cells, and macrophages. Activation of these receptors promotes the activation of effector immune cells, thereby enhancing their uptake of tumor antigens and improving their immune activation effect. Therefore, bacteria, as natural immune adjuvants, have a natural advantage in constructing bacterial vector-based in situ vaccine drugs. The key to this strategy lies in the bacteria's ability to efficiently bind to in situ tumor antigens with different charges and be taken up and presented by dendritic cells (DCs).

[0004] Metabolic labeling of unnatural sugars offers an ideal option for achieving this goal because it can label proteins by attaching unnatural chemical tags (chemical tags that do not exist in organisms). Unnatural sugars with azido groups can serve as competitive substrates for natural monosaccharides, integrating into the protein's glycan chain through the post-translational modification process of protein glycosylation, thereby achieving intracellular protein labeling. Therefore, tumor antigens can be metabolically labeled using unnatural sugars, enabling the binding of engineered bacteria to antigens and constructing bacteria-based in situ vaccines. Summary of the Invention

[0005] 1. The technical problem to be solved:

[0006] To address the aforementioned technical problems and achieve the goal of treating solid tumors, this invention utilizes engineered bacteria and a non-natural sugar composition to prepare a drug for treating solid tumors. This composition significantly improves the therapeutic effect on solid tumors by enhancing the immune response to tumor antigens.

[0007] 2. Technical Solution: This invention provides a composition for treating solid tumors, wherein one of its components, engineered bacteria, is prepared as follows: After culturing the bacteria in LB liquid medium for 12-16 h, they were incubated with cycloalkynes linked to NHS esters to obtain the engineered bacteria.

[0008] Preferably, the culture conditions are 30~37 ℃ and 150~220 rpm oscillation. More preferably, the culture conditions are 37 ℃ and 220 rpm oscillation.

[0009] Preferably, the incubation conditions are 22~37℃ for 15~30 min, more preferably 25℃ for 20 min.

[0010] Preferably, the bacteria are Gram-negative bacteria, including Salmonella and Escherichia coli.

[0011] Preferably, the cyclic alkyne linked to the NHS ester is one or more of DBCO, OCT, DIBO, TMTH, DIFO, BCN, and BARAC.

[0012] Preferably, the LB medium is formulated as follows: 0.5-1.5% sodium chloride (mass fraction) + 0.5-1.5% peptone + 0.25-0.75% yeast extract, more preferably 1% sodium chloride (mass fraction) + 1% peptone + 0.5% yeast extract.

[0013] Preferably, the culture is carried out in a shaking incubator.

[0014] The composition provided by the present invention comprises, as a second non-natural sugar, one or more of Ac4ManNAz, Ac4GalNAz, and Ac4GlcNAz in any combination.

[0015] The method of applying the composition of the present invention consists of three steps: A) Tumor antigen labeling: intratumoral injection of non-natural sugar-labeled antigens; B) Radiotherapy; C) Intratumoral injection of engineered bacteria; Preferably, the interval between each radiotherapy session shall not exceed 3 days, and the dose of a single radiotherapy session shall not exceed 25 Gy.

[0016] Preferably, the engineered bacteria injected into the tumor are administered no more than three times, with each dose not exceeding 5 × 10⁶ CFU.

[0017] The present invention also provides the use of the composition drug prepared by the above method in the treatment of solid tumors.

[0018] Under the method for preparing engineered bacteria described in this invention, the bacteria can maintain a viability rate of over 80%. When the prepared bacteria are incubated with labeled antigens, the bacteria can specifically bind to the labeled antigens, enhance the presentation of antigens by dendritic cells, and improve the level of antigen-activated immune responses.

[0019] 3. Beneficial effects: This invention successfully prepared engineered bacteria capable of specifically binding to labeled antigens by modifying the bacterial surface with cycloalkynes. When combined with the non-natural sugars described in this invention, it significantly increased the antigen-presenting capacity of dendritic cells, thereby demonstrating significant therapeutic efficacy in treating solid tumors. Attached Figure Description

[0020] Figure 1 The results show the bacterial viability and modification efficiency of Salmonella VNP20009 after modification with DBCO-PEG4 NHS in Example 1. Figure 1 a is a schematic diagram of the preparation process; Figure 1 b represents the bacterial viability and modification efficiency after incubation with different concentrations of DBCO-PEG4 NHS; Figure 1 c is a representative flow cytometry image of Salmonella modified with 320 μg / mL DBCO-PEG4 NHS; Figure 1 d is a confocal microscope image of Salmonella modified with 320 μg / mL DBCO-PEG4 NHS; Figure 1 e represents the modification efficiency of different concentrations of BCN-PEG4 NHS after incubation with bacteria.

[0021] Figure 2 This is the result of labeling tumor antigens with non-natural sugar metabolism in Example 2. Figure 2 a represents the in-gel fluorescence analysis of B16F10 protein, a non-natural sugar-labeled protein; Figure 2 b is a confocal imaging image of B16F10 cells labeled with non-natural sugars in vitro; Figure 2 c is Figure 2 The statistical chart for b. Figure 2 d represents the types of B16F10 tumor antigens that are not naturally occurring sugar metabolism markers and their isoelectric point distribution.

[0022] Figure 3 This refers to the bacterial-specific binding marker antigen modified with DBCO in Example 3. Figure 3 a is a tumor antigen derived from B16F10 that specifically binds to bacteria; Figure 3 b represents the bacterial-specific binding marker pattern antigen ovalbumin; Figure 4 This is an evaluation of the immune activation effect of the composition in Example 4. Figure 4 a is a schematic diagram of the experimental design. Figure 4 b indicates that the application of the composition enhanced antigen presentation by dendritic cells in tumor-draining lymph nodes; Figure 4 c indicates that the application of the composition promoted the infiltration of antigen-specific T cells into the tumor. Figure 5 This demonstrates the effect of the composition in Example 5 on treating melanoma. Figure 5 a is a schematic diagram of the experimental design. Figure 5 b represents the growth curve of the tumor on the treated side after the application of the composition; Figure 5 c represents the growth curve of the distal tumor after application of the composition; Figure 5 d represents the survival curve of tumor-bearing mice; Figure 6 This is an immunological characterization of the tumor in mice after the composition was applied in Example 6. Figure 6 a and b represent the proportions of CD8+ T cells and CD4+ T cells in the tumor on the treated side after application of the composition, respectively. Figure 6 c and d represent the proportions of CD8+ T cells and CD4+ T cells in the distal tumor after the application of the composition, respectively. Detailed Implementation

[0023] The present invention will now be described in detail with reference to the accompanying drawings.

[0024] As attached Figure 1 To be continued Figure 6 As shown, The cell cultures described in the following examples all used the same culture medium and conditions. The culture medium consisted of: low-glucose DMEM + 10% fetal bovine serum + 100 U / ml penicillin + 100 µg / ml streptomycin. The culture conditions were: static culture at 37 ℃ in a 5% CO2 saturated humidity incubator, with the medium changed every 2-3 days thereafter.

[0025] The bacterial cultures described in the following examples all used the same culture medium and conditions. The culture medium consisted of 1% sodium chloride (mass fraction) + 1% peptone + 0.5% yeast extract. The culture conditions were: incubation at 37°C in a shaking incubator at a speed of 220 rpm for 12–16 hours.

[0026] Example 1: NHS ester-linked cycloalkyne-modified Salmonella VNP20009 NHS, as a highly reactive cross-linking agent, can chemically react with amino groups on the bacterial surface to form covalent ester bonds. This reaction is commonly used for the chemical modification and functionalization of biomolecules. Based on this principle, the inventors utilized the reaction of DBCO-PEG4 NHS and BCN-PEG4 NHS with amino groups on the bacterial surface to engineer bacteria and prepare engineered bacteria. The specific experimental methods are as follows: Bacteria were inoculated at a 1:1000 ratio into LB medium and cultured for 12-16 hours. The bacterial cells were then collected by centrifugation. The cells were resuspended in PBS buffer, and the bacterial OD value was measured using a spectrophotometer. Based on the OD value, the cells were diluted to 5 × 10⁷ CFU / mL. One mL of the bacteria was added to different concentrations of DBCO-PEG₄ NHS or BCN-PEG₄ NHS and incubated at 25°C for 20 min. After centrifugation, the cells were washed once with PBS. The resulting bacteria were the engineered bacteria.

[0027] To determine the efficiency and viability of the engineered cycloalkyne bacteria, a portion of the engineered bacteria was diluted and plated for colony counting. Another portion was incubated with AZDye 488-Azide probe at a final concentration of 20 μM at 37°C for 1 hour, followed by flow cytometry and confocal microscopy analysis.

[0028] The results show that: Figure 1 As shown in b and c, the use of 320 μg / mL DBCO-PEG4 NHS engineered bacteria showed high efficiency (~60%), and under these conditions, the bacteria maintained a viability of over 80%. Confocal microscopy imaging results indicated that the DBCO groups remained stably present on the bacterial surface for 48 hours. Figure 1 d). The modification efficiency of BCN-PEG4 NHS is relatively low, with a modification efficiency of 40% at 320 ug / mL ( Figure 1 e).

[0029] Example 2: Metabolic labeling of tumor antigens using non-natural sugars B16F10 cells were seeded at a density of 30% in 10cm cell culture dishes. After complete cell adhesion, 50µM of non-natural sugars (Ac4ManNAz, Ac4GalNAz, Ac4GlcNAz) were added. After 48 hours, cell lysates were collected, and copper-catalyzed azide-alkyne cycloaddition (CuAAC) was performed using the alkyne-Cy5 probe. The reaction products were then subjected to SDS-PAGE gel electrophoresis. After electrophoresis, in-gel fluorescence imaging was performed using the ChemiDoc MP Imaging System (Bio-Rad).

[0030] For confocal imaging experiments, cells were seeded at a density of 3 × 10⁴ cells per well onto coverslips in 6-well plates and co-cultured with 50 μM Ac₄GalNAz for 48 h. After incubation, cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) solution. Subsequently, cells were incubated with 100 μL of freshly premixed CuAAc reaction mixture (containing 50 μM CuSO₄, 300 μM BTTAA, 50 μM alkyne-Cy₅, and 2.5 mM sodium ascorbate) at room temperature for 10 min. Next, cell nuclei were stained with Hoechst 33342 (20 μM). Finally, the cells were washed, and coverslips were mounted on microscope slides for observation and imaging using a confocal laser scanning microscope (CLSM, Olympus FV3000).

[0031] The results showed that non-natural sugars could effectively metabolize proteins labeled in B16F10 cells. The efficiency of different non-natural sugars in labeling proteins varied. Figure 2 (a~c). Proteomics analysis revealed the presence of multiple identified tumor neoantigens in the labeled proteins (Figure d).

[0032] Example 3: Engineered bacteria can specifically bind to metabolically labeled antigens. Furthermore, the inventors verified the ability of engineered bacteria to bind tumor antigens. The specific method is as follows: B16F10 cells were seeded at a density of 30% in 10cm cell culture dishes. After complete cell adhesion, 50µM of non-natural sugars (Ac4ManNAz, Ac4GalNAz, Ac4GlcNAz) were added and the cells were cultured for 48 hours. Cell lysates were collected and incubated with biotin-NHS ester at room temperature for 30 minutes. Free biotin-NHS was removed by ultrafiltration using a 3K MWCO filter (Millipore). 8mg of biotin-labeled cell lysate was incubated with 2×10⁸ CFU of engineered bacteria at 37°C for 2 hours, and the bacterial pellet was collected by centrifugation. The bacterial-antigen complex pellet was then lysed in RIPA lysate at 95°C for 15 minutes, and the resulting product was analyzed by Western blotting. Streptavidin-horseradish peroxidase (HRP) was used to detect the N3-labeled protein bound by the bacteria.

[0033] To verify the ability to bind the pattern antigen ovalbumin, lysates of B16-OVA cells treated with azide were collected. 8 mg of B16-OVA cell lysates were incubated with 2 × 10⁸ CFU of engineered bacteria at 37°C for 2 hours, and the bacterial pellet was collected by centrifugation. Subsequently, the bacterial-antigen complex pellet was lysed in RIPA lysate at 95°C for 15 minutes. The resulting product was analyzed by Western blotting, and the proteins bound to the bacteria were analyzed using OVA antibodies.

[0034] The results showed that the engineered bacteria significantly bound to a large amount of azide-labeled B16F10 tumor antigen (B16F10). Figure 3 a) and pattern antigen OVA ( Figure 3 b).

[0035] Example 4 Evaluation of the in vivo immune activation effect of the composition B16-OVA mouse model construction: BALB / c female mice (6-8 weeks old) used in the experiment were purchased from the Comparative Medicine Center of Yangzhou University and bred according to IACUC-NJU requirements. The fur on both sides of the mouse abdomen was removed, and a suspension of 0.1 mL of 5 × 10⁶ B16F10 tumor cells / mL was injected subcutaneously into the right abdomen. Three days later, a suspension of 0.1 mL of 5 × 10⁶ B16F10 tumor cells / mL was injected subcutaneously into the left abdomen.

[0036] When the tumor on the right side grows to approximately 100 mm³, patients will be randomly assigned to different groups. The grouping and treatment plan are as follows: Figure 4 As shown in Figure a, after three consecutive days of injection of non-natural sugar, engineered bacteria were injected intratumorally into tumor-bearing mice at a dose of 5 × 10⁵ CFU, and the radiotherapy dose was 8 Gy / fraction. On days 16 and 21 after administration, tumor drainage lymph nodes and tumor tissue were collected, respectively, and the ratio of antigen-presenting dendritic cells (DCs) and antigen-specific T cells was analyzed by flow cytometry.

[0037] The results showed that the application of the composition significantly promoted the presentation of antigens by dendritic cells. Figure 4 b), further promoting tumor infiltration of antigen-specific T cells ( Figure 4 c).

[0038] Example 5: Experiment on the inhibition of melanoma growth by combination of composition and radiotherapy A B16F10 melanoma model was constructed according to the method described in Example 4. When the right-sided tumor grew to approximately 100 mm³, the tumors were randomly assigned to groups, and the grouping and treatment regimens were as follows. Figure 5As shown in Figure a, after three consecutive days of injection of non-natural sugar, engineered bacteria were injected intratumorally into tumor-bearing mice at a dose of 5 × 10⁵ CFU, and the radiotherapy dose was 8 Gy / fraction. The tumor volume V was calculated using the formula: V = D × d² / 2, where D is the long diameter of the tumor and d is the short diameter. During the treatment period, the size of both tumors and the weight of the mice were recorded daily, and tumor growth curves were plotted (e.g., ...). Figure 5 b and Figure 5 (as shown in c).

[0039] The results showed that application of the composition after radiotherapy significantly inhibited the treatment side ( Figure 5 b) and remote ( Figure 5 c) Tumor growth. Notably, the inhibitory effect of unmodified wild-type bacteria was significantly worse than that of engineered bacteria. Furthermore, a statistical analysis of the survival of tumor-bearing mice after treatment showed that intratumoral injection of engineered bacteria after radiotherapy prolonged the survival time of tumor-bearing mice. Figure 5 d).

[0040] Example 6: Detection of T cell infiltration in tumor tissue after application of the composition Based on the bilateral tumor efficacy experiment of B16F10 described in Example 5, mice were euthanized by cervical dislocation on day 7 after intratumoral administration. Tumor tissue from both sides of each group of mice was collected, ground, and centrifuged to prepare a tumor single-cell suspension. Anti-CD3 FITC, anti-CD8 PE, and anti-CD4 APC antibodies were added, and the suspension was incubated at 4°C for 30 min. Cells were then collected by centrifugation. The proportion of T cells in the tumor tissue was analyzed using flow cytometry.

[0041] The results showed that the composition significantly increased CD8+ T cells in both lateral and distal tumors treated with the drug. Figure 6 a and c) and CD4+ T cells ( Figure 6 The ratio of b to d).

[0042] Although the present invention has been disclosed above with reference to preferred embodiments, these are not intended to limit the invention. Any person skilled in the art can make various changes or modifications without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be defined by the scope of the claims of this application.

Claims

1. A composition of engineered bacteria and non-natural sugars for treating solid tumors, characterized in that, The composition consists of engineered bacteria modified with a bioorthogonal group and one or more azide-based non-natural sugars.

2. The composition for treating solid tumors according to claim 1, characterized in that, The bacteria in question are Gram-negative.

3. The composition for treating solid tumors according to claim 1, characterized in that, The engineered bacteria are NHS ester-linked cycloalkynes modified with bacteria. The preparation method includes the following steps: culturing the bacteria in LB liquid medium for 12-16 h, followed by incubation with NHS ester-linked cycloalkynes to obtain the engineered bacteria.

4. The composition for treating solid tumors according to claim 1, characterized in that, The azide-based non-natural sugar is one or more of Ac4ManNAz, Ac4GalNAz, and Ac4GlcNAz, or any combination thereof.

5. The preparation method according to claim 3, characterized in that, The LB medium is formulated as follows: 0.5-1.5% sodium chloride (mass fraction) + 0.5-1.5% peptone + 0.25-0.75% yeast extract.

6. The preparation method according to claim 3, characterized in that, The culture conditions are 30~37 ℃, with shaking culture at 150~220 rpm / min.

7. The preparation method according to claim 3, characterized in that, The cyclic alkynes are one or more of DBCO, OCT, DIBO, TMTH, DIFO, BCN, and BARAC, and any combination thereof.

8. The preparation method according to claim 3, characterized in that, The incubation conditions are 22~37℃ for 15~30 minutes.

9. The composition for treating solid tumors according to claim 1, characterized in that, The solid tumors mentioned are melanoma, lung cancer, gastric cancer, breast cancer, liver cancer, pancreatic cancer, and colorectal cancer.

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