Construction method, product and application of lactic acid-consuming genetic engineering strain

Abstract

The invention belongs to the field of gene engineering, and particularly relates to a construction method of a lactic acid-consuming gene engineering strain, a related product and application of the lactic acid-consuming gene engineering strain in tumor treatment. Probiotics with tumor targeted colonization capability are taken as an original strain, CRISPR-Cas9 mediated homologous recombination transformation is carried out, glk is knocked out and an lldD gene is inserted, ldhA is knocked out and a dld gene is inserted, and the genes are driven by a pJ23119 promoter. The modified engineering bacteria can be colonized in tumor tissues in a targeted manner, efficiently metabolize lactic acid in a tumor microenvironment and effectively relieve local acidosis, so that immune cell activation is promoted, and the Treg cell mediated immunosuppression effect is weakened. In animal models of melanoma and colon cancer, when the engineering bacterium is combined with an anti-PD-1 antibody for use, a remarkable synergistic tumor inhibition effect is shown, obvious systemic toxicity is not observed, and a safe and effective new strategy is provided for tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a method for constructing a genetically engineered strain that consumes lactic acid, as well as the product and its application. Background Technology

[0002] Even under oxygen-rich conditions, tumor cells tend to rapidly break down glucose and produce large amounts of lactic acid via glycolysis, a phenomenon known as the Warburg effect. Lactic acid accumulation in the tumor microenvironment (TME) plays a crucial role in tumor immune escape and treatment resistance. First, high concentrations of lactic acid can inhibit the differentiation and function of T cells and natural killer (NK) cells, hinder the upregulation of intracellular activated T cell nuclear factor 1 (NFAT1), and consequently reduce the production of interferon-gamma (IFN-γ), weakening the body's tumor immune surveillance capabilities. Second, lactic acid accumulation leads to intracellular acidification and impaired energy metabolism, further inhibiting immune cell proliferation, disrupting anti-tumor immune responses, and indirectly weakening the efficacy of immune checkpoint inhibitors. Furthermore, lactic acid remodels the immunosuppressive microenvironment through multiple pathways, including interfering with innate immune signaling, promoting tumor cell DNA damage repair, inhibiting tumor suppressor protein activity, and inducing the infiltration and polarization of immunosuppressive cells. Therefore, regulating lactic acid levels in the TME holds promise for alleviating tumor-induced immunosuppression and enhancing the effectiveness of immunotherapy.

[0003] Lactic acid, as a key immunosuppressive metabolite and signaling molecule in the tumor microenvironment, has become an important intervention target for cancer treatment. A series of novel therapeutic strategies targeting lactate production, transport, and its downstream biological effects (promoting tumor angiogenesis, maintaining tumor stem cell characteristics, and promoting tumor invasion and metastasis) have been proposed and have shown great potential in early clinical studies. For example, the small molecule inhibitor SBI-477, targeting the MondoA-TXNIP signaling axis, can reduce its impact on CD8 by blocking lactate-driven metabolic-immune cross-regulation. + The inhibition of T cell killing function also weakens the immunosuppressive activity of Tregs; the small molecule inhibitor AZD3965, which targets monocarboxylic acid transporter 1 (MCT1), blocks lactate efflux, leading to lactate accumulation in tumor cells, interfering with the activity of various enzymes and key signaling pathways, and ultimately inhibiting tumor growth; in addition, the nanodelivery system PMLR, which carries lactate oxidase (LOX) and glycolysis inhibitor 3PO, uses LOX to catalyze lactate in the tumor into pyruvate and hydrogen peroxide, achieving in-situ clearance of lactate.

[0004] The aforementioned strategies all enhance the efficacy of tumor immunotherapy by regulating lactate levels, but they still have significant limitations. Traditional small molecule inhibitors lack tumor-specific targeting capabilities, potentially leading to lactate metabolism disorders in normal tissues and causing severe off-target toxicity. Furthermore, LOX-based nanosystems are highly oxygen-dependent; in the hypoxic microenvironment prevalent within tumors, enzyme catalytic efficiency is significantly limited, making it difficult to achieve effective lactate clearance and sustained inhibition of tumor growth. In summary, existing anti-lactate therapy strategies still face multiple challenges, including insufficient targeting, limited regulatory efficiency, and potential systemic toxicity, severely hindering their clinical translation. Therefore, there is an urgent need to develop novel therapeutic systems that can precisely target tumor sites, adapt to the characteristics of the tumor microenvironment, and achieve sustained and efficient lactate clearance to overcome the limitations of current tumor metabolic interventions. Summary of the Invention

[0005] To address the aforementioned shortcomings, this invention provides a genetically engineered strain that combines tumor targeting, highly efficient lactate clearance, and biosafety. This engineered strain uses a probiotic with tumor-targeting colonization capabilities as the original strain and employs CRISPR-Cas9-mediated homologous recombination technology to knock out lactate synthesis-related genes. glk and ldhA Simultaneously insert lactate catabolism gene lldD and dld It can specifically colonize tumor tissues and efficiently consume lactic acid, reversing the acidification of the tumor microenvironment and the state of immunosuppression. When used in combination with anti-PD-1 antibodies, it exhibits a significant synergistic tumor-suppressing effect and has no obvious systemic toxicity, providing a safe and effective new strategy for tumor treatment.

[0006] The technical solution of this invention is as follows: On the one hand, this invention provides a method for constructing a genetically engineered strain that consumes lactic acid, using a probiotic with tumor-targeting colonization ability as the starting strain, and achieving the following modifications through gene editing: (1) Knockout of lactate biosynthesis-related genes glk Simultaneously insert lactate catabolism gene lldD ; (2) Knockout of lactate biosynthesis-related genes ldhA Simultaneously insert lactate catabolism gene dld ; (3) The gene editing described herein uses homologous recombination to... lldD and dld The gene is integrated into a specific site in the genome of the originating strain.

[0007] Specifically, the homologous recombination described in step (3) can be mediated by the CRISPR-Cas system.

[0008] Preferably, the CRISPR-Cas system can be a CRISPR-Cas9 system.

[0009] Specifically, the homologous recombination is achieved through the following steps: a repair template containing a homologous arm is co-transformed with an sgRNA plasmid into Cas9 competent cells of the starting strain, and after screening with two antibiotics, the plasmid is removed by passage culture to obtain the target strain.

[0010] More specifically, the repair template can be a DNA fragment constructed using seamless cloning technology.

[0011] Preferably, the seamless cloning is glk The repair template corresponding to gene knockout includes glk Upstream and downstream homologous arms and lldD Gene, ldhA The repair template corresponding to gene knockout includes ldhA Upstream and downstream homologous arms and dld Gene.

[0012] Preferably, the glk The sgRNA sequence of the gene is SEQ ID NO.29. ldhA The sgRNA sequence of the gene is SEQ ID NO.30.

[0013] More specifically, the dual antibiotics may be kanamycin and chloramphenicol.

[0014] Specifically, step (3) is described dld and lldD Gene expression can be driven by constitutive promoters.

[0015] Preferably, the constitutive promoter may be the pJ23119 promoter.

[0016] Specifically, the probiotics with tumor-targeting colonization capabilities can be Escherichia coli, Salmonella, Clostridium, or Bifidobacterium.

[0017] Preferably, the probiotic with tumor-targeting colonization ability can be Escherichia coli.

[0018] In another aspect, the present invention provides a lactic acid-consuming genetically engineered strain obtained by the aforementioned construction method.

[0019] In another aspect, the present invention provides a drug comprising the aforementioned genetically engineered strain.

[0020] Specifically, the concentration of the genetically engineered strain can be 1×10⁻⁶. 6 -1×10 12 CFU / mL.

[0021] Preferably, the concentration of the genetically engineered strain can be 1×10⁻⁶. 7 -1×10 10 CFU / mL.

[0022] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0023] Preferably, the pharmaceutically acceptable excipients for injection are selected from one or more of solvents, osmotic pressure regulators, pH regulators, antioxidants, suspending agents, and complexing agents.

[0024] Preferably, the suspending agent is selected from one or more of sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, gelatin, povidone, aluminum monostearate, and sodium alginate.

[0025] Preferably, the solvent is selected from one or more of water for injection, sterile vegetable oil for injection, ethanol, propylene glycol, and polyethylene glycol.

[0026] Preferably, the osmotic pressure regulator is selected from one or more of sodium chloride, glucose, and mannitol.

[0027] Preferably, the pH adjuster is selected from one or more of hydrochloric acid, sodium hydroxide, phosphate buffer, and citrate buffer.

[0028] Preferably, the antioxidant is selected from one or more of sodium sulfite, sodium bisulfite, sodium metabisulfite, and vitamin C.

[0029] Specifically, the dosage form of the drug is an injection.

[0030] In another aspect, the present invention provides the application of the aforementioned genetically engineered strains in the preparation of drugs for treating tumors.

[0031] Specifically, the tumor includes, but is not limited to, melanoma or colon cancer.

[0032] Specifically, the drug is used in combination with an anti-PD-1 antibody.

[0033] Preferably, the combined administration frequency is consistent.

[0034] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0035] Preferably, the pharmaceutically acceptable excipients for injection are selected from one or more of solvents, osmotic pressure regulators, pH regulators, antioxidants, suspending agents, and complexing agents.

[0036] Preferably, the suspending agent is selected from one or more of sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, gelatin, povidone, aluminum monostearate, and sodium alginate.

[0037] Preferably, the solvent is selected from one or more of water for injection, sterile vegetable oil for injection, ethanol, propylene glycol, and polyethylene glycol.

[0038] Preferably, the osmotic pressure regulator is selected from one or more of sodium chloride, glucose, and mannitol.

[0039] Preferably, the pH adjuster is selected from one or more of hydrochloric acid, sodium hydroxide, phosphate buffer, and citrate buffer.

[0040] Preferably, the antioxidant is selected from one or more of sodium sulfite, sodium bisulfite, sodium metabisulfite, and vitamin C.

[0041] Specifically, the dosage form of the drug is an injection.

[0042] The beneficial effects of this invention are as follows: This invention utilizes rationally designed bacterial engineering to achieve targeted lactate consumption within the tumor microenvironment (TME) using Lac-EcN. In B16-F10 melanoma and MC-38 colon cancer mouse models, Lac-EcN exhibited excellent colonization ability, metabolic regulation function, and antitumor activity. Data shows that a single intratumoral injection of 5 × 10⁶ cells / mL... 6 CFU's Lac-EcN can reduce lactate levels in the tumor microenvironment (TME) by 40–65% within 24 hours, while simultaneously increasing the number of tumor-infiltrating T cells by more than 50%. Lac-EcN effectively alleviates the acidification state of the TME and promotes CD8... + The nuclear translocation of NFAT1 in T cells and the inhibition of NFAT1 nuclear translocation in Treg cells directly validate the bidirectional regulatory role of lactate clearance on the immune microenvironment. More importantly, when Lac-EcN was used in combination with anti-PD-1 antibodies, the tumor inhibition rate reached 75% in the B16-F10 melanoma mouse model; while in the MC-38 colon cancer mouse model, 50% of the mice achieved complete tumor regression, and no significant toxicity was observed. In conclusion, Lac-EcN not only achieves precise regulation of the TME but also significantly enhances the efficacy of immune checkpoint therapy, providing a new approach for developing metabolic-immune combined anti-tumor therapy strategies. Attached Figure Description

[0043] Figure 1 A schematic diagram illustrating the construction of Lac-EcN and its immunotherapy mechanism.

[0044] Figure 2The agarose gel electrophoresis was used to verify the gene editing. In diagram a, we show the verification of the lldD gene replacing the glk gene (glk - 1850 bp, lldD - 2129 bp). In diagram b, we show the verification of the dld gene replacing the ldhA gene (ldhA - 1642 bp, dld - 2422 bp). In diagram c, we show a schematic diagram of the homologous arms of the glk gene and its PCR primers. In diagram d, we show a schematic diagram of the homologous arms of the ldhA gene and its PCR primers.

[0045] Figure 3 Multi-omics analysis and lactate consumption capacity assessment of Lac-EcN. a) Transcriptomic analysis of Lac-EcN and EcN. RNA was extracted from Lac-EcN and EcN and sequenced and analyzed using transcriptome sequencing technology. b) Proteomic analysis of Lac-EcN and EcN. Lac-EcN and EcN were pretreated, and their total proteome was analyzed by LC-MS / MS. Volcano plots show the results of differential abundance analysis between Lac-EcN and EcN (two-tailed Welch t-test) (a, b). Red and green dots represent proteins with significantly different abundances between the two strains (P < 0.05, log2 |fold change| > 1). n = 3. c) Schematic diagram of the Lac-EcN glycolysis pathway. Color coding indicates log2 / (fold change) measured by LC-MS / MS. Arrow directions between enzymes are labeled according to the KEGG database. d represents the assessment of lactic acid concentration in the supernatant of Lac-EcN and EcN bacteria. n = 3. P-values ​​are presented graphically and derived using a two-tailed t-test (d). *p < 0.05. Data are expressed as mean ± standard error.

[0046] Figure 4 To assess the colonization ability and immunomodulatory activity of Lac-EcN in B16-F10 tumors. Where 'a' represents the number of bacteria colonizing B16-F10 tumors at different time points. 5 × 10⁻⁶ 6CFU EcN or Lac-EcN were injected into B16-F10 tumors. Tumors were collected and homogenized after 24, 72, or 120 h, and bacterial counts were measured using the CFU counting method. The line represents the average bacterial count per gram of tumor. n = 3. b shows the lactate content of different groups of B16-F10 tumors after 24 h. EcN or Lac-EcN was injected into B16-F10 tumors. Tumors were collected and homogenized after 24 h, and lactate levels were determined by LC-MS / MS. n = 3. c shows the pH value of different groups of B16-F10 tumors after 24 h. Tumors were extracted 24 h after colonization with EcN or Lac-EcN in B16-F10 tumors, and intracellular fluorescence intensity was detected using the BCECF AM probe. The results show the mean fluorescence intensity (MFI) of different groups. n = 3. d shows the histopathological analysis of tumor tissue. Hematoxylin and eosin (H&E) staining and CD3 immunohistochemical analysis were performed on B16-F10 tumors in the control group and tumors treated with EcN or Lac-EcN 120 h later. Arrows indicate CD3. + T cells. Scale bar, 100 μm. e, f, g represent the immune infiltration status of B16-F10 tumors in different groups 24 h after bacterial injection. CD4, CD8, and Foxp3 levels in B16-F10 tumor immune infiltration were analyzed by flow cytometry 24 h after EcN or Lac-EcN injection. + The proportion of regulatory T cells. n = 5. P-values ​​are presented graphically and derived by two-tailed t-tests (a) and one-way ANOVA (b, c, e-g). ns indicates no significant difference; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Data are expressed as mean ± standard error.

[0047] Figure 5 This is an example of a gating strategy in flow cytometry.

[0048] Figure 6This diagram illustrates the antitumor immune response of Lac-EcN in B16-F10 tumors. Figure a shows a schematic diagram of treatment in B16-F10 tumor-bearing mice, illustrating the time points and treatment methods. *it* represents intratumoral injection, and *ip* represents intraperitoneal injection. Figure b shows the lactate content of B16-F10 tumors in different groups 24 hours after a single treatment. Tumors were collected and homogenized 24 hours after a single treatment, and lactate levels were measured by LC-MS / MS. n = 5. Figure c shows the pH value of B16-F10 tumors in different groups 24 hours after a single treatment. Tumors were extracted 24 hours after a single treatment, and intracellular fluorescence intensity was detected using the BCECF AM probe. The results show the mean fluorescence intensity (MFI) of different groups. n = 5. Figure d (left) shows the detection of CD8 by Western blotting. + Expression of NFAT1 protein in T cell nuclei. The right figure shows CD8 expression. + The relative expression level of NFAT1 in T cell nuclei. n=3. e. The left figure shows the expression of NFAT1 protein in Treg cell nuclei detected by Western blotting. e. The right figure shows the relative expression level of NFAT1 in Treg cell nuclei. n=3. f. Evaluation of in vivo antitumor activity. B16-F10 tumor-bearing mice were treated according to (a). The arrows indicate the time points of each treatment. The left figure shows the mean growth curve of B16-F10 tumors (error bars represent standard errors). The right figure shows the survival curve of mice; where "number of surviving mice / total number of mice" represents the proportion of surviving mice. n = 8. P-values ​​are presented in graphical form and obtained by one-way ANOVA (be), two-way ANOVA (f, left), or two-tailed Mantell-Cox log-rank test (f, right). ns indicates no significant difference; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Data are expressed as mean ± standard error.

[0049] Figure 7 Health assessment of B16-F10 tumor-bearing mice. a) Tumor growth curves for individual mice in different groups. n = 8. b) Body weight curves for B16-F10 tumor-bearing mice. After establishing the tumor model, mice received appropriate treatments according to their groups, and body weight was measured periodically during the treatment. Data are expressed as mean ± standard error (SEM). c) Changes in tumor size after bacterial treatment. After 30 days of treatment, tumors in different groups of mice were resected and compared. d) H&E staining of major organs in mice. Scale bar, 100 μm.

[0050] Figure 8This study aimed to evaluate the in vivo immune activation effect of Lac-EcN. Here, ad represents the expression levels of Foxp3, Nrp1, CD44, and PD-1 in Treg cells. After two treatments, the expression of Foxp3, Nrp1, CD44, and PD-1 in tumor cells of mice in different treatment groups was analyzed by flow cytometry. n = 5. eh represents CD8+. + Expression levels of TNF-α, IFN-γ, IL-2, and GrB in T cells. After two treatments, the expression levels of TNF-α, IFN-γ, IL-2, and GrB in tumor cells of mice in different treatment groups were analyzed by flow cytometry. n = 5. P-values ​​are presented graphically and derived through one-way ANOVA (ah). ns indicates no significant difference; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Data are expressed as mean ± standard error.

[0051] Figure 9 This study describes the antitumor effect of Lac-EcN in a T-cell deficiency model. Where a represents the effect in C57BL / 6N wild-type mice and CD3e... - / - The number of bacteria colonizing B16-F10 tumors in mice. B16-F10 tumors were established in two mouse species by intratumoral injection of 5 × 10⁶ CFU EcN or Lac-EcN. Tumors were harvested and homogenized 24 h later, and bacterial counts were measured using the CFU counting method. n = 3. b represents the growth curve of the B16-F10 tumor. (The last sentence appears to be incomplete and possibly refers to a different topic.) - / - B16-F10 tumors were established in mice, and the mice were treated twice weekly as shown in the figure. Data are expressed as mean ± standard error. n = 10. P-values ​​are presented graphically and derived by a two-tailed t-test (a) and two-way ANOVA (b). ns indicates no significant difference in the data.

[0052] Figure 10 This section assesses the in vivo efficacy of systemic drug administration. Figure a illustrates the treatment strategy of tail vein administration to B16-F10 tumor-bearing mice. The figure shows the treatment time points and methods. iv, intravenous injection; ip, intraperitoneal injection. Figures b and c show the bacterial distribution in the tumor and various tissues. 5 × 10⁵ cells were injected into the mice via the tail vein. 7 CFU EcN or Lac-EcN. One week later, tumor, heart, liver, spleen, lung and kidney samples were collected, homogenized and diluted to the same concentration (10). -5(b) The tissue was spread onto LB medium. Bacterial count was measured using a CFU assay (c). The bar chart represents the mean CFU value per gram of tissue. n = 3. d) shows the histopathological analysis of the tumor tissue. After 10 days of treatment, H&E staining and CD3 immunohistochemical analysis were performed on the control group and on B16-F10 tumors treated with EcN or Lac-EcN. Arrows indicate CD3. + T cells. Scale bar, 100 μm. e represents in vivo antitumor activity assessment. B16-F10 tumor-bearing mice were treated according to (a). Arrows indicate the time points of each treatment. The left plot shows the mean growth curve of B16-F10 tumors (error bars represent standard errors). The right plot shows the survival curve of mice; where "number of surviving mice / total number of mice" represents the proportion of surviving mice. n = 10. P-values ​​are presented graphically and derived by two-way ANOVA (e, left) or two-tailed Mantell-Cox log-rank test (e, right). ns indicates no significant difference; ***p<0.001; ****p<0.0001. Data are expressed as mean ± standard error.

[0053] Figure 11 This study investigated the effect of tail vein injection of bacteria on the health of B16-F10 tumor-bearing mice. In the figure, a represents the tumor growth curves of individual mice in different groups. n = 10. b represents the body weight curves of B16-F10 tumor-bearing mice. After establishing the tumor model, mice received appropriate treatments according to their groups, and their body weight was measured periodically. Data are expressed as mean ± standard error.

[0054] Figure 12 This diagram illustrates the antitumor immune response of Lac-EcN in MC-38 tumors. Figure a shows a schematic diagram of the treatment strategy for MC-38 tumor-bearing mice. This figure shows the time points and treatment methods. *it*, intratumoral injection; *ip*, intraperitoneal injection. Figure b shows the lactate content of MC-38 tumors in different groups after 24 h. Tumors were extracted and homogenized 24 h after treatment, and lactate levels were measured by LC-MS / MS. n = 3. Figure c shows the pH value of MC-38 tumors in different groups after 24 h. Tumors were extracted 24 h after treatment, and intracellular fluorescence intensity was detected using the BCECF AM probe. The results show the mean fluorescence intensity (MFI) of different groups. n = 3. Figure d shows the tumor histopathological analysis. H&E staining and CD3 immunohistochemical analysis were performed on MC-38 tumors from the control group and tumors treated with EcN or Lac-EcN 120 h later. Arrows indicate CD3. +T cells. Scale bar, 100 μm. e represents in vivo antitumor activity assessment. MC-38 tumor-bearing mice were treated according to (a). Arrows indicate the time points of each treatment. The left figure shows the mean growth curve of B16-F10 tumors (error bars represent standard errors). The right figure shows the survival curve of mice; where "number of surviving mice / total number of mice" represents the proportion of surviving mice. n = 10. P-values ​​are presented graphically and derived by one-way ANOVA (b, c), two-way ANOVA (e, left), or two-tailed Mantell-Cox log-rank test (e, right). ns indicates no significant difference; **p<0.01; ****p<0.0001. Data are expressed as mean ± standard error.

[0055] Figure 13 This study investigated the effect of bacterial combined with antibody therapy on the health of MC-38 tumor-bearing mice. In the figures, a represents tumor growth curves for individual mice in different groups (n = 10). b represents the body weight curves of MC-38 tumor-bearing mice. Mice with MC-38 tumors were treated accordingly, and their body weight was continuously monitored. Data are expressed as mean ± standard error. c represents the change in tumor size after bacterial treatment. After 30 days of treatment, tumors in different groups of mice were resected and compared. Detailed Implementation

[0056] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0057] 1. The bacterial strains, cell lines, and experimental mice of this invention Escherichia coli Nissle 1917 (EcN) was purchased from Hangzhou Baosai Plasmid & Strains Resource Co., Ltd. (Hangzhou, China) and cultured in LB medium at 37°C with continuous shaking at 220 rpm. B16-F10 melanoma cells (CL-0319) and MC-38 colon cancer cells (CL-0972) were purchased from Wuhan Pronosai Life Sciences Co., Ltd. These two cell lines were cultured in 1640 medium and DMEM medium (Gibco), respectively, both supplemented with 10% (v / v) fetal bovine serum and antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin). Cells were incubated at 37°C in a 5% CO2 incubator, and routine mycoplasma testing was performed to ensure the culture system was free of contamination. 6-8 week old Cd3e cells... - / - Mice and C57BL / 6 mice (both male and female) were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. (Nanjing, China) and housed at the Specific Pathogen Free (SPF) Grade Laboratory Animal Center of the Hefei National Science Center for Big Health. The environmental conditions for the laboratory animals were controlled as follows: temperature 20-26℃ (68-79°F), humidity 30%-70%, 12-hour light-dark cycle (6:00 AM to 6:00 PM), and free access to food and water. All animal experimental protocols were approved by the Laboratory Animal Management Committee of the Hefei National Science Center for Big Health.

[0058] 2. The antibodies used in this invention are shown in Table 1: Table 1. All antibodies used in this invention

[0059] 3. Main experimental methods of this invention (1) Laboratory animals C57BL / 6 mice and Cd3e - / - Mice were purchased from Jiangsu GemPharmatech Co., Ltd. Animals were housed in a standardized barrier environment with controlled conditions of 20 ± 2℃ and 55 ± 8% relative humidity, maintaining a 12-hour light / dark cycle. Five mice were housed in each ventilated cage, and routinely provided with sterile food and water. Mice underwent daily health checks to ensure animal welfare throughout the experiment. Female mice used in the experiment were 6–10 weeks old. All animal experimental protocols were approved by the Experimental Animal Management Committee of the Hefei National Science Center for Big Health. Tumor growth monitoring strictly adhered to ethical endpoint standards, and no tumors exceeded 2 cm in diameter, ensuring that the maximum permissible tumor endpoint was not reached.

[0060] (2) Construction and gene editing of Lac-EcN Escherichia coli Nissle 1917 strain was purchased from Hangzhou Baosai Plasmid and Strains Resource Company and cultured in LB medium at 37℃ and 220 rpm with continuous shaking. To block the glucose metabolism pathway and enhance lactate utilization, a fourfold modification of the core glycolysis pathway on the EcN chromosome was performed using the Red homologous recombination method: knocking out the glucokinase gene. glk and lactate dehydrogenase gene ldhA Simultaneously, it affects the D-lactate dehydrogenase gene. dld and L-lactic acid dehydrogenase gene lldD Functional replacements are performed to promote efficient uptake and oxidative metabolism of lactic acid.

[0061] (3) Bacterial proteome LC-MS analysis Bacteria were cultured to the logarithmic growth phase (OD). 600 (≈ 0.6), take 1 ml of bacterial culture, centrifuge at 10000 rpm for 10 min at 4℃, carefully discard the supernatant, and retain the bacterial pellet. Add 100 μL of HEGX lysis buffer (containing 20 mM HEPES-KOH, 0.8 M NaCl, 1 mM EDTA, 10% glycerol and 0.2% Triton X-100) to resuspend the bacterial cells, mix thoroughly, and then sonicate for 15 min. After lysis, centrifuge at 10000 rpm for 10 min at 4℃, and collect the supernatant as the soluble protein extract. Determine the protein concentration using the BCA protein concentration assay kit (YEASEN, 20201ES76). Based on the results, take an equal volume of protein sample, add 5× SDS-PAGE loading buffer, and boil at 100℃ for 10 min to fully denature. Separate the sample by 12% SDS-PAGE gel electrophoresis (120 V, approximately 12 min). Coomassie Brilliant Blue staining was then performed to visualize the protein bands. After staining, the background was removed using destaining solution until the bands were clearly visible. The target protein band was excised from the gel and then placed in a 1.5 ml centrifuge tube for analysis. The sample was sealed and frozen for storage, and then sent to the Institute of Big Health, Hefei Comprehensive National Science Center for mass spectrometry analysis.

[0062] (4) Bacterial metabolomics LC-MS / MS analysis Bacteria were cultured to the logarithmic growth phase. 1 ml of bacterial culture was centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant was carefully discarded, and the bacterial pellet was retained. The bacterial cells were gently washed three times with pre-chilled PBS to remove any residual culture medium. The bacterial pellet was rapidly frozen on dry ice, and 2 ml of 80% (v / v) methanol (pre-chilled to -80 °C) was added for metabolite extraction. The sample was sonicated for 15 min, followed by incubation at -80 °C for 2 h to fully lyse the cells and stabilize the metabolites. After extraction, the sample was centrifuged at 14,000 × g for 20 min at 4 °C. The supernatant containing metabolites was transferred to a new 1.5 ml centrifuge tube (placed on dry ice). Care was taken to avoid contact with bottom particles during transfer. The extract was dried into particles using a SpeedVac vacuum concentrator at room temperature. The dried sample was sealed and stored at -80 °C for subsequent analysis. All samples were ultimately analyzed by the Institute of Big Health, Hefei Comprehensive National Science Center, using an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) system for targeted metabolomics analysis.

[0063] (5) In vitro lactate consumption experiment of Lac-EcN EcN and Lac-EcN were cultured to the logarithmic growth phase, and 1×10⁻⁶ samples were taken. 9 CFU bacteria were inoculated into 200 ml M9 medium (Sigma-Aldrich, M6030), and 400 μl glucose and 200 μl lactic acid were added. The culture was carried out at 37°C with shaking. Samples were taken at different time points, and the residual lactic acid concentration in the medium was detected using a lactic acid content kit (Jianglai Biotechnology, JL-T1068).

[0064] (6) Tumor tissue homogenization and CFU count Tumor tissue was extracted from C57BL / 6 mice, weighed, and washed with pre-cooled PBS to remove surface blood and residue. The tumor was then placed in a 1.5 ml centrifuge tube, 500 µl of PBS was added, and the tissue was mechanically homogenized using a tissue homogenizer. The homogenate was then filtered through a 70 μm sieve. The resulting homogenate was serially diluted 10-fold, and 100 µl was spread evenly on LB agar plates. The plates were incubated at 37°C for 12 h until colonies were clearly visible. The colony-forming units (CFU) at each dilution were counted. Based on the dilution factor and inoculum volume, the bacterial colony-forming units (CFU / g) per gram of tumor tissue were calculated to assess the bacterial colonization level within the tumor.

[0065] (7) LC-MS / MS analysis of lactic acid in tumor homogenate Tumor tissue (approximately 50 mg) was collected from each treatment group and mixed with 500 μl of pre-chilled 50% acetonitrile-water at 4°C. 200 μL of the homogenate was transferred to a new Eppendorf tube, an internal standard was added, and 300 μl of pre-chilled acetonitrile was added to precipitate the protein. The mixture was vortexed and sonicated for 5 min. The sample was then frozen at -20°C for 30 min and centrifuged at 13,000 rpm for 15 min at 4°C. The supernatant was filtered through a 0.22 μm filter and analyzed using a UPLC Xevo TQ-S triple quadrupole mass spectrometer (Waters, USA).

[0066] (8) Intratumoral pH detection Tumor-bearing mice were rapidly euthanized, and tumor tissue was aseptically removed. The tissue was thoroughly ground, filtered through a 40 μm cell filter, and a single-cell suspension was collected. The suspension was centrifuged at 300 × g for 5 min, the supernatant was discarded, and the cells were washed once with PBS. After resuspending the cells, the BCECFAM fluorescent probe was added, and the cells were incubated at 37°C in the dark for 30 min. After incubation, the cells were washed twice with PBS to remove unloaded probe. Fluorescence signals were detected using a Cytoflex LX flow cytometer, and data analysis was performed using CytExpert software. Intracellular pH was determined based on fluorescence intensity.

[0067] (9) H&E staining and CD3 immunohistochemical analysis Mice were rapidly euthanized, and target tissues (including tumors, heart, liver, spleen, lungs, and kidneys) were aseptically removed intact. The tissues were immediately cut into small pieces no larger than 1 cm³ to ensure sufficient penetration of the fixative. The cut tissues were immersed in 4% paraformaldehyde and fixed at 4 °C for 24 h to maintain tissue morphology and antigen integrity. To detect T cell infiltration in tumor tissues, immunohistochemistry was used. Paraffin sections were stained with a polyclonal anti-CD3E antibody (ABclonal, A19017) to label T cells. H&E staining and CD3 immunohistochemistry experiments were performed by the Institute of Big Health, Hefei Comprehensive National Science Center. Staining results were observed using the TG panoramic multispectral tissue scanning quantitative analysis system (TissueGnostics GmbH).

[0068] (10) Flow cytometry Tumor-bearing mice were rapidly euthanized, and tumor tissue was aseptically extracted, homogenized using a tissue homogenizer, and then filtered through a 40 μm cell sieve. The filtrate was collected, centrifuged at 300×g for 5 min, the supernatant was discarded, and the cells were washed once with PBS and resuspended to prepare a single-cell suspension. To eliminate interference from dead cells, staining was performed using eBioscience™ 7-AAD active dye (ThermoFisher). For intracellular marker staining, T cells were fixed and permeabilized with 4% paraformaldehyde, and then incubated with antibody dyes that specifically bind to the corresponding targets under light-protected conditions. Samples were collected using a Cytoflex LX flow cytometer, and data were analyzed using CytExpert software.

[0069] (11) Experiment on combined treatment with anti-PD-1 antibody and bacterial administration B16-F10 melanoma cells and MC-38 colon cancer cells were purchased from the cell bank of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. B16-F10 melanoma cells were cultured in RPMI-1640 medium (containing 10% FBS, 1% penicillin-drug antibody, 1% NEAA, and 1% GlutaMax), while MC-38 mouse colon cancer cells were cultured in DMEM medium (containing 10% FBS, 1% penicillin-drug antibody, 1% NEAA, 1% GlutaMax, 1% sodium pyruvate, and 1% HEPES). Cells were cultured at 37 ℃ in a 5% CO2 incubator, and routine mycoplasma testing was performed to ensure the culture system was free of contamination. When cells were in good logarithmic growth phase, they were digested with trypsin and resuspended in PBS. After adjusting to an appropriate concentration, 5 × 10⁶ cells were subcutaneously seeded on the backs of C57BL / 6 wild-type mice. 5 One tumor cell. Six to nine days after inoculation, when the tumor volume reaches 50-80 mm... 3 Treatment intervention began at that time. For the intratumoral injection group, 5×10 6 One EcN or Lac-EcN bacterium was resuspended in 50 µl PBS and injected directly into the tumor, twice a week for a total of four treatments. Mice receiving tail vein injection received a single injection of 5 × 10⁵ bacteria. 7 Two bacteria were administered to mice undergoing combination therapy. Simultaneously with the bacterial injection, mice were given an intraperitoneal injection of 200 µg of anti-PD-1 antibody (Leinco Technologies, Product No. P372) twice weekly for a total of four treatments. Tumor volume and mouse weight were measured periodically during treatment to assess efficacy and toxicity. All experimental procedures were conducted in accordance with animal ethics guidelines.

[0070] (12) Western blot detection of nuclear protein NFAT1 Tumor-bearing mice were rapidly euthanized, and tumor tissue was aseptically extracted and prepared into a single-cell suspension through mechanical grinding and enzymatic digestion. After staining with specific antibodies, CD8+ cells were sorted using a BD LSRFortessa™ X-20 flow cytometer. + T cells and Foxp3 + T cells. After sorting, cells were washed once with PBS, then resuspended in pre-chilled ATAC lysis buffer containing a protease inhibitor (Roche, 4693159001) and a phosphatase inhibitor A (ABclonal, RM02997). Cells were incubated on ice for 30 min, centrifuged to collect the cell pellet, resuspended in cell lysis buffer, and then subjected to sonication to disrupt the nuclei. The resulting supernatant was the nucleoprotein extract. Proteins were separated using SDS-PAGE, transferred to a membrane, blocked, incubated with primary and secondary antibodies, and visualized using chemiluminescence. Protein band signal intensity was semi-quantitatively analyzed using Fiji software.

[0071] (13) Quantitative and statistical analysis Statistical analysis was performed using GraphPad Prism software. A two-tailed unpaired Student t-test was used to compare two groups, and analysis of variance (ANOVA) was used to compare multiple groups (> two groups). The Kaplan-Meier method was used to analyze survival rates, and the log-rank test was used to analyze statistical differences between survival curves. A p-value <0.05 was considered statistically significant. Unless otherwise noted, the data shown in the figures are mean ± standard error.

[0072] Example 1: Construction and Verification of Lac-EcN 1.1 Construction of Lac-EcN All sequence information is listed in Tables 2 and 3. For glk and ldhA The sgRNA of the gene was analyzed using the CHOPCHOP tool (website: https: / / chopchop.cbu.uib.no / )design.

[0073] Table 2 Oligonucleotide Sequences

[0074] Table 3 Sequence information of genetic elements

[0075] glk gene knockout and lldD gene insertion: Using EcN genomic DNA as a template, the glk homologous arm and the target gene were amplified using primers glk-19-F and glk-19-R. Subsequently, they were ligated into the pUC19 vector (Miaoling plasmid P0368) using seamless cloning technology to construct the glk-pUC19 vector (containing the ampicillin resistance gene, the sequence of which was verified by sequencing). The construction process of the 23119-lldD fragment is as follows: the pJ23119 promoter was amplified using pJ23119 plasmid (Miaoling plasmid P55980) as a template, and the lldD gene was amplified using EcN DNA as a template. The two were then seamlessly ligated to form the 23119-lldD fragment. This fragment was inserted into the linearized glk-pUC19 vector to obtain the glk-23119-lldD-pUC19 vector. The vector was amplified using primers glk-up-F and glk-down-R to obtain the repair template, which was then purified for subsequent transformation experiments.

[0076] ldhA gene knockout and dld gene insertion: The homologous arm of the ldhA gene was amplified using primers ldhA-19-F and ldhA-19-R, and cloned into the pUC19 vector to construct the ldhA-pUC19 vector. A 23119-dld fragment containing the dld gene was constructed using a similar method and inserted into the ldhA-pUC19 vector to obtain the ldhA-23119-dld-pUC19 vector. The resulting vector was amplified and purified to obtain the repair template required for ldhA gene editing.

[0077] Competent cell preparation and Cas9 plasmid transformation: *E. coli* strain Nissle1917 was activated and cultured at 37℃. Single clones were inoculated into 5 ml LB broth, and the next day, 1% were transferred to 50 ml LB broth. OD was then calculated. 600 Collect the bacterial cells when they reach approximately 0.8 μL, wash them three times with pre-cooled 10% (v / v) glycerol solution, and finally resuspend them in 2 mL of 10% (v / v) glycerol solution to obtain competent cells. Add 10 μL of Cas9 plasmid to the prepared competent cells, place them on ice for 5 min, electroporate at 2500 V, add 1 mL of LB medium, and incubate at 30 °C for 1 h. Then, plate them on Kans resistant plates to obtain the EcN-Cas9 strain. The activation culture and competent cell preparation steps are the same as above. After washing with glycerol three times, resuspend the bacterial cells in 2 mL of 10% glycerol to obtain competent cells for subsequent gene editing experiments.

[0078] Gene editing and acquisition of the Lac-EcN strain: The glk repair template and glk-sgRNA plasmid were co-electroplated and introduced into EcN-Cas9 competent cells. The bacterial culture was plated on double-antibiotic plates containing kanamycin (Kan) and chloramphenicol (Cm) and incubated at 37°C for 16 h for colony selection. Positive clones were picked and passaged in antibiotic-free LB medium to remove the plasmid, obtaining the intermediate strain EcN:Δglk::P23119-lldD. Competent cells of this intermediate strain were prepared, and the ldhA repair template and ldhA-sgRNA plasmid were co-transformed into them. The cells were again plated on double-antibiotic plates containing Kan and Cm for screening of double-antibiotic colonies. After continuous passage culture to remove the plasmid, the Lac-EcN strain was finally obtained.

[0079] 1.2 Identification of Lac-EcN The Lac-EcN strain was validated using PCR (polymerase chain reaction) and sequencing technologies. Using Lac-EcN genomic DNA as a template, PCR amplification was performed using primers glk-up-F / glk-down-R, yielding a 2129 bp product (the wild-type EcN amplification product was 1850 bp), confirming that the glk gene had been successfully replaced by the lldD gene. Using Lac-EcN genomic DNA as a template, PCR amplification was performed using primers ldhA-up-F / ldhA-down-R, yielding a 2422 bp product (the wild-type EcN amplification product was 1642 bp), confirming that the ldhA gene had been successfully replaced by the dld gene. PCR amplification of Lac-EcN was performed using internal primers glk-ter-F / R for the glk gene and ldhA-ter-F / R for the ldhA gene. No products were detected in either case (the corresponding amplification products in wild-type EcN were 832 bp and 745 bp, respectively), indicating that the glk and ldhA genes have been completely knocked out. The integration accuracy and sequence integrity of the 23119-lldD and 23119-dld fragments were verified by first-generation sequencing, confirming that no mutations occurred.

[0080] First-generation sequencing and gel electrophoresis results validated the gene editing effect, showing that glk and ldhA were successfully knocked out, and lldD and dld were precisely inserted. Figure 2 Next, the gene expression levels of Lac-EcN were detected by RNA-seq. The results showed that, compared to EcN, the expression levels of glk and ldhA genes in Lac-EcN were significantly downregulated, while the expression levels of lldD and dld genes were significantly upregulated. Figure 3(a) The results of mass spectrometry-based proteomics analysis were consistent with those of transcriptomics, and the expression levels of other enzymes in the metabolic pathway were not significantly affected. Figure 3 (b and c in the text).

[0081] 1.3Lac-EcN in vitro lactate consumption experiment EcN and Lac-EcN were cultured to the logarithmic growth phase, and 1×10⁻⁶ samples were taken. 9 CFU bacteria were inoculated into 200 ml of M9 medium, and 2 mM glucose and 1 mM lactic acid were added. The medium was then cultured at 37°C with shaking. Samples were taken at different time points, and the concentration of residual lactic acid in the medium was detected using a lactic acid content kit.

[0082] The results are as follows Figure 3 As shown in d, after 6.5 h of in vitro culture, the residual lactic acid concentration in the Lac-EcN group (0.37 mg / ml) was significantly lower than that in the EcN group (0.68 mg / ml). Within a certain time period, the lactic acid consumption efficiency of Lac-EcN was approximately 46% higher than that of EcN. Therefore, Lac-EcN achieved the expected modification of the lactic acid metabolism pathway and is expected to become an engineered probiotic for clearing lactic acid.

[0083] 1.4 Tumor colonization ability of Lac-EcN Inject 5×10 into the tumor of B16-F10 tumor-bearing mice 6 Tumor samples were collected at 24, 72, and 120 hours after CFU injection into EcN or Lac-EcN. The bacterial count per gram of tumor tissue was determined using the CFU counting method. Colony count analysis showed that Lac-EcN retained tumor colonization capacity comparable to the original strain. Specifically, the bacterial load in tumor tissue reached 10^6 bacteria per gram 24 hours after injection. 9 CFU / g, and maintained at 10 for up to 120 h. 8 CFU / g ( Figure 4 (a) The results showed that genetic engineering did not affect the survival and colonization ability of bacteria in tumors, and Lac-EcN maintained the good tumor colonization characteristics of EcN.

[0084] 1.5Lac-EcN's ability to improve the acidic tumor microenvironment Inject 5×10 into the tumor of B16-F10 tumor-bearing mice 6 Lactate concentration in tumor homogenates was measured by LC-MS / MS 24 h after intratumoral injection of CFU-EcN or Lac-EcN. Quantitative analysis by LC-MS / MS showed that, 24 h after intratumoral injection, the lactate content in tumor tissue homogenates injected with Lac-EcN was reduced by approximately 62% compared to the control group. Figure 4(b) To further investigate the effect of lactate clearance on the pH of the tumor tissue (TME), the pH changes in tumor tissue were monitored by flow cytometry using the 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) pH fluorescent probe. The results showed that the fluorescence intensity of the Lac-EcN treatment group was significantly higher than that of the control group (b). Figure 4 The value of c) indicates that the pH value within the tumor tissue is relatively high, and the acidity is significantly alleviated. These results suggest that Lac-EcN can efficiently consume lactic acid accumulated in tumor tissue, thereby reversing the acidic microenvironment of the tumor and creating favorable conditions for restoring anti-tumor immune function.

[0085] Example 2: Lac-EcN enhances anti-tumor immune response To evaluate the effect of Lac-EcN-mediated lactate clearance on tumor-infiltrating immune cells, histopathological analysis and immune cell phenotype identification were performed on tumor tissues after treatment. CD3 immunohistochemical results showed that, 120 h after bacterial treatment, compared with the control group and the wild-type EcN treatment group, the Lac-EcN treatment group had significantly higher levels of CD3 in the tumor. + The infiltration density of T cells increased significantly ( Figure 4 (d) This result was obtained by using flow cytometry to analyze CD4+ in isolated tumor cells. + and CD8 + Quantitative detection of T cell count confirmed this. Figure 4 e and f in Figure 5 This indicates that it can effectively promote T cell recruitment and activation. At the same time, the proportion of Tregs in tumors decreased by approximately 54% after Lac-EcN treatment. Figure 4 (g in the text). The above results indicate that Lac-EcN not only reduces lactate levels and improves the acidic environment through metabolic intervention, but also reshapes the tumor immune microenvironment, enhances the infiltration of effector T cells and inhibits the accumulation of Tregs, thereby alleviating immunosuppression.

[0086] Example 3: Study on the therapeutic effect and mechanism of Lac-EcN combined with anti-PD-1 antibody on B16-F10 tumor mice. 3.1 Intratumoral injection of Lac-EcN synergistically activates tumor immune response with anti-PD-1 antibody 5×10 5 One B16-F10 melanoma cell was subcutaneously inoculated into C57BL / 6 mice, and treatment of tumor-bearing mice began on day 9. Mice carrying B16-F10 tumors received four treatments, each time with an intratumoral injection of 5 × 10⁶ cells. 6CFU bacteria, or intraperitoneal injection of 200 μg anti-PD-1 antibody, or a combination of both, were administered. Tumors were collected 24 hours after the start of treatment for LC-MS / MS analysis. Results showed that Lac-EcN, alone or in combination with anti-PD-1 antibody, reduced lactate concentration and improved the acidic environment, while no such effect was observed with anti-PD-1 antibody alone. Figure 6 (b and c in the original text). This result indicates that Lac-EcN is a key factor in improving tumor lactate accumulation and acidic microenvironment, while anti-PD-1 antibodies have no direct metabolic regulatory role in this process.

[0087] Subsequently, the antitumor efficacy of each treatment group was evaluated. Tumor volume dynamic monitoring results showed that, compared with the PBS control group, EcN injection alone had no significant inhibitory effect on tumor growth, indicating that the wild-type strain lacks intrinsic antitumor activity. Treatment with Lac-EcN alone significantly delayed tumor progression, and approximately 40% of mice exhibited significant rejection of B16-F10 tumors. This may be because Lac-EcN depletes lactate in the TME, making it unable to meet the lactate requirements for tumor growth. Notably, after Lac-EcN combined with anti-PD-1 antibody treatment, approximately 75% of mice showed significant tumor rejection, and some mice achieved long-term complete remission. In contrast, only 37% and 30% of mice treated with anti-PD-1 antibody alone or in combination with EcN, respectively, showed significant tumor rejection. These results were validated in tumor growth curves and mouse survival analysis. Figure 6 f in Figure 7 (a) The above results indicate that Lac-EcN not only possesses independent tumor-suppressing capabilities but also significantly enhances the efficacy of immune checkpoint blockade therapy, demonstrating its great potential as a metabolic-immune modulator.

[0088] 3.1 The immunomodulatory mechanism by which Lac-EcN and anti-PD-1 antibodies exert synergistic effects Previous studies have shown that lactate enters Treg cells via MCT1, promoting the translocation of the transcription factor NFAT1 into the nucleus, thereby inducing PD-1 expression and enhancing its immunosuppressive function; conversely, it inhibits PD-1 expression in CD8+ cells. + In T cells, a high lactate environment inhibits PD-1 expression and weakens its activation. Therefore, Lac-EcN may inhibit PD-1 expression and weaken its activation by affecting Treg and CD8. + The co-regulation of T cells eliminated the immunosuppressive state, thereby enhancing the efficacy of PD-1 blockade therapy. To confirm this mechanism, CD8+ cells were isolated from tumor tissues of B16-F10 tumor-bearing mice after two treatments. + T cells and Foxp3 + CD4 +T cells were analyzed, and their nuclear proteins were extracted. The expression level of NFAT1 in the nucleus was detected by Western blot. Results showed that, compared with the control group, the expression level of CD8+ in the Lac-EcN combined with anti-PD-1 antibody treatment group was significantly higher. + The level of NFAT1 in the T cell nucleus increased by approximately 35%. Figure 6 (d) and the NFAT1 content in the nucleus of Treg cells decreased by about 30% ( Figure 6 (e). This result indicates that Lac-EcN successfully reversed lactate-mediated abnormal nuclear translocation of NFAT1 by clearing lactate from the TME, thereby weakening the immunosuppressive function of Treg cells and promoting CD8+. + The activation and effector capacity of T cells, this regulatory mechanism provides a molecular basis for the synergistic effect of Lac-EcN and PD-1 blockade therapy.

[0089] 3.2 Evaluation of the effect of combination therapy on tumor immune activation To further analyze the effects of Lac-EcN combined with anti-PD-1 antibody therapy on the functional status of immunosuppressive Treg cells and effector T cells, the expression changes of Treg cell surface functional markers and CD8 were systematically evaluated. + The secretion levels of key T cell effector molecules were analyzed. Phenotypic analysis of immune cells using flow cytometry revealed that in mice treated with a combination of Lac-EcN and antibodies, the expression levels of the immunosuppressive markers Neuropilin-1 (Nrp1) and CD44 on the surface of Treg cells decreased by approximately 60% and 48%, respectively, while the expression level of the immune checkpoint molecule PD-1 decreased by approximately 40%. Figure 9 Figure 8 This result indicates that under the dual pressure of sustained lactate metabolism intervention and immune attack, Treg cells enter a state of dysfunction or pre-exhaustion, and their immunosuppressive capacity is subsequently weakened. Simultaneously, it was found that the combination therapy significantly promoted the release of several key effector molecules. CD8 in TME... + The levels of tumor necrosis factor-α (TNF-α), IFN-γ, interleukin-2 (IL-2), and granzyme B (GrB) secreted by T cells were significantly increased by 40%-60%. Figure 8 These molecules (e.g., eh) play a central role in mediating tumor cell killing, promoting T cell proliferation, enhancing antigen presentation, and activating innate immune responses, indicating a robust activation of the overall immune system function. These results demonstrate that Lac-EcN combined with PD-1 blockade therapy not only reverses the abnormal activation state of Treg cells through metabolic intervention, promoting their dysfunctional transformation, but also significantly enhances CD8... +T cells exert their effector function, releasing a variety of key cytokines and cytotoxic molecules, and comprehensively activating the anti-tumor immune response.

[0090] 3.3 Biosafety assessment of EcN and Lac-EcN bacteria in vivo First, the weight changes of tumor-bearing mice were monitored throughout the entire experimental period. The results showed that the weight of mice in both the control group and the bacterial antibody treatment group remained within the normal range without significant fluctuations throughout the entire experimental period. Figure 7 (b) indicates that the bacterial treatment did not cause significant systemic toxicity or metabolic disturbances. Furthermore, histopathological analysis was performed on the major organs (including the heart, liver, spleen, lungs, and kidneys) of the mice. Figure 7 As shown in d, the structures of all organs and tissues remained intact, cell morphology was normal, and no inflammatory cell infiltration, necrosis, or other abnormal pathological changes were observed. These results indicate that Lac-EcN exhibits good in vivo biocompatibility after intratumoral administration, and can achieve targeted tumor colonization without interfering with normal physiological functions or damaging vital organs.

[0091] 3.4 T cell-deficient mouse model to verify synergistic therapeutic effect To investigate whether the function of Lac-EcN in enhancing PD-1 blockade therapy depends on functional T cells, CD3ε gene knockout (CD3e) was selected. - / - Mice were used as a T-cell deficiency model. First, the tumor colonization ability of EcN and Lac-EcN in immunodeficient hosts was evaluated. Results showed that in CD3e... - / - In mice, both intratumorally injected EcN and Lac-EcN effectively colonized B16-F10 tumor tissue, and their colonization levels were not significantly different from those in wild-type mice. Figure 9 (a) indicates that the bacteria's tumor targeting and in vivo survival are not affected by the host's T cell status. Similar to the aforementioned treatment strategy, CD3e... - / - The mice received four treatments. Dynamic monitoring of tumor volume showed that, in the absence of T cells, the anti-PD-1 antibody completely lost its anti-tumor effect; while Lac-EcN treatment alone or in combination with anti-PD-1 antibody could delay tumor growth to some extent, it failed to induce a significant tumor rejection response. Figure 9(b) It is speculated that even with reduced lactate concentration in the TME, mice lacking T cells are still unable to initiate an effective anti-tumor immune response. In this case, Lac-EcN may only slightly delay tumor growth through non-immune mechanisms such as nutrient competition. This result indicates that Lac-EcN's tumor colonization ability is independent of T cells, but its anti-tumor effect, especially its synergistic effect with anti-PD-1, is highly dependent on functional T cells. T cells are not only the target of PD-1 immune checkpoint therapy but also the core effector population for Lac-EcN to activate anti-tumor immunity through metabolic reprogramming. This finding highlights the crucial role of immune system integrity in metabolic intervention therapy and further confirms the core mechanism by which Lac-EcN drives its anti-tumor effect by activating T cell immunity.

[0092] Example 4: Intravenous injection of Lac-EcN enhances tumor-targeted and immunotherapy efficacy. 4.1 Tail vein injection of bacteria can achieve targeted tumor colonization. B16-F10 melanoma-bearing mice were injected via tail vein with 5×10 7 CFU EcN or Lac-EcN, and the distribution of bacteria in the body is tested one week later. For example... Figure 10 As shown in b and c, both strains effectively colonized tumor tissue, while their bacterial content was extremely low in major organs such as the heart, liver, spleen, lungs, and kidneys. This indicates that despite systemic administration via tail vein injection, EcN and its derivative strain Lac-EcN retain their natural tumor tropism, enabling them to specifically colonize tumor tissue after systemic administration, overcoming the limitations of intratumoral injection. Ten days after a single tail vein injection, histopathological analysis of the tumors showed that, compared with the PBS control group or the wild-type EcN treatment group, the Lac-EcN treatment group had significantly higher levels of CD3+ in the tumors. + The density of T-cell infiltration was significantly increased, and no inflammatory response was observed. Figure 10 (d) indicates that even with systemic administration, the engineered bacteria can still effectively promote T cell recruitment and improve the immunosuppressive microenvironment.

[0093] 4.2 Tail vein injection of Lac-EcN and anti-PD-1 antibody can exert a synergistic anti-tumor effect. B16-F10 melanoma-bearing mice were injected once via tail vein with 5×10 7 CFU EcN or Lac-EcN, combined with four doses of anti-PD-1 antibody therapy, or four doses of antibody therapy alone. Dynamic monitoring of tumor volume showed that a single intravenous injection of 5×10⁻⁶ tumor cells... 7CFU Lac-EcN bacteria combined with four doses of anti-PD-1 antibody significantly inhibited tumor growth, with approximately 50% of mice exhibiting marked tumor rejection. In contrast, antibody injection alone or in combination with EcN did not show similar significant efficacy. Figure 10 e in Figure 11 (a) No significant fluctuations in mouse body weight were observed during the treatment process. Figure 11 (b) in the middle.

[0094] In summary, Lac-EcN administered via tail vein injection not only targets and accumulates in tumor tissue but also effectively activates local anti-tumor immune responses and exhibits a significant synergistic effect with PD-1 blockade therapy. More importantly, a single bacterial injection can induce sustained immune activation, suggesting its advantage of long-lasting efficacy.

[0095] Example 5: Lac-EcN bacteria and PD-1 blockade synergistically promote MC-38 tumor rejection response To assess whether the Lac-EcN-mediated metabolic immunotherapy strategy has universality, rather than being limited to the B16-F10 melanoma model, it was extended to another tumor model with different biological characteristics. 5 × 10⁶ mice were subcutaneously injected with the drug. 5 MC-38 mouse colon cancer cells, such as Figure 13 As shown in a, mice carrying MC-38 tumors were treated four times with either EcN or Lac-EcN bacteria in combination with anti-PD-1 antibody. LC-MS / MS analysis showed that 24 hours after intratumoral injection, the lactate concentration in the tumor tissue of the Lac-EcN-treated group was reduced by approximately 40% compared to the control group. Figure 12 (b) , while BCECF-AM fluorescent probe detection showed a significant increase in the pH value of TME ( Figure 12 (c) This confirms that it still possesses highly efficient lactate clearance capacity in the MC-38 model, effectively alleviating acidosis. Further histopathological analysis of tumor tissue showed that, 120 h after treatment, a large number of tumor-infiltrating CD3 groups were observed within the tumors of the Lac-EcN group. + T cells ( Figure 12 (d) It can also promote immune cell recruitment in MC-38 tumors. In the long-term efficacy evaluation, the tumor growth curves showed significant differences among the groups. In the Lac-EcN bacteria combined with anti-PD-1 antibody treatment group, about 70% of the mice showed obvious tumor rejection. Notably, about 50% of the mice achieved complete tumor regression. Figure 12 e in Figure 13The above results indicate that the therapeutic effect of Lac-EcN is not limited to melanoma; it also has anti-tumor therapeutic effects in the MC-38 colon cancer model, and the therapeutic effect is even more significant.

[0096] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A method for constructing a genetically engineered strain that consumes lactic acid, characterized in that, Using probiotic strains with tumor-targeting colonization capabilities as the starting strain, the following modifications were achieved through gene editing: (1) Knockout of lactate biosynthesis-related genes glk Simultaneously insert lactate catabolism gene lldD ; (2) Knockout of lactate biosynthesis-related genes ldhA Simultaneously insert lactate catabolism gene dld ; (3) The gene editing described herein uses homologous recombination to... lldD and dld The gene is integrated into a specific site in the genome of the originating strain.

2. The construction method according to claim 1, characterized in that, The homologous recombination in step (3) is mediated by the CRISPR-Cas system.

3. The construction method according to claim 2, characterized in that, The CRISPR-Cas system is the CRISPR-Cas9 system.

4. The construction method according to claim 3, characterized in that, The homologous recombination is achieved through the following steps: a repair template containing a homologous arm and an sgRNA plasmid are co-transformed into Cas9 competent cells of the starting strain. After screening with two antibiotics, the plasmid is removed by passage culture to obtain the target strain.

5. The construction method according to claim 3, characterized in that, The repair template is a DNA fragment constructed using seamless cloning technology.

6. The construction method according to claim 5, characterized in that, The seamless cloning is glk The repair template corresponding to gene knockout includes glk Upstream and downstream homologous arms and lldD Gene, ldhA The repair template corresponding to gene knockout includes ldhA Upstream and downstream homologous arms and dld Gene.

7. The construction method according to claim 3, characterized in that, The glk The sgRNA sequence of the gene is SEQ ID NO.

29. ldhA The sgRNA sequence of the gene is SEQ ID NO.

30.

8. The construction method according to claim 4, characterized in that, The two antibiotics are kanamycin and chloramphenicol.

9. The construction method according to claim 1, characterized in that, Step (3) dld and lldD Gene expression is driven by constitutive promoters.

10. The construction method according to claim 9, characterized in that, The constitutive promoter is the pJ23119 promoter.

11. The construction method according to claim 1, characterized in that, The probiotics with tumor-targeting colonization capabilities are Escherichia coli, Salmonella, Clostridium, or Bifidobacterium.

12. The construction method according to claim 11, characterized in that, The probiotic with tumor-targeting colonization ability is Escherichia coli.

13. The lactate-consuming genetically engineered strain obtained by the construction method according to any one of claims 1-12.

14. A drug, characterized in that, It includes the genetically engineered strain as described in claim 13.

15. The medicament according to claim 14, characterized in that, The concentration of the genetically engineered strain is 1×10⁻⁶. 6 -1×10 12 CFU / mL.

16. The medicament according to claim 14 or 15, characterized in that, It also includes pharmaceutically acceptable excipients.

17. The medicament according to claim 16, characterized in that, The drug is in the form of an injection.

18. The use of the genetically engineered strain of claim 13 in the preparation of a medicament for treating tumors.

19. The application according to claim 18, characterized in that, The tumor is either melanoma or colon cancer.

20. The application according to claim 18, characterized in that, The drug is used in combination with an anti-PD-1 antibody.

21. The application according to claim 20, characterized in that, The combined administration frequency is consistent.

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