An engineered bacterium strain with strong tumor targeting and intratumoral controllable drug expression and a preparation method and application thereof

Abstract

This invention discloses an engineered bacterial strain with strong tumor targeting and controllable intratumoral drug expression, its preparation method, and its applications. An ST engineered bacterial strain stably expressing OmpA-SpyTag on its outer membrane was obtained through genetic modification. A SpyCatcherΔ-targeting peptide fusion protein (e.g., multiple targeting peptide 4×RGD) was conjugated to its surface to obtain an ST / SC-targeting peptide engineered bacterial strain. This engineered strain, under the recognition and adhesion of the targeting peptide, is efficiently retained in tumor tissue while greatly avoiding off-target migration to healthy organs. The strain colonizing the tumor can continuously proliferate and induce intratumoral expression of antitumor protein drugs through quorum sensing, including: HtrA protein expression to enhance extracellular polysaccharide-mediated immunogenicity and activate immune cells; and therapeutic nanobody expression to synergistically enhance antitumor immunity. This method achieves durable and potent tumor suppression through targeted immune remodeling. This engineered bacterium can achieve more efficient, durable, and safe tumor-targeted therapy.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an engineered bacterial strain that strongly targets tumors and allows for controllable intratumoral drug expression, its preparation method, and its applications. Background Technology

[0002] Bacterial-mediated cancer therapy is a promising strategy for cancer immunotherapy. These bacteria can specifically proliferate within tumors and remodel the tumor microenvironment through their immunogenic components. Furthermore, genetically engineered bacteria can serve as multifunctional carriers for delivering anti-tumor cytokines, nucleic acid drugs, or antibody drugs (L. Wu et al., 2022, Advanced Drug Delivery Reviews, 114363; SY Kwon et al., 2024, Nat RevClin Oncol, 569–589). Despite its promising prospects, this therapy faces significant challenges in clinical application: the inherent proliferative capacity of bacteria and strong immune stimulation may pose toxicity risks; migration to normal tissues can lead to side effects; and even with dose restrictions, the therapy may not be effective within the safe therapeutic window—a problem that has led to the termination of several clinical trials (I. Yelin et al., 2019, Nat Med, 1728–1732). For example, in patients with metastatic melanoma, attenuated Salmonella typhimurium failed to induce tumor regression due to insufficient targeting ability (JF Toso et al., 2002, J Clin Oncol, 142–152; DM Heimann et al., 2003, J Immunother, 179–180). Furthermore, off-target effects and uncontrolled premature release of immune-activating drugs can also damage healthy tissue (L. Wu et al., 2022, Advanced Drug Delivery Reviews, 114363; E. Kolaczkowska et al., 2015, Nat Commun, 6673; SY Kwon et al., 2024, Nat Rev Clin Oncol, 569–589). Therefore, improving the tumor-targeting efficiency of bacteria, enhancing their accumulation and retention within the tumor, and achieving controlled drug release are crucial for maximizing anticancer efficacy.

[0003] Current strategies to enhance bacterial targeting primarily involve displaying specific functional peptides on their outer membranes, such as FimH (Y. Zhang et al., 2022, J Immunother Cancer), CD20-targeting antibodies (PE Massa et al., 2013, Blood, 705–714), and CDH17 nanobodies (X. Xu et al., 2024, Adv Sci (Weinh), e2401905). These modifications often utilize outer membrane proteins (OMPs) (A. Beygmoradi et al., 2023, Int J Biol Macromol, 123407), specific transmembrane signal peptides (Y. Zhang et al., 2022, J Immunother Cancer; QW Chen et al., 2024, Nano Lett, 10362–10371), or click chemistry modifications (L. Wang et al., 2022, Adv Mater, e2106669). This was achieved (Z. Geng et al., 2021, Nat Commun, 6584; Y. Xiao et al., 2024, Signal Transduct Target Ther, 272). Specifically, by displaying an arginine-glycine-aspartic acid (RGD) peptide on the bacterial surface, its high affinity for integrin αvβ3 significantly enhanced the bacterial accumulation capacity in tumor tissues (L. Wu et al., 2024, Advanced Materials, 2406140; S.-H. Park et al., 2016, Theranostics, 1672–1682; Z. Cong et al., 2022, Adv). (Mater, e2201042). However, existing modification strategies still have significant limitations: First, fusion expression based on outer membrane proteins or signal peptides easily leads to protein misfolding, steric hindrance, and low expression levels, requiring optimization for different structures, which is complex and has limited effectiveness; Second, bioconjugation methods such as click chemistry often require the use of organic solvents, which can easily cause irreversible damage to bacterial activity, thus significantly affecting the final therapeutic effect. In addition, the efficacy of traditional bacterial antitumor strategies is generally limited, mainly because although single bacterial therapy can target tumors and induce local immune inflammation, its effect is often constrained by multiple mechanisms of the tumor microenvironment. For example, tumor cells can mediate immune resistance through upregulation of immune checkpoints, resulting in insufficient intensity and limited scope of antitumor response, making it difficult to effectively inhibit tumor growth and metastasis.Therefore, relying solely on bacteria often fails to achieve ideal therapeutic effects. The synergistic effect of in situ expressed antitumor protein drugs (such as immunomodulatory factors or pro-apoptotic molecules) must be utilized to significantly improve treatment efficacy. Such combined strategies, by enhancing the targeted killing ability of bacteria against tumors and synergizing the pharmacological mechanisms of antitumor proteins, hold the promise of overcoming existing limitations in bacterial therapy, thereby achieving more efficient and stable antitumor effects.

[0004] To address the aforementioned challenges, this invention provides a method for constructing engineered bacterial strains with strong tumor targeting and controllable intratumoral drug expression, and its applications. Through genetic modification, this invention stably expresses the OmpA-SpyTag fusion protein on the bacterial outer membrane. Then, using the SpyTag / SpyCatcher system, modular covalent binding with the SpyCatcherΔ-targeting peptide fusion protein is achieved, thereby rapidly and precisely modifying the bacterial surface. SpyTag / SpyCatcher is a gene-encoded click chemistry tool that enables efficient self-assembly of proteins in vitro through the formation of heteropeptide bonds (B. Zakeri et al., 2012, Proc NatlAcad Sci USA, E690–697; AR Sutherland et al., 2019, Chembiochem, 319–328; N. Kasaraneni et al., 2017, mBio). The targeting peptide can be flexibly linked to the N-terminus or C-terminus of SpyCatcherΔ, and multivalent targeting units can be constructed using linkers, significantly enhancing the binding affinity. Taking RGD peptides as an example, this invention successfully constructed a SpyCatcherΔ-4×RGD fusion protein. After co-incubation with engineered bacteria, a strain displaying multivalent RGD on its surface, SpyTag / SpyCatcherΔ-4×RGD, was obtained. This strain achieves efficient intratumoral enrichment and colonization by leveraging the specific binding of RGD to the highly expressed integrin αvβ3 on tumor cells, significantly inhibiting off-target risks. Based on obtaining this novel engineered bacterial strain, this invention further utilizes its initial high tumor targeting and sustained intratumoral proliferation characteristics to express antitumor effector proteins based on quorum sensing promoters, including: HtrA protein expression, enhancing extracellular polysaccharide-mediated immunogenicity and activating immune cells; and antitumor protein drug expression, synergistically enhancing antitumor immunity. This method achieves durable and potent tumor suppression through targeted immune remodeling. Ultimately, this engineered bacterium can achieve more efficient, durable, and safe tumor-targeted therapy. In mouse tumor models, this triple engineering modification endows the bacteria with strong tumor targeting, growth inhibition, and excellent biosafety. The engineered bacterial strain developed in this invention, which exhibits strong tumor targeting and controllable intratumoral drug expression, demonstrates the potential of a gene editing and click chemistry-based synthetic modification strategy. This strategy can simplify the introduction of functional enhancements, personalize the delivery of targeted peptides to anticancer drugs, and improve the biological characteristics of bacterial strains in biomedical applications. Summary of the Invention

[0005] The purpose of this invention is to provide an engineered bacterial strain with strong tumor targeting and controllable intratumoral drug expression, its preparation method, and its anti-tumor application. Specifically, based on *Bacillus subtilis* with a deficient htrA gene, the gene expressing the outer membrane protein OmpA in the *Bacillus subtilis* genome is knocked out using CRISPR-Cas9, and the pTD103(Cm) plasmid continuously expressing OmpA-SpyTAg is used in the *Bacillus subtilis* strain to achieve the display of the SpyTag protein on the bacterial outer membrane, thus constructing the AISI-ST strain. A fusion protein containing a SpyCatcher Δ-multivalent targeting peptide is constructed, and through the covalent binding between SpyTag and SpyCatcher, the fusion protein is displayed on the bacterial outer membrane, obtaining an engineered bacterial strain capable of plug-and-play targeting peptide modification on the outer membrane surface, and producing a tumor-targeting engineered strain displaying multivalent targeting peptides on the bacterial outer membrane surface. Subsequently, the prepared novel engineered strain and its combined application are used in a tumor treatment strategy.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: In the first aspect, the present application provides an engineered attenuated Salmonella with strong tumor targeting and controllable intratumoral drug expression.

[0007] Secondly, this application provides a method for preparing engineered attenuated Salmonella with strong tumor targeting and controllable intratumoral drug expression.

[0008] The first aspect of this application provides an engineered attenuated Salmonella strain with strong tumor targeting and controllable intratumoral drug expression. This engineered attenuated Salmonella strain with strong tumor targeting and controllable intratumoral drug expression is obtained from a chassis strain through the following modifications: (1) AISI strain construction: HtrA protein and outer membrane protein OmpA gene were knocked out in the genome of Salmonella Typhimurium VNP20009 by CRISPR-Cas9; (2) Anchoring unit: OmpA-SpyTag fusion protein located on the bacterial outer membrane, wherein SpyTag is displayed on the bacterial surface; (3) Connecting unit: an isopeptide bond formed by covalent binding of SpyTag and SpyCatcherΔ; (4) Targeting unit: a multivalent targeting peptide fused with the SpyCatcherΔ protein; wherein the multivalent targeting peptide contains at least two targeting peptide fragments that specifically bind to receptors on the surface of tumor cells, and the multivalent targeting peptide is displayed on the bacterial surface through the linker unit, so that the adhesion ability of the engineered strain to tumor cells is significantly enhanced compared with the strain modified with a single-valent targeting peptide. (5) Introducing an expression cassette regulated by a quorum sensing system, the expression cassette induces the expression of therapeutic proteins when bacterial density increases. The therapeutic proteins are selected from one or a combination of two of HtrA proteins and anti-tumor proteins. Anti-tumor proteins include, but are not limited to, endostatin (CN115992083A), anti-PD1 nanobody (CN114958698A), anti-PDL1 nanobody (CN114736840A), TNF-α nanobody (CN117089506A), IFN-β (CN118126918A), etc.

[0009] Further, in step (1), the pTAT plasmid carrying homologous arms flanking the gene is introduced into the VNP and works in conjunction with the pCas plasmid to disrupt gene loci on the genome through allele exchange. The nucleotide sequences of the homologous arms of the pTAT-HtrA-VNP plasmid are shown in SEQ ID NO:1 and SEQ ID NO:2, and the nucleotide sequences of the homologous arms of the pTAT-OmpA-VNP plasmid are shown in SEQ ID NO:3 and SEQ ID NO:4. After removing the plasmid, the ΔhtrA-VNP (AISI) chassis strain is obtained.

[0010] In step (2), the amino acid sequence of the OmpA-SpyTag fusion protein is shown in SEQ ID NO:5, and the amino acid sequence of the mutant OmpA-SpyTag fusion protein is shown in SEQ ID NO:6; the SpyCatcherΔ-multivalent targeting peptide fusion protein comprises, from the N-terminus to the C-terminus, a first targeting peptide unit, a SpyCatcherΔ protein, and a second targeting peptide unit; the targeting peptide units are linked by a linker, and the fusion protein comprises 2-6 targeting peptide units; The chassis strains are original, mutant, or engineered strains of attenuated Salmonella typhimurium, Escherichia coli, Bifidobacterium, BCG, Shigella, Klebsiella pneumoniae, or Pseudomonas aeruginosa with htrA gene defects.

[0011] Further, in step (4), the targeting peptide unit is RGD peptide, the SpyCatcherΔ-multivalent targeting peptide fusion protein is SpyCatcherΔ-RGD, and the amino acid sequence of SpyCatcherΔ-RGD is shown in SEQ ID NO:7; the amino acid sequence of SpyCatcherΔ-multivalent targeting peptide fusion protein SpyCatcherΔ-RGD×2 is shown in SEQ ID NO:8; the amino acid sequence of SpyCatcherΔ-multivalent targeting peptide fusion protein SpyCatcherΔ-RGD×4 is shown in SEQ ID NO:9; the RGD peptide unit is an arginine-glycine-aspartic acid tripeptide sequence, which can specifically recognize the integrin αvβ3 highly expressed on the surface of tumor cells; the chassis of the strain is the attenuated Salmonella Typhimurium AISI with htrA gene deficiency, and the engineered strain is named AISI-ST / SC-RGD×4.

[0012] Furthermore, in step (4), the multivalent tumor-targeting peptide is a tetravalent RGD peptide, wherein the N-terminus and C-terminus of SpyCatcherΔ are respectively linked to the RGD peptide via rigid linkers, and are tandemly linked via flexible linkers to form an SC-RGD×4 structure; the incubation ratio of SpyCatcherΔ-targeting peptide fusion protein to bacteria is 0.1-3 mg:10. 6 -10 8 CFU, incubation temperature is 20-40℃, incubation time is 0.5-5 hours.

[0013] Furthermore, in step (5), the expression cassette regulated by the quorum sensing system contains both the HtrA protein encoding gene and the anti-tumor protein encoding gene, both of which are controlled by the LuxI promoter. The HtrA protein encoding gene is expressed within the bacteria, and the anti-tumor protein encoding gene contains the pelB signal peptide to achieve secretory expression. The anti-tumor proteins include, but are not limited to, endostatin (CN115992083A), anti-PD1 nanobody (CN114958698A), anti-PDL1 nanobody (CN114736840A), TNF-α nanobody (CN117089506A), IFN-β (CN118126918A), etc. The expression cassette regulated by the quorum sensing system also contains the Axe-Txe toxin-antitoxin system to prevent plasmid loss.

[0014] The second aspect of this application provides a method for preparing an engineered attenuated Salmonella strain with strong tumor targeting and controllable intratumoral drug expression, comprising the following steps: (1) The OmpA gene in the genome of a chassis strain with a defective htrA gene was knocked out using the CRISPR-Cas9 system to construct the ΔOmpA chassis strain; (2) Construct and transform plasmid pTD103(Cm)-OmpA-GS-SpyTag-GS to obtain engineered bacterial strains with SpyTag displayed on the outer membrane; (3) Express and purify SpyCatcher Δ-targeting peptide fusion protein; (4) The strain obtained in step (2) is co-incubated with the fusion protein obtained in step (3), and the surface modification is completed by covalent binding of SpyTag-SpyCatcher; (5) Construct and transfer the quorum sensing regulatory plasmid pTD103-LuxI-antitumor effector protein to obtain an engineered strain with controllable intratumoral drug expression.

[0015] The second aspect of this application provides the use of an engineered attenuated Salmonella strain in the preparation of antitumor drugs.

[0016] Furthermore, tumors include one or more of the following: melanoma, lung cancer, lymphoma, bladder cancer, liver cancer, colorectal cancer, and breast cancer.

[0017] Furthermore, the route of administration of the drug is selected from any one of intraperitoneal injection, intravenous injection, oral instillation, intratumoral injection, or bladder instillation; the dosage of the drug is 10. 4 -10 7 CFU / mouse, administered once or multiple times.

[0018] Furthermore, the drug is also used in combination with one or more of the following: chemotherapy drugs, immune checkpoint inhibitors, radiotherapy, photothermal therapy, CAR-T cell therapy, or other bacterial therapies.

[0019] Beneficial Effects: The engineered bacterial strain described in this invention, characterized by strong tumor targeting and controllable intratumoral drug expression, exerts its anti-tumor effects through multiple synergistic mechanisms. This strain utilizes a surface-expressed SpyTag protein to bio-sponge with the SpyCatcherΔ-RGD×4 fusion protein, thereby specifically recognizing and binding to the highly expressed αvβ3 integrin on tumor cells, achieving tumor-targeted adhesion. In the tumor microenvironment, increased bacterial density activates the quorum sensing system, inducing HtrA protein expression, which in turn promotes extracellular polysaccharide (EPS) synthesis via the HtrA–Lon–RcsA axis. The increased EPS directly activates macrophages through the TLR4-NFκB signaling pathway, driving them towards the M1 anti-tumor phenotype and enhancing the overall immune response. Simultaneously, the quorum sensing system also regulates the secretion of anti-tumor proteins, such as anti-PD1 nanobodies (PD1nb), blocking the PD1-PDL1 immunosuppressive pathway and reversing T cell dysfunction. The synergistic effect of HtrA and anti-tumor proteins significantly enhances the infiltration of anti-tumor immune cells (such as M1 macrophages, mature dendritic cells, and CD8⁺T cells) while inhibiting pro-tumor cells (such as M2 macrophages and CD4⁺Tregs), ultimately achieving highly efficient tumor suppression.

[0020] Compared with the prior art, the present invention has the following advantages: (1) Strong tumor targeting: By modifying the surface of bacteria with multivalent RGD peptides (such as RGD×4) through the SpyTag / SpyCatcher system, the adhesion ability of bacteria to tumor cells and the enrichment efficiency within the tumor are significantly improved. This modification method has higher tumor targeting than the traditional single RGD peptide modification. (2) Reduced non-specific accumulation: The enrichment of RGD-modified strains in tumors is significantly increased, while the accumulation in non-target organs (such as liver and spleen) is reduced, thereby reducing potential side effects. (3) Precise intratumoral drug release: The expression of HtrA protein and anti-tumor protein (such as PD1nb) is regulated by the quorum sensing system to ensure that these therapeutic proteins are expressed and secreted only under high-density bacterial conditions inside the tumor, thereby reducing side effects on normal tissues. This dynamic regulation mechanism improves the precision and safety of treatment. (4) Enhanced anti-tumor immune response: HtrA protein activates macrophages and promotes their polarization into the anti-tumor M1 type by increasing the level of bacterial extracellular polysaccharides (EPS). Meanwhile, PD1nb reverses immunosuppression by blocking the PD1-PDL1 pathway, further enhancing the anti-tumor immune response. This dual regulatory mechanism significantly enhances the anti-tumor effect of the strain. (5) Synergistic anti-tumor effect: The simultaneous expression and secretion of HtrA protein and anti-tumor proteins (such as PD1nb) produce a synergistic effect within the tumor, enhancing the anti-tumor immune response, including increasing anti-tumor immune cells (such as M1 macrophages, mature dendritic cells and CD8+). +The proportion of T cells was reduced, while the number of pro-tumor immune cells (such as M2 macrophages and CD4 cells) was decreased. + The proportion of Tregs was increased. This synergistic effect improved the overall antitumor ability of the strain. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram illustrating the construction and therapeutic mechanism of the octopus-inspired triple-engineered bacteria (OITE strain) of the present invention.

[0023] Figure 2 The SpyTag protein structure of the present invention is inserted into the third transmembrane domain of OmpA. The SpyTag-DA protein structure is used as a blank control.

[0024] Figure 3 This is a schematic diagram of the molecular structure of the SpyCatcherΔ-eGFP fusion protein obtained in this invention.

[0025] Figure 4 Different amounts of SpyCatcher Δ-eGFP (0.5 / 1 / 2 / 3 / 4 mg) and AISI-ST (curve 1) or AISI-SD (curve 2) strains of the present invention were used to test different amounts of SpyCatcher Δ-eGFP (0.5 / 1 / 2 / 3 / 4 mg) and strain 10. 8 The graph shows the change in total fluorescence intensity of the strain after 1 h of CFU co-incubation. n = 3. AISI-SD represents AISI-SpyTag DA.

[0026] Figure 5 The present invention uses 3 mg SpyCatcherΔ-eGFP and 10 μg of AISI-ST (curve 1) or AISI-SD (curve 2) strains. 8 The graph shows the changes in total fluorescence intensity of the strains after co-incubation with CFU for different times (0.01, 0.5, 1, 2, 4, or 6 hours). n=3. AISI-SD represents AISI-SpyTag DA.

[0027] Figure 6 This is a predicted structural diagram of the SpyCatcherΔ-RGD×4 fusion protein of the present invention.

[0028] Figure 7This figure shows the number of bacteria adhering to the bottom of a culture dish after co-culturing different engineered bacterial strains of the present invention in a random field of view after the dish was coated with αvβ3 protein. n=8, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The right figure shows representative images of the adhesion of different bacteria to αvβ3 protein, scale bar = 20µM. Engineered bacterial strains stably expressing red fluorescent protein (RFP) were used. The group supplemented with bovine serum albumin (BSA) at the bottom served as the control group. 1 and 3 represent AISI-ST / SC-RGE, and 2 and 4 represent AISI-ST / SC-RGD×4.

[0029] Figure 8 This is a comparison of bacterial titers in tumor tissue 12 hours after treatment with different engineered bacterial strains of the present invention, n=5. 1 represents AISI-ST / SC-RGE, 2 represents AISI-ST / SC-RGD×1, 3 represents AISI-ST / SC-RGD×2, and 4 represents AISI-ST / SC-RGD×4. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0030] Figure 9 This is a graph comparing bacterial titers in the liver, spleen, and other normal organs (including the heart, lungs, and kidneys) 12 hours after treatment with different engineered bacterial strains of the present invention, n=5. 1 represents AISI-ST / SC-RGD, 2 represents AISI-ST / SC-RGD×1, 3 represents AISI-ST / SC-RGD×2, and 4 represents AISI-ST / SC-RGD×4. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0031] Figure 10 This graph shows the ratio of colony-forming units (CFU) in tumors to those in the liver and spleen per gram of tissue 12 hours after treatment with different engineered bacterial strains of the present invention. The average ratios of tumor / spleen (left) and tumor / liver (right) are shown.

[0032] Figure 11 This graph shows the changes in bacterial density and bioluminescent flux in mouse tumors 18, 36, and 72 hours after treatment with different engineered bacterial strains of the present invention. The chassis engineered bacterial strain AISI-L-ST, used for density-controlled bioluminescence, was employed. n = 6, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 1 represents the AISI-L-ST / SC-RGE strain, and 2 represents the AISI-L-ST / SC-RGD×4 strain.

[0033] Figure 12 These are representative animal images taken 36 hours after treatment with different engineered bacterial strains of the present invention, n = 6. 1 represents the AISI-L-ST / SC-RGE strain, and 2 represents the AISI-L-ST / SC-RGD×4 strain.

[0034] Figure 13 This diagram illustrates the controllable extracellular polysaccharide (EPS)-mediated virulence restoration pathway of *AISI-ST* *Chaetomium*, a strain of this invention. The expression of the htrA gene is controlled by the LuxI promoter, causing the bacterial HtrA protein to be re-expressed only at high population densities. This novel strain is named AISI-H-ST. The strain AISI-B-ST, carrying the corresponding blank plasmid, serves as a control.

[0035] Figure 14 This is an immunoblotting analysis of HtrA expression in different bacterial strains under high bacterial density conditions in vitro, according to the present invention. 1 represents AISI-B-ST strain, and 2 represents AISI-H-ST strain.

[0036] Figure 15 This is a comparison chart of EPS levels among different strains under the same bacterial count conditions according to the present invention. n=6, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 1 represents AISI-B-ST strain, and 2 represents AISI-H-ST strain.

[0037] Figure 16 This diagram illustrates the co-culture of macrophages activated by different strains of the present invention with B16-F10-GFP tumor cells (left). After 12 hours of co-culture, the relative fluorescence intensity differences of tumor cells in different groups were compared (right). n=5, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 1 represents the blank group, 2 represents the saline group, 3 represents the AISI-B-ST strain, and 4 represents the AISI-H-ST strain.

[0038] Figure 17 The image shows tumor growth curves in mice treated with different engineered bacterial strains according to this invention. n = 7, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 1 represents the saline group, 2 represents AISI-B-ST / SC-RGD×4, 3 represents AISI-H-ST / SC-RGE, and 4 represents AISI-H-ST / SC-RGD×4.

[0039] Figure 18Survival curves of mice treated with different engineered bacterial strains according to this invention are shown. Mice were euthanized at the humane endpoint. n = 7, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 1 represents the saline group, 2 represents AISI-B-ST / SC-RGD×4, 3 represents AISI-H-ST / SC-RGD×4, and 4 represents AISI-H-ST / SC-RGD×4.

[0040] Figure 19 This is a schematic diagram of the pathway of the engineered AISI-HtrA&PD1nb-SpyTag (AISI-HP-ST strain) based on AISI-ST chassis bacteria. The expression of HtrA and the secretion of PD1nb are regulated by the LuxI promoter.

[0041] Figure 20 This invention provides an immunoblotting analysis of the relationship between bacterial density and PD1nb expression in the AISI-HP-ST strain.

[0042] Figure 21 This is a graph showing the content of PD1nb secreted in the supernatant of AISI-HP-ST strain at OD600 = 0.8, as determined by immunoblotting analysis in this invention. The bar chart analyzes the relationship between bacterial density and PD1nb expression in AISI-HP-ST strain. 1 represents OD600 = 0.2, 2 represents OD600 = 0.4, 3 represents OD600 = 0.6, and 4 represents OD600 = 0.8.

[0043] Figure 22 The values ​​represent the protein expression levels of HtrA and PD1nb in different strains of this invention. 1 represents AISI-B-ST, 2 represents AISI-H-ST, and 3 represents AISI-HP-ST.

[0044] Figure 23 This is a tumor growth curve of mice treated with different engineered bacterial strains according to the present invention. n = 7, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 1 represents the saline group, 2 represents AISI-B-ST / SC-RGD×4, 3 represents AISI-H-ST / SC-RGD, 4 represents AISI-PD1nb-ST / SC-RGD×4, and 5 represents AISI-HP-ST / SC-RGD×4.

[0045] Figure 24On day 16 after treatment with different engineered bacterial strains of this invention, tumors in mice were photographed (left image) and weighed (right image). Scale bar = 10 mm, n = 7, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 1 represents the saline group, 2 represents AISI-B-ST / SC-RGD×4, 3 represents AISI-H-ST / SC-RGD, 4 represents AISI-PD1nb-ST / SC-RGD×4, and 5 represents AISI-HP-ST / SC-RGD×4.

[0046] Figure 25 This is a survival curve of mice treated with different engineered bacterial strains according to the present invention. Mice were euthanized when they reached the humane endpoint. n = 7, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 1 represents the saline group, 2 represents AISI-B-ST / SC-RGD×4, 3 represents AISI-H-ST / SC-RGD, 4 represents AISI-PD1nb-ST / SC-RGD×4, and 5 represents AISI-HP-ST / SC-RGD×4.

[0047] Figure 26 The total CD8 count of mouse tumors treated with different engineered bacterial strains according to this invention. + Non-CD8 T cells + Effector T cells (CD8) + GzmB - ), proliferative CD8 + Effector T cells (CD8) + GzmB + Ki67 + ) and non-proliferative CD8 + Effector T cells (CD8) + GzmB + Ki67 - The percentage chart.

[0048] Figure 27 For the present invention Figure 26 Percentage bar chart analysis. Detailed Implementation

[0049] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply.

[0050] The first aspect of this application provides an engineered attenuated Salmonella strain with strong tumor targeting and controllable intratumoral drug expression. This engineered attenuated Salmonella strain with strong tumor targeting and controllable intratumoral drug expression is obtained from a chassis strain through the following modifications: (1) AISI strain construction: HtrA protein and outer membrane protein OmpA gene were knocked out in the genome of Salmonella Typhimurium VNP20009 by CRISPR-Cas9; (2) Anchoring unit: OmpA-SpyTag fusion protein located on the bacterial outer membrane, wherein SpyTag is displayed on the bacterial surface; (3) Connecting unit: an isopeptide bond formed by covalent binding of SpyTag and SpyCatcherΔ; (4) Targeting unit: a multivalent targeting peptide fused with the SpyCatcherΔ protein; wherein the multivalent targeting peptide contains at least two targeting peptide fragments that specifically bind to receptors on the surface of tumor cells, and the multivalent targeting peptide is displayed on the bacterial surface through the linker unit, so that the adhesion ability of the engineered strain to tumor cells is significantly enhanced compared with the strain modified with a single-valent targeting peptide. (5) Introducing an expression cassette regulated by a quorum sensing system, the expression cassette induces the expression of therapeutic proteins when bacterial density increases. The therapeutic proteins are selected from one or a combination of two of HtrA proteins and anti-tumor proteins. Anti-tumor proteins include, but are not limited to, endostatin (CN115992083A), anti-PD1 nanobody (CN114958698A), anti-PDL1 nanobody (CN114736840A), TNF-α nanobody (CN117089506A), IFN-β (CN118126918A), etc.

[0051] In some embodiments, in step (1), the pTAT plasmid carrying homologous arms flanking the gene is introduced into the VNP and works in conjunction with the pCas plasmid to disrupt gene loci on the genome through allele exchange. The nucleotide sequences of the homologous arms of the pTAT-HtrA-VNP plasmid are shown in SEQ ID NO:1 and SEQ ID NO:2, and the nucleotide sequences of the homologous arms of the pTAT-OmpA-VNP plasmid are shown in SEQ ID NO:3 and SEQ ID NO:4. After removing the plasmid, the ΔhtrA-VNP (AISI) chassis strain is obtained.

[0052] In step (2), the amino acid sequence of the OmpA-SpyTag fusion protein is shown in SEQ ID NO:5, and the amino acid sequence of the mutant OmpA-SpyTag fusion protein is shown in SEQ ID NO:6; the SpyCatcherΔ-multivalent targeting peptide fusion protein comprises, from the N-terminus to the C-terminus, a first targeting peptide unit, a SpyCatcherΔ-protein, and a second targeting peptide unit; the targeting peptide units are linked by a linker, and the fusion protein contains 2-6 targeting peptide units; The chassis strains are original, mutant, or engineered strains of attenuated Salmonella typhimurium, Escherichia coli, Bifidobacterium, BCG, Shigella, Klebsiella pneumoniae, or Pseudomonas aeruginosa with htrA gene defects.

[0053] In some embodiments, in step (4), the targeting peptide unit is RGD peptide, the SpyCatcherΔ-multivalent targeting peptide fusion protein is SpyCatcherΔ-RGD, and the amino acid sequence of SpyCatcherΔ-RGD is shown in SEQ ID NO:7; the amino acid sequence of the SpyCatcherΔ-multivalent targeting peptide fusion protein SpyCatcherΔ-RGD×2 is shown in SEQ ID NO:8; the amino acid sequence of the SpyCatcherΔ-multivalent targeting peptide fusion protein SpyCatcherΔ-RGD×4 is shown in SEQ ID NO:9; the RGD peptide unit is an arginine-glycine-aspartic acid tripeptide sequence, which can specifically recognize the integrin αvβ3 highly expressed on the surface of tumor cells; the chassis of the strain is an attenuated Salmonella Typhimurium AISI with htrA gene deficiency, and the engineered strain is named AISI-ST / SC-RGD×4.

[0054] In some embodiments, in step (4), the multivalent tumor-targeting peptide is a tetravalent RGD peptide, wherein the N-terminus and C-terminus of SpyCatcherΔ are respectively linked to the RGD peptide via rigid linkers and tandemly connected via flexible linkers to form an SC-RGD×4 structure; the incubation ratio of the SpyCatcherΔ-targeting peptide fusion protein to bacteria is 0.1-3 mg:10. 6 -10 8 CFU, incubation temperature is 20-40℃, incubation time is 0.5-5 hours.

[0055] In some embodiments, in step (5), the expression cassette regulated by the quorum sensing system simultaneously contains an HtrA protein-coding gene and an anti-tumor protein-coding gene, both of which are controlled by the LuxI promoter. The HtrA protein-coding gene is expressed within bacteria, and the anti-tumor protein-coding gene contains the pelB signal peptide for secretory expression. The anti-tumor protein includes, but is not limited to, endostatin (CN115992083A), anti-PD1 nanobody (CN114958698A), anti-PDL1 nanobody (CN114736840A), TNF-α nanobody (CN117089506A), IFN-β (CN118126918A), etc. The expression cassette regulated by the quorum sensing system also includes an Axe-Txe toxin-antitoxin system to prevent plasmid loss.

[0056] The second aspect of this application provides a method for preparing an engineered attenuated Salmonella strain with strong tumor targeting and controllable intratumoral drug expression, comprising the following steps: (1) The OmpA gene in the genome of a chassis strain with a defective htrA gene was knocked out using the CRISPR-Cas9 system to construct the ΔOmpA chassis strain; (2) Construct and transform plasmid pTD103(Cm)-OmpA-GS-SpyTag-GS to obtain engineered bacterial strains with SpyTag displayed on the outer membrane; (3) Express and purify SpyCatcher Δ-targeting peptide fusion protein; (4) The strain obtained in step (2) is co-incubated with the fusion protein obtained in step (3), and the surface modification is completed by covalent binding of SpyTag / SpyCatcher; (5) Construct and transfer the quorum sensing regulatory plasmid pTD103-LuxI-antitumor effector protein to obtain an engineered strain with controllable intratumoral drug expression.

[0057] The third aspect of this application provides the application of an engineered attenuated Salmonella strain in the preparation of antitumor drugs.

[0058] In some embodiments, the tumor includes one or more of the following: melanoma, lung cancer, lymphoma, bladder cancer, liver cancer, colorectal cancer, and breast cancer.

[0059] In some embodiments, the route of administration of the drug is selected from any one of intraperitoneal injection, intravenous injection, oral instillation, intratumoral injection, or bladder instillation; the dosage of the drug is 10. 4 -10 7 CFU / mouse, administered once or multiple times.

[0060] Furthermore, the drug is also used in combination with one or more of the following: chemotherapy drugs, immune checkpoint inhibitors, radiotherapy, photothermal therapy, CAR-T cell therapy, or other bacterial therapies. Example

[0061] The first aspect of this application of the present invention provides an engineered attenuated Salmonella strain with strong tumor targeting and controllable intratumoral drug expression. This engineered attenuated Salmonella strain with strong tumor targeting and controllable intratumoral drug expression is obtained from a chassis strain through the following modifications: (1) Construction of AISI strain: The HtrA protein and outer membrane protein OmpA gene were knocked out in the genome of Salmonella Typhimurium VNP20009 using CRISPR-Cas9. The sgRNA and PAM sequences of the htrA and OmpA genes were designed to construct the PTAT plasmid. The pTAT plasmid carrying homologous arms flanking the genes was introduced into VNP and worked in conjunction with the pCas plasmid to disrupt gene loci on the genome through allele exchange. The nucleotide sequences of the homologous arms of the pTAT-HtrA-VNP plasmid are shown in SEQ ID NO:1 and SEQ ID NO:2, and the nucleotide sequences of the homologous arms of the pTAT-OmpA-VNP plasmid are shown in SEQ ID NO:3 and SEQ ID NO:4. After removing the plasmid, the ΔhtrA-VNP (AISI) chassis strain was obtained.

[0062] (2) Anchoring unit: OmpA-SpyTag fusion protein located on the bacterial outer membrane, wherein SpyTag is displayed on the bacterial surface; the amino acid sequence of OmpA-SpyTag fusion protein is shown in SEQ ID NO:5, and the amino acid sequence of mutant OmpA-SpyTag fusion protein is shown in SEQ ID NO:6; the SpyCatcherΔ-multivalent targeting peptide fusion protein contains a first targeting peptide unit, a SpyCatcherΔ protein and a second targeting peptide unit from the N-terminus to the C-terminus; the targeting peptide units are linked by a linker, and the fusion protein contains 2-6 targeting peptide units; The chassis strains are original, mutant, or engineered strains of attenuated Salmonella typhimurium, Escherichia coli, Bifidobacterium, BCG, Shigella, Klebsiella pneumoniae, or Pseudomonas aeruginosa with htrA gene defects.

[0063] (3) Connecting unit: an isopeptide bond formed by covalent binding of SpyTag and SpyCatcherΔ; (4) Targeting unit: a multivalent targeting peptide fused with SpyCatcherΔ protein; wherein the multivalent targeting peptide contains at least two targeting peptide fragments that specifically bind to receptors on the surface of tumor cells, and the multivalent targeting peptide is displayed on the bacterial surface through the linker unit, thereby significantly enhancing the adhesion ability of the engineered strain to tumor cells compared with the strain modified with a single-valent targeting peptide; the targeting peptide unit is an RGD peptide, the SpyCatcherΔ-multivalent targeting peptide fusion protein is SpyCatcherΔ-RGD, and the amino acid sequence of SpyCatcherΔ-RGD is shown in SEQ ID NO:7; the amino acid sequence of the SpyCatcherΔ-multivalent targeting peptide fusion protein SpyCatcherΔ-RGD×2 is shown in SEQ ID NO:8; the amino acid sequence of the SpyCatcherΔ-multivalent targeting peptide fusion protein SpyCatcherΔ-RGD×4 is shown in SEQ ID NO:8. As shown in NO:9; the RGD peptide unit is an arginine-glycine-aspartic acid tripeptide sequence, which can specifically recognize integrin αvβ3, which is highly expressed on the surface of tumor cells; the chassis of the strain is an attenuated Salmonella typhimurium AISI with a defective htrA gene, and the engineered strain is named AISI-ST / SC-RGD×4.

[0064] (5) Introducing an expression cassette regulated by a quorum sensing system. The expression cassette induces the expression of therapeutic proteins when bacterial density increases. The therapeutic proteins are selected from one or a combination of two of HtrA proteins and anti-tumor proteins. Anti-tumor proteins include, but are not limited to, endostatin (CN115992083A), anti-PD1 nanobody (CN114958698A), anti-PDL1 nanobody (CN114736840A), TNF-α nanobody (CN117089506A), IFN-β (CN118126918A), etc. Example

[0065] The SpyCatcherΔ-RGD fusion protein was obtained by constructing BL21(DE3) strains expressing SpyCatcherΔ-RGD fusion proteins of different valence states (such as SC-RGD×1, SC-RGD×2 and SC-RGD×4), expanding the culture, purifying the SpyCatcherΔ-RGD fusion protein with Ni-NTA agarose resin and ultrafiltration tubes, and finally determining the concentration, dispensing, and storing at -80℃ for later use.

[0066] The engineered, attenuated Salmonella strain with strong tumor targeting is an AISI-ST strain expressing OmpA-SpyTag on its outer membrane, combined with purified SpyCatcherΔ fusion protein at a ratio of 0.1-3 mg: 102. 6 -10 8CFU was co-incubated at 20-40℃ for 0.5-5 hours. The fusion protein was displayed on the bacterial outer membrane through covalent binding between SpyTag and SpyCatcher, resulting in engineered attenuated Salmonella with a plug-and-play system. Then, SpyCatcherΔ-RGD×4 was used instead of SpyCatcherΔ-eGFP to construct an engineered attenuated Salmonella with strong tumor targeting.

[0067] The present invention relates to a method for preparing engineered bacterial strains with strong tumor targeting and controllable intratumoral drug expression, and their application in antitumor therapy.

[0068] The engineered bacterial strain with strong tumor targeting and controllable intratumoral drug expression was constructed by transferring pTD103-LuxI-LuxCDABE, which is quorum sensing-regulated, into the AISI-ST chassis strain to create the AISI-L-ST strain. In vivo animal imaging showed that the AISI-L-ST / SC-RGD×4 strain initiated bioluminescence earlier than the AISI-L-ST strain. After confirming the acquisition of an engineered attenuated Salmonella strain with strong tumor targeting and controllable intratumoral drug expression, HtrA protein and PD1nb antitumor protein were used instead of LuxCDABE to construct an engineered bacterial strain with strong tumor targeting and controllable intratumoral drug expression.

[0069] The present invention relates to an engineered bacterial strain with strong tumor targeting and controllable intratumoral drug expression, and its application in anti-tumor treatment.

[0070] Compared to unmodified Salmonella, the attenuated Salmonella modified with RGD peptide, after intravenous injection, showed significantly higher tumor targeting efficiency of the AISI-ST / SC-RGD×4 strain than the AISI-ST / SC-RGE strain. For example, the tumor / spleen ratio of the AISI-ST / SC-RGD×4 strain was 65.7-fold and the tumor / liver ratio was 74.9-fold, while the corresponding ratios of the AISI-ST / SC-RGE strain were lower.

[0071] Compared to attenuated Salmonella modified with RGD peptide, the engineered bacterial strain with strong tumor targeting and controllable intratumoral drug expression significantly increased the number of necrotic cells and infiltrating immune cells within the tumor. Furthermore, the AISI-HP-ST / SC-RGD×4 strain induced superior tumor suppression, with an average tumor weight of 0.146 grams, equivalent to 16.7% of the saline group. After three days of treatment, the tumor necrosis area exceeded 60%, and the survival rate of tumor-bearing mice increased to 100%.

[0072] The therapeutic dose of engineered bacterial strains with strong tumor targeting and controllable intratumoral drug expression is 10 mg / L of engineered bacterial strain administered via tail vein injection. 4 -10 7CFU / mouse, administered as a single or multiple doses, where the unit is CFU / mouse. The engineered bacterial strains with strong tumor targeting and controllable intratumoral drug expression can be administered via intraperitoneal injection, intravenous injection, oral instillation, intratumoral injection, or bladder instillation.

[0073] The bacterial preparations include glycerol bacteria, lyophilized bacterial powder, etc., and the targeted peptide fusion proteins include lyophilized protein powder, protein solution, etc. The administration methods include intravenous injection, oral gavage, intraperitoneal injection, intravesical instillation, intratumoral injection, etc.

[0074] The following describes the implementation of a synergistic antitumor strategy using attenuated Salmonella Typhimurium AISI strain with htrA gene deficiency as a representative chassis strain, RGD as a representative targeting peptide, and quorum sensing-mediated simultaneous expression of HtrA protein and anti-PD1 nanobody as a representative synergistic antitumor strategy. However, it should be clearly stated that this system is not limited to AISI strain, but also includes RGD targeting peptide, HtrA protein, endostatin, anti-PD1 nanobody, anti-PDL1 nanobody, TNF-α nanobody, and IFN-β. Example

[0075] This invention discloses a method for preparing an engineered bacterial strain with strong tumor targeting and controllable intratumoral drug expression, and its anticancer application. The method for preparing the tumor-targeting engineered attenuated Salmonella involves stably expressing the SpyTag polypeptide on the bacterial outer membrane of engineered attenuated Salmonella AISI-ST. The display of the SpyTag polypeptide allows for transient linkage with the SpyCatcher Δ-multivalent targeting peptide fusion protein, achieving plug-and-play engineered modification of Salmonella AISI. This enhances AISI's recognition and enrichment in tumor tissues, enabling faster colonization at tumor sites. Modification of Salmonella AISI with the targeting peptide improves the efficiency of targeted delivery of Salmonella for tumor therapy, significantly inhibiting tumor growth and prolonging survival time in mice.

[0076] The present invention provides a method for preparing a tumor-targeting engineered attenuated Salmonella, comprising the following steps: (1) Knocking out the htrA gene in the genome of Salmonella Typhimurium VNP20009 using CRISPR-Cas9. Designing the sgRNA and PAM sequences of the HtrA gene, constructing the plasmid PTAT-HtrA-VNP, transforming the recombinant plasmid into competent Escherichia coli cells, plating for verification, picking positive clones and performing sequencing verification. Simultaneously, preparing VNP20009 competent cells, performing electroporation of the pCas plasmid under specific conditions, verifying the electroporation results, and constructing the pCas-VNP strain. Then, arabinose-induced λ-red recombinase production and competent cell preparation were performed on the pCas-VNP strain, and the extracted plasmid PTAT-OmpA-VNP was electroporated into pCas-VNP, plated for culture, and single colonies were picked for PCR verification. IPTG was added to the verified strain and passaged overnight, the PTAT plasmid was removed, streaked, and single colonies were picked for preservation. The pCas plasmid was removed by overnight incubation at 37°C. Single bacteria were picked and preserved. ΔhtrA-VNP (AISI) chassis strains were obtained.

[0077] The gene for the outer membrane protein OmpA was knocked out of the AISI genome using CRISPR-Cas9. The sgRNA and PAM sequences of the OmpA gene were designed, and the plasmid PTAT-OmpA-VNP was constructed. This recombinant plasmid was transformed into competent *E. coli* cells, plated for verification, and positive clones were picked for sequencing verification. Simultaneously, AISI competent cells were prepared, and the pCas plasmid was electroporated under specific conditions. The electroporation results were verified, and the pCas-AISI strain was constructed. Then, the pCas-AISI strain was subjected to arabinose-induced λ-red recombinase production and competent cell preparation. The extracted plasmid PTAT-OmpA-VNP was electroporated into pCas-AISI, plated, and single colonies were picked for PCR verification. IPTG was added to the correctly verified strain and passaged overnight to remove the PTAT plasmid. The cells were streaked, and single colonies were picked for preservation. The cells were then incubated overnight at 37°C to remove the pCas plasmid, and single colonies were picked and preserved. The ΔOmpA-AISI chassis strain was obtained.

[0078] The plasmid pTD103(Cm)-OmpA-GS-SpyTag-GS was designed and constructed. The OmpA-GS-SpyTag-GS gene was inserted into the pTD103(Cm) plasmid backbone containing the p15A origin of replication (10-15 copies per cell). The expression of the target gene OmpA-GS-SpyTag-GS was driven by the J23100 promoter, and the B0010 terminator was used to prevent transcriptional readthrough. Chloramphenicol resistance (CmR) was used as a selection marker. The extracted plasmid pTD103(Cm)-OmpA-GS-SpyTag-GS was transformed into ΔOmpA-AISI chassis strain, plated, and single colonies were picked for PCR verification. After streak culture, single colonies were picked for preservation.

[0079] A protein purification plasmid expressing SpyCatcherΔ-RGD fusion protein was constructed, in which the RGD peptide was linked to the N-terminus and C-terminus of SpyCatcherΔ via a linker, forming different oligomeric forms of the RGD peptide (such as SC-RGD×1, SC-RGD×2 and SC-RGD×4). First, *E. coli* strains containing plasmids expressing the SpyCatcherΔ-RGD fusion protein were inoculated into LB medium for activation, then transferred to expansion medium for culture. When the bacterial density reached the logarithmic growth phase, a suitable inducer was added, and expression was induced at 4-25°C for 12-72 hours. After induction, the bacterial cells were collected by centrifugation, resuspended in lysis buffer, and then lysed by sonication. After centrifugation to remove bacterial debris, a crude supernatant was obtained. The supernatant was then passed through a pre-equilibrated affinity chromatography column to specifically bind the target protein to the column. Non-specifically bound proteins were removed with elution buffer containing 100 mM imidazole, and finally, the high-purity target protein was eluted with elution buffer containing 150 mM imidazole. The purified protein was further concentrated by ultrafiltration tubes with appropriate molecular weight cutoff, and finally, the concentration was determined, aliquoted, and stored at -80°C for later use.

[0080] To construct engineered attenuated Salmonella strains with strong tumor targeting, the AISI-ST strain expressing OmpA-SpyTag on its outer membrane was combined with the purified fusion protein SpyCatcherΔ-eGFP at a ratio of 0.1-3 mg: 102. 6 -10 8CFU was co-incubated at 20-40℃ for 0.5-5 h. The fusion protein was displayed on the bacterial outer membrane via covalent binding between SpyTag and SpyCatcher, yielding engineered attenuated Salmonella with a plug-and-play system. Flow cytometry (BD FACSCanto II) and fluorescence microscopy were used to detect the GFP fluorescence intensity on the bacterial surface to confirm the successful construction of the engineered attenuated Salmonella with the plug-and-play system. Furthermore, SpyCatcherΔ-RGD×4 was used instead of SpyCatcherΔ-eGFP to construct engineered attenuated Salmonella with strong tumor targeting.

[0081] To detect the adhesion of different bacterial strains to surfaces coated with recombinant αvβ3 integrin protein, recombinant mouse integrin protein αVβ3 (TGAV&ITGB3) heterodimer protein (MCE, HY-P700761) was diluted to 10 μg / ml in 50 mM carbonate buffer (pH 9.6), and the diluted solution was spread onto individual 96-well plates (100 μl / well) and incubated overnight at 4°C. After washing, 3% BSA was added to each well, the plates were blocked, and incubated at 37°C for 3 hours. After washing, 100 μl of different serially diluted bacterial strains were added to each well. After standing at 37°C for 30 minutes, the wells were washed with PBST, and the bacteria adhering to the wells were photographed and counted using a fluorescence microscope.

[0082] To detect the expression levels of density-regulated intratumoral protein drugs, the pTD103-LuxI-LuxCDABE plasmid was designed and constructed. The LuxCDABE gene encoding quorum sensing-regulated expression was inserted into the pTD103luxI plasmid backbone. This plasmid contains a pBR322 origin of replication (500-700 copies per cell), a pLux promoter driving target gene expression, a B0010 terminator to prevent transcriptional readthrough, and an Axe-Txe toxin-antitoxin system to prevent plasmid loss. Kanamycin resistance (KanR) was used for selection. The extracted pTD103-LuxI-LuxCDABE plasmid was transformed into AISI-ST chassis strain, plated, and single colonies were picked for PCR verification. After streak culture, single colonies were picked for preservation to construct the AISI-L-ST strain. In vivo imaging in animals showed that the AISI-L-ST / SC-RGD×4 strain initiated bioluminescence earlier than the AISI-L-ST strain. At 36 hours after administration, the intratumoral bioluminescence intensity of the AISI-L-ST / SC-RGD×4 strain was 27.8 times that of the AISI-L-ST strain.

[0083] To investigate the engineered attenuated Salmonella strains capable of controlling intratumoral drug expression by inducing HtrA protein expression through quorum sensing activation via density increase, which in turn promotes extracellular polysaccharide (EPS) synthesis via the HtrA–Lon–RcsA axis, AISI-H-ST and AISI-B-ST strains (AISI-ST strains transformed with corresponding blank plasmids were used as controls) were designed and constructed according to the description in (6). EPS concentration was determined by phenol-sulfuric acid colorimetry using a polysaccharide quantitative kit (ZCIBIO, ZC-S0885, Shanghai, China). Single colonies of different strains were picked from agar plates and cultured overnight at 37°C. An equal volume of bacterial culture (OD600 = 1.0) was centrifuged, and the precipitate was collected and resuspended in EPS extraction reagent (Solarbio, EX1750, China). The resuspended solution was incubated in an 80°C water bath for 6 hours. After centrifugation, the supernatant containing the extracted EPS was collected. The absorbance (OD490) of each sample at 490 nm was measured using a kit, and the polysaccharide content was calculated based on a standard curve to determine that the EPS level of AISI-H-ST was higher than that of AISI-B-ST.

[0084] To construct an engineered attenuated Salmonella strain with controllable intratumoral drug expression, the AISI-HP-ST strain was designed and constructed according to the description in (6). In vitro, the expression of HtrA and PD1nb in different strains under high bacterial density was analyzed by Western blotting. It was confirmed that the engineered attenuated Salmonella strain with controllable intratumoral drug expression could restore HtrA protein expression through quorum sensing induction, enhance immunogenicity, and further express antitumor proteins such as PD1nb.

[0085] The present invention relates to the engineered bacterial strains with strong tumor targeting and controllable intratumoral drug expression, and their application in anti-tumor treatment.

[0086] Compared to unmodified Salmonella, attenuated Salmonella modified with RGD peptides, after intravenous injection, achieves tumor-targeted adhesion by specifically recognizing and binding to αvβ3 integrin, which is highly expressed on tumor cells. The tumor-targeting efficiency of the AISI-ST / SC-RGD×4 strain is significantly higher than that of the AISI-ST / SC-RGE strain. For example, the tumor / spleen ratio of the AISI-ST / SC-RGD×4 strain is 65.7-fold and the tumor / liver ratio is 74.9-fold, while the corresponding ratios of the AISI-ST / SC-RGE strain are lower.

[0087] The plug-and-play engineered attenuated Salmonella strains demonstrated by SpyTag / SpyCatcher can be quickly and easily modified with RGD peptides, which is a key factor in improving the targeting of this type of drug.

[0088] Compared to attenuated Salmonella modified with RGD peptides, the engineered bacterial strain described above, which exhibits strong tumor targeting and controllable intratumoral drug expression, increases bacterial density in the tumor microenvironment, activating the quorum sensing system, inducing HtrA protein expression, and subsequently promoting extracellular polysaccharide (EPS) synthesis via the HtrA–Lon–RcsA axis. The increased EPS directly activates macrophages through the TLR4-NFκB signaling pathway, driving them towards the M1 anti-tumor phenotype and enhancing the overall immune response.

[0089] Fusing target proteins with bacterial outer membrane proteins (such as OmpA) facilitates their display on bacterial surfaces. This invention hypothesizes that, through a similar strategy, SpyTag proteins can be anchored to the bacterial outer membrane, allowing them to splice with SpyCatcher-modified target proteins via simple incubation. This invention designs the AISI strain, an attenuated Salmonella chassis strain with an htrA gene deficiency, exhibiting excellent safety. This invention deletes the OmpA gene from the genome of the AISI strain and introduces a plasmid to continuously express the OmpA-SpyTag protein, thereby creating the AISI-SpyTag engineered strain (abbreviated as AISI-ST, e.g., Figure 1 SpyTag was inserted into the third outward-projecting loop of OmpA in the AISI strain. Figure 2 AISI-SD strain, in which the inserted SpyTag was replaced with a SpyTag-DA structure (a SpyTag mutant that does not bind to SpyCatcher), served as a control. Figure 2 Then, this invention evaluated the splicing efficiency of the SpyCatcherΔ-eGFP fusion protein in the AISI-ST strain, where SpyCatcherΔ lacks 21 residues at the N-terminus and 14 residues at the C-terminus to improve splicing accuracy. Figure 3 The SpyCatcher Δ-eGFP protein is effectively spliced ​​with the AISI-ST strain in a dose- and time-dependent manner to produce the AISI-ST / SC-eGFP strain. Figure 4 , Figure 5 This invention confirms that combining 3 mg of SpyCatcher Δ-eGFP with 10 mg of SpyCatcher Δ-eGFP... 8 Incubation of CFU strains for 1 hour results in a uniform and saturated distribution of engineered proteins on the bacterial outer membrane. Figure 4 , Figure 5 Each bacterium exhibits approximately 4.706 × 10⁻⁶ on its surface. 8 3.428 × 10⁶ eGFP molecules (approximately 3.428 × 10⁶ per bacterium) -5 (ng protein). Recombinant proteins can be uniformly integrated into the bacterial surface, ensuring sufficient contact between the protein and the receptor on the target cell.

[0090] This invention aims to express an RGD peptide fused with SpyCatcherΔ and splice it onto the bacterial outer surface. Previous poly-RGD structures, particularly tetrameric RGD structures, have shown superior in vivo tumor-targeting properties, but are primarily chemically synthesized. Therefore, this invention develops a bioexpressible tetrameric RGD-SpyCatcherΔ fusion protein (SC-RGD×4) to achieve similar effects. Figure 6 , Figure 7 The RGD peptide is linked to the N-terminus and C-terminus of the SpyCatcherΔ protein using a rigid linker, and a second RGD peptide is extended outward from the RGD peptide using a flexible linker to ensure overall stability. Figure 6 These proteins are related to 10 8 CFU was incubated with the AISI-ST strain for 1 hour to create AISI-ST / SC-RGD×4. These engineered strains were confirmed by recombinant αvβ3 integrin adhesion assays to increase bacterial binding to tumor cells through interaction with αvβ integrins, which are abundant on the surface of tumor cells. Figure 8 ).

[0091] Establishment of a mouse model of B16F10 subcutaneous solid tumors: B16F10 cells were cultured continuously in DMEM medium supplemented with 10% fetal bovine serum. Tumor cells were then collected and subcutaneously injected into 6-week-old female C57 mice via the axillary region (5 × 10⁶ cells per mouse). 5 (Cells). Subsequent experiments were conducted after initial tumor formation in mice (approximately 5-7 days).

[0092] This invention hypothesizes that AISI-ST / SC-RGD strains possessing multiple RGD peptides will exhibit enhanced tumor recognition and enrichment in vivo. This invention involves intravenously injecting AISI-ST / SC-RGD×4 into mice with subcutaneous xenografts of B16F10 cells. Each strain was engineered to continuously express a luminescent reporter gene (LuxCDABE) for real-time tracking. The AISI-ST / SC-RGD×1 strain showed a 2.48-fold increase in titer in melanoma, the AISI-ST / SC-RGD×2 strain a 5.07-fold increase, and the AISI-ST / SC-RGD×4 strain a 9.87-fold increase. Figure 8 Tumor targeting efficiency is defined as the ratio of bacterial titer within a tumor to bacterial titer in a normal organ (such as the liver or spleen). The AISI-ST / SC-RGD×4 strain showed the most significant increase in tumor targeting, with a 65.7-fold increase in tumor / spleen efficiency and a 74.9-fold increase in tumor / liver efficiency in the melanoma model. Figure 9 , Figure 10This enhanced targeting is attributed to increased bacterial enrichment within the tumor and reduced non-target distribution.

[0093] Compared to the AISI-ST strain, the intravenously injected AISI-ST / SC-RGD×4 strain achieved earlier and more precise intratumoral enrichment by utilizing the tumor-specific recognition and adhesion properties of the RGD peptide. However, the high intratumoral titer of the AISI-ST / SC-RGD×4 strain did not correspond to an increased anticancer effect. Therefore, this invention further suggests that re-expressing the HtrA protein through a quorum sensing system can enhance the anticancer effect of the AISI-ST / SC-RGD×4 strain. To verify this hypothesis, this invention designed the AISI-pLuxI-LuxCDABE-ST strain (abbreviated as AISI-L-ST), a quorum sensing-mediated luminescent strain. The loss prevention element Axe-Txe was inserted into the plasmid to ensure the persistence of the strain's engineered phenotype. In vivo imaging showed that, compared to the AISI-L-ST strain, the AISI-L-ST / SC-RGD×4 strain initiated bioluminescence earlier. Figure 11 , Figure 12 36 hours after administration, the intratumoral bioluminescence intensity of the AISI-L-ST / SC-RGD×4 strain was 27.8 times that of the AISI-L-ST strain. Figure 11 , Figure 12 ).

[0094] Next, this invention designed the AISI-pLuxI-HtrA-ST strain (abbreviated as AISI-H-ST), in which quorum sensing triggers HtrA protein expression ( Figure 13 AISI-ST strains transfected with the corresponding blank plasmid were designated AISI-B-ST and used as a control. Western blot analysis confirmed the expression of HtrA protein in AISI-H-ST strains. Figure 14 The HtrA protein indirectly increases the level of bacterial extracellular polysaccharides (EPS) through the HtrA–Lon–RcsA axis. Therefore, the EPS level of the AISI-H-ST strain was significantly higher than that of the AISI-B-ST strain. Figure 15 Indirect co-culture of macrophages activated by AISI-H-ST strain with tumor cells significantly inhibited tumor cell growth. Figure 16 The present invention further tested the AISI-H-ST / SC-RGD×4 strain in a B16F10 mouse melanoma model, confirming that both AISI-H-ST / SC-RGD×4 strains produced significant antitumor effects, while the inactive AISI-B-ST / SC-RGD×4 strain did not. Figure 17This highlights that quorum-sensing-based HtrA reexpression increases the strain's antitumor potential. This is due to its faster intratumoral enrichment ( Figure 17 The AISI-H-ST / SC-RGD×4 strain showed particularly significant antitumor effects. Compared with saline treatment, treatment with the AISI-H-ST / SC-RGD×4 (AISI-H-ST / SCR4) strain also significantly prolonged the survival time of tumor-bearing mice. Figure 18 This study confirmed that the AISI-H-ST / SC-RGD×4 strain, which specifically restored HtrA expression through quorum sensing, enhanced the anticancer effect of the engineered strain.

[0095] MetaCore gene network analysis showed that the AISI-H-ST / SCR4 strain induced stronger antigen presentation and T cell activation, suggesting a potential synergistic effect with immune checkpoint blockade therapy. Therefore, this invention tested various anti-tumor proteins, including but not limited to endostatin (CN115992083A), anti-PD1 nanobody (CN114958698A), anti-PDL1 nanobody (CN114736840A), TNF-α nanobody (CN117089506A), and IFN-β (CN118126918A). All of these anti-tumor proteins have been tested by our team in Salmonella for their secretion expression, anti-tumor efficacy, and toxicity. Here, we only use the PD1 nanobody as an example, presenting the AISI-HtrA&PD1nb-ST / SC-RGD×4 strain (hereinafter referred to as AISI-HP-ST / SCR4) that expresses the HtrA protein and secretes the anti-PD1 nanobody (PD1nb) under high bacterial density. Figure 19 The experimental results showed that PD1nb expression levels increased with increasing strain density, and was subsequently secreted into the culture medium via the Sec-dependent secretion pathway mediated by the pelB signal peptide. Figure 20 , Figure 21 PD1nb expression does not affect the simultaneous expression of HtrA by the strain at high density. Figure 22 This secreted PD1nb blocks the PD1–PDL1 pathway, reversing immunosuppression and inhibiting tumor growth. Tumor growth monitoring showed that the AISI-HP-ST / SCR4 group had significantly reduced tumor volume and weight, with a mean tumor weight of 0.146 grams, significantly lower than other groups, equivalent to 16.7% of the saline group. Figure 23 , Figure 24Furthermore, in the AISI-H-ST / SCR4 and AISI-P-ST / SCR4 (AISI-PD1nb-ST / SC-RGD×4) groups, less than 50% of mice survived beyond day 35, while in the AISI-HP-ST / SCR4 group, 100% of mice survived. Figure 25 Overall, these results highlight the potent antitumor effect of the AISI-HP-ST / SCR4 strain through the simultaneous expression of HtrA and PD1nb. Other antitumor proteins, such as endostatin (CN115992083A), anti-PD1 nanobody (CN114958698A), anti-PDL1 nanobody (CN114736840A), TNF-α nanobody (CN117089506A), and IFN-β (CN118126918A), can also be expressed simultaneously with HtrA in the AISI-HP-ST / SCR4 strain, producing a potent antitumor effect similar to PD1nb, thus demonstrating the versatility of this method.

[0096] To explore the antitumor mechanism mediated by the engineered bacterial strain AISI-HP-ST / SCR4, this invention conducted a detailed analysis of the tumor microenvironment (TME) in mice bearing B16–F10 tumors 72 hours after drug administration. Specifically, these findings highlight the multifaceted antitumor activity of the AISI-HP-ST / SCR4 strain, which promotes M1-type TAM polarization, enhances DC maturation, and amplifies CD8+. + T cell effector functions coordinate a favorable immune environment. Bacteria expressing HtrA strongly activate CD8. + T cells were activated, but their proliferation was not triggered. Conversely, bacteria secreting PD1nb effectively induced cell proliferation, despite relatively weak activation of CD8+ T cells. Figure 26 , Figure 27 In the AISI-HP-ST / SCR4 group, which simultaneously produces HtrA and PD1nb, CD8 + The proportion of Teffs was significantly increased. More importantly, approximately 36.5% of these cells were proliferating, a proportion 4.73 times higher than that in the AISI-H-ST / SCR4 group. Figure 26 , Figure 27 The simultaneous expression of PD1nb and HtrA by the AISI-ST / SCR4 strain indeed led to synergistic antitumor immune activation. Overall, an engineered bacterial strain with potent tumor targeting and controllable intratumoral drug expression produced a favorable antitumor effect through robust antitumor immune activation.

[0097] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.

Claims

1. An engineered attenuated Salmonella strain with strong tumor targeting and controllable intratumoral drug expression, characterized in that: The engineered attenuated Salmonella strain with strong tumor targeting and controllable intratumoral drug expression was obtained from a chassis strain through the following modifications: (1) AISI strain construction: HtrA protein and outer membrane protein OmpA gene were knocked out in the genome of Salmonella Typhimurium VNP20009 by CRISPR-Cas9; (2) Anchoring unit: OmpA-SpyTag fusion protein located on the bacterial outer membrane, wherein SpyTag is displayed on the bacterial surface; (3) Connecting unit: an isopeptide bond formed by covalent binding of SpyTag and SpyCatcherΔ; (4) Targeting unit: a multivalent targeting peptide fused with the SpyCatcherΔ protein; wherein the multivalent targeting peptide contains at least two targeting peptide fragments that specifically bind to receptors on the surface of tumor cells, and the multivalent targeting peptide is displayed on the bacterial surface through the linker unit, so that the adhesion ability of the engineered strain to tumor cells is significantly enhanced compared with the strain modified with a single-valent targeting peptide. (5) Introducing a quorum sensing system-regulated expression cassette, which induces the expression of a therapeutic protein when bacterial density increases, wherein the therapeutic protein is selected from one or a combination of two of HtrA protein and anti-tumor protein drugs (immunomodulatory factors).

2. The engineered attenuated Salmonella strain according to claim 1, characterized in that: In step (1), the pTAT plasmid carrying homologous arms to both sides of the gene is introduced into the VNP and works together with the pCas plasmid to disrupt the gene loci on the genome through allele exchange; the nucleotide sequences of the homologous arms of the pTAT-HtrA-VNP plasmid are shown in SEQ ID NO:1 and SEQ ID NO:2, and the nucleotide sequences of the homologous arms of the pTAT-OmpA-VNP plasmid are shown in SEQ ID NO:3 and SEQ ID NO:

4. After removing the plasmid, the ΔhtrA-VNP chassis strain is obtained.

3. The engineered attenuated Salmonella strain according to claim 1, characterized in that: In step (2), the amino acid sequence of the OmpA-SpyTag fusion protein is shown in SEQ ID NO:5, and the amino acid sequence of the mutant OmpA-SpyTag fusion protein is shown in SEQ ID NO:6; the SpyCatcherΔ-multivalent targeting peptide fusion protein contains a first targeting peptide unit, a SpyCatcherΔ protein, and a second targeting peptide unit from the N-terminus to the C-terminus; the targeting peptide units are linked by a Linker, and the fusion protein contains 2-6 targeting peptide units.

4. The chassis strain is an original strain, mutant strain, or engineered strain of attenuated Salmonella typhimurium, Escherichia coli, Bifidobacterium, BCG, Shigella, Klebsiella pneumoniae, or Pseudomonas aeruginosa with htrA gene deficiency.

5. The engineered attenuated Salmonella strain according to claim 1, characterized in that: The targeting peptide unit is RGD peptide, the SpyCatcherΔ-multivalent targeting peptide fusion protein is SpyCatcherΔ-RGD, and the amino acid sequence of SpyCatcherΔ-RGD is shown in SEQ ID NO:7; the amino acid sequence of the SpyCatcherΔ-multivalent targeting peptide fusion protein SpyCatcherΔ-RGD×2 is shown in SEQ ID NO:8; the amino acid sequence of the SpyCatcherΔ-multivalent targeting peptide fusion protein SpyCatcherΔ-RGD×4 is shown in SEQ ID NO:9; the RGD peptide unit is an arginine-glycine-aspartic acid tripeptide sequence, which can specifically recognize integrin αvβ3 highly expressed on the surface of tumor cells; the chassis of the strain is an attenuated Salmonella Typhimurium with a defective htrA gene, and the engineered strain is named AISI-ST / SC-RGD×4.

6. The engineered attenuated Salmonella strain according to claim 1, characterized in that: In step (4), the multivalent tumor-targeting peptide is a tetravalent RGD peptide, wherein the N- and C-termini of SpyCatcherΔ are connected to RGD peptides through rigid Linkers, respectively, and are connected in series through a flexible Linker to form a SC-RGDx4 structure; the incubation ratio of the SpyCatcherΔ-targeting peptide fusion protein and bacteria is 0.1-3 mg: 10 6 -10 8 CFU, the incubation temperature is 20-40℃, and the incubation time is 0.5-5 hours.

7. The engineered attenuated Salmonella strain according to claim 1, characterized in that: In step (5), the expression cassette regulated by the quorum sensing system contains both the HtrA protein encoding gene and the anti-tumor protein encoding gene, both of which are controlled by the LuxI promoter. The HtrA protein encoding gene is expressed within the bacteria, and the anti-tumor protein encoding gene contains the pelB signal peptide for secretory expression. The anti-tumor protein includes, but is not limited to, endostatin, anti-PD1 nanobody, anti-PDL1 nanobody, TNF-α nanobody, and IFN-β. The expression cassette regulated by the quorum sensing system also contains the Axe-Txe toxin-antitoxin system to prevent plasmid loss.

8. A method for preparing an engineered attenuated Salmonella strain with strong tumor targeting and controllable intratumoral drug expression as described in any one of claims 1-5, characterized in that... Includes the following steps: (1) The htrA and OmpA genes in the VNP20009 genome were knocked out using the CRISPR-Cas9 system to construct the AISI chassis bacteria; (2) Construct and transform plasmid pTD103(Cm)-OmpA-GS-SpyTag-GS to obtain engineered bacterial strains with SpyTag displayed on the outer membrane; (3) Express and purify SpyCatcher Δ-targeting peptide fusion protein; (4) The strain obtained in step (2) is co-incubated with the fusion protein obtained in step (3), and the surface modification is completed by covalent binding of SpyTag / SpyCatcher; (5) Construct and transfer the quorum sensing regulatory plasmid pTD103-LuxI-antitumor effector protein to obtain an engineered strain with controllable intratumoral drug expression.

9. The use of the engineered attenuated Salmonella strain according to any one of claims 1-6 in the preparation of antitumor drugs.

10. The application according to claim 7, characterized in that: The tumors include one or more of the following: melanoma, lung cancer, lymphoma, bladder cancer, liver cancer, colorectal cancer, and breast cancer.

11. The application according to claim 7, characterized in that: The route of administration of the drug is selected from any one of intraperitoneal injection, intravenous injection, oral instillation, intratumoral injection, or bladder instillation; the dosage of the drug is 10. 4 -10 7 CFU / mouse, administered once or multiple times.

12. The application according to claim 9, characterized in that: The drug is also used in combination with one or more of the following: chemotherapy drugs, immune checkpoint inhibitors, radiotherapy, photothermal therapy, CAR-T cell therapy, or other bacterial therapies.

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