Methods of using engineered lactobacillus bacteria to treat cancer

Abstract

The present invention relates to engineered lactobacilli. In particular, engineered lactobacilli carrying prodrugs on their surface. The cleavable linker within the prodrug allows for targeted release of the drug. Methods, compositions and kits for treating cancer are also described herein.

Description

[0001] This application claims priority to filings 10202400039Q and 10202400040Q, filed January 5, 2024, the contents and elements of which are incorporated herein by reference for all purposes.

[0002] sequence list

[0003] This application contains a sequence list that has been filed together with it and is hereby incorporated in its entirety by reference. The .xml copy created on January 2, 2025 is named 008686016 and has a size of 12,515 bytes. Technical Field

[0004] The present invention generally relates to methods for treating cancer, and more particularly to methods for treating cancer using Lactobacillus bacteria that have been engineered to exhibit capture domains for binding drugs. Background Technology

[0005] Chemotherapy plays a crucial role in the treatment of various cancers, particularly in reducing distant metastasis. However, chemotherapy regimens are known for their poor bioavailability at certain tumor sites, significant systemic toxicity, and low patient tolerability. Prolonged chemotherapy is intolerable for many patients, and thus, distant metastasis has become a leading cause of treatment failure in many cancers. Therefore, there is a need for drug delivery vehicles capable of targeted delivery and local release of anticancer agents to improve the effectiveness of cancer treatment and allow for the de-escalation of chemotherapy regimens.

[0006] Prodrugs are pharmacologically inactive molecules that can be converted into active drugs in vivo through chemical or enzymatic reactions. Higher selectivity of prodrugs can be achieved by leveraging the unique physiological conditions of the tumor microenvironment (TME). In this context, cytotoxic drugs are modified with chemical groups that respond to TME signals (cue) and released upon reaching the tumor in a site-specific manner. This is achieved by recognizing tumor-specific markers, such as antigens or receptors targeted by antibody-conjugated drugs (ADCs). To date, 14 ADCs have received market approval from the U.S. Food and Drug Administration (FDA) for cancer treatment, marking a significant advance in targeted cancer therapy (Fu et al., 2022).

[0007] However, current prodrug strategies have limited target specificity. For example, TME signals cannot be clearly distinguished from normal tissues, leading to significant off-target effects in prodrugs. Furthermore, the macromolecular nature of conjugated tumor-targeting carriers often complicates the pharmacokinetic profile of prodrugs in circulation, affecting their biodistribution, metabolism, and clearance. In the case of ADCs, conjugating antibody carriers to the original drug significantly increases molecular size, impairing drug penetration and bioavailability in tumors (Xu et al., 2015). The long half-life of the carrier antibody also prolongs the clearance of the payload drug in vivo, impairing liver and kidney function (Khera et al., 2018). In addition to systemic toxicity caused by chemotherapy, systemic administration of ADCs can introduce toxicity from the antibody component, thereby inducing immune responses and causing severe secondary damage, leading to nephrotoxicity in patients (Mecklenburg et al., 2018). Several preclinical studies have also revealed similar complications and side effects with various nanomaterial carriers (Cheng et al., 2021).

[0008] The aim is to overcome or mitigate at least one of the above problems, or at least provide a useful alternative.

[0009] The present invention was designed based on the above considerations. Summary of the Invention

[0010] To overcome limitations stemming from limited treatment specificity and associated complications, the inventors discovered and utilized the intrinsic interactions between bacteria and cancer cells within the tumor microbiome for precise prodrug delivery. The inventors hypothesized that they could precisely locate and engineer symbiotic microorganisms with cancer-binding capabilities to deliver prodrugs precisely to specific cancer sites. The payload prodrug can be conjugated to a bacterial carrier via a chemical linker to ensure site-specific release of the original drug. Using nasopharyngeal carcinoma (NPC) as a representative cancer model, symbiotic microorganisms with the ability to bind to cancer cells were identified and characterized, and subsequently engineered to achieve precise delivery of chemotherapeutic agents to cancer sites. NPC is the most common head and neck cancer, with high incidence in southern China, Southeast Asia, and North Africa, affecting over 133,000 people and causing over 80,000 deaths in 2020 alone (Tang et al., 2016). By 2040, the number of NPC cases and deaths worldwide is projected to rise to approximately 179,000 and 114,000, respectively (Zhang et al., 2023). Chemotherapy for NPC is associated with severe toxicity and administration difficulties, making prolonged chemotherapy intolerable for patients (McDowell et al., 2020; Wang et al., 2019).

[0011] In one aspect, this document discloses a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of engineered Lactobacillus bacteria carrying a drug, wherein the drug binds to a capture domain displayed on the surface of the bacteria. In some embodiments, the bacteria are derived from cancerous tissue or tissue capable of developing cancer. In a preferred embodiment, the bacteria are the Lactobacillus plantarum (Lp) species. In some embodiments, the Lp is strain WCFS1. In some embodiments, the bacteria contain an OppA polypeptide on the surface of the bacteria. The OppA polypeptide may be an exogenous polypeptide not expressed by non-engineered bacteria or an endogenous polypeptide expressed by non-engineered bacteria. The OppA polypeptide may be a purified or semi-purified formulation added to the bacteria and coated on the surface of the bacteria. The OppA polypeptide may be covalently or non-covalently attached to the surface of the bacteria. The OppA polypeptide may be attached to the cell wall or cell membrane of the bacteria. The bacteria may contain one or more OppA polypeptides. In some embodiments, the OppA polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequences shown in SEQ ID NO: 1-7.

[0012] In some embodiments, the drug comprises a tag capable of binding a capture domain. In some embodiments, the capture domain is a biotin-binding polypeptide, and the tag is biotin. In some embodiments, the biotin-binding polypeptide is streptavidin. In some embodiments, the capture domain comprises an anchoring domain for anchoring to the bacterial surface. In some embodiments, the anchoring domain is a bacterial transmembrane domain. In some embodiments, the bacterial transmembrane domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 8.

[0013] In some embodiments, the drug is a prodrug comprising a cleavable entity. In some embodiments, the prodrug is activated near cancer cells or cancerous tissue through the cleavage of the cleavable entity. In some embodiments, the cleavable entity is a redox-sensitive entity. In some embodiments, the bacteria are administered via mucosa, around the tumor, or within the tumor.

[0014] In some embodiments, the cancer is carcinoma. In some embodiments, the cancer is nasopharyngeal carcinoma, oral cancer, lung cancer, bladder cancer, stomach cancer, colorectal cancer, skin cancer, breast cancer, or ovarian cancer. In a preferred embodiment, the cancer is nasopharyngeal carcinoma.

[0015] In a second aspect, this paper discloses an engineered Lactobacillus bacterium carrying a cancer drug for treating a subject’s cancer, wherein the drug binds to a capture domain displayed on the surface of the bacterium.

[0016] In a third aspect, this document discloses the use of engineered Lactobacillus bacteria carrying cancer drugs in the manufacture of medicaments for treating cancer in subjects, wherein the drugs bind to capture domains displayed on the surface of the bacteria.

[0017] In a fourth aspect, this document discloses a method for delivering a drug to cancer cells, the method comprising contacting the cancer cells with a Lactobacillus bacterium carrying the drug, wherein the drug binds to a capture domain displayed on the surface of the bacterium.

[0018] In a fifth aspect, this document discloses a method for detecting cancer in a subject, the method comprising administering to the subject an effective amount of engineered Lactobacillus bacteria carrying a detectable marker, wherein the detectable marker binds to a capture domain displayed on the surface of the bacteria.

[0019] In a sixth aspect, this document discloses a method for labeling cancer cells, the method comprising contacting the cancer cells with a Lactobacillus bacterium carrying a detectable label, wherein the detectable label binds to a capture domain displayed on the surface of the bacterium.

[0020] In a seventh aspect, this document discloses an engineered *Lactobacillus* bacterium comprising a) a heterologous nucleic acid molecule encoding a polypeptide containing a capture domain displayed on the surface of the bacterium; and b) a heterologous portion binding to the capture domain. In some embodiments, the bacterium is *Lactobacillus plantarum* (Lp). In some embodiments, the Lp strain is WCFS1. In some embodiments, the bacterium contains an OppA polypeptide on its surface. The OppA polypeptide may be an exogenous polypeptide not expressed by non-engineered bacteria or an endogenous polypeptide expressed by non-engineered bacteria. The OppA polypeptide may be a purified or semi-purified formulation added to the bacteria and coated on the surface of the bacteria. The OppA polypeptide may be covalently or non-covalently attached to the surface of the bacteria. The OppA polypeptide may be attached to the cell wall or cell membrane of the bacteria. The bacteria may contain one or more OppA polypeptides. In some embodiments, the OppA polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequences shown in SEQ ID NO: 1-7.

[0021] In some embodiments, the heterologous portion includes a tag capable of binding a capture domain. In some embodiments, the capture domain includes a biotin-binding polypeptide, and the tag is biotin. In some embodiments, the biotin-binding polypeptide is streptavidin. In some embodiments, the heterologous portion is a drug. In some embodiments, the drug is a prodrug containing a cleavable entity. In some embodiments, the heterologous portion includes a detectable marker. In some embodiments, the bacteria also contain an OppA polypeptide on the surface of the bacteria.

[0022] In an eighth aspect, this document discloses a composition comprising the *Lactobacillus* bacteria of the seventh aspect. In some embodiments, the composition comprises one or more *Lactobacillus* bacteria.

[0023] In the ninth aspect, this document discloses either the Lactobacillus bacteria of the seventh aspect or the composition of the eighth aspect, for use as a pharmaceutical agent.

[0024] In a tenth aspect, this document discloses a method for preparing engineered Lactobacillus bacteria according to the seventh aspect, the method comprising: a) expressing in the bacteria a polypeptide containing a capture domain displayed on the surface of the bacteria; and b) contacting the bacteria with a heterologous portion capable of binding the capture domain.

[0025] In the eleventh aspect, this document discloses a kit containing bacteria according to the seventh aspect. In some embodiments, the kit comprises a first container containing a first composition according to the eighth aspect and a second container containing a second composition according to the eighth aspect, wherein the drug carried by the bacteria of the first composition contains a different drug than the drug carried by the bacteria of the second composition.

[0026] In one embodiment, the *Lactobacillus* bacteria are also engineered to produce and / or carry a drug for intranasal drug delivery to the olfactory region, brain, and CNS. In a preferred embodiment, the drug is a prodrug. In some embodiments, the bacteria are engineered to secrete the drug, preferably at an intranasal site (e.g., olfactory epithelium) after intranasal administration. The bacteria may comprise one or more expression constructs encoding a heterologous drug molecule (e.g., a recombinant peptide or polypeptide) or a component of a biosynthetic pathway required to synthesize the heterologous drug molecule. The expression construct may comprise one or more nucleotide sequences encoding a peptide or polypeptide, the one or more nucleotide sequences being operatively linked to one or more of the same or different promoters capable of directing the expression of the sequence in a *Lactobacillus* host. The promoters used according to the method of the invention are preferably constitutively expressed in the bacteria. The use of constitutive promoters avoids the need to provide inducers or other regulatory signals to cause expression to occur. The promoters may also be inducible promoters, preferably conditionally inducible promoters found at intranasal sites. The promoters may be homologous to the bacteria used, i.e., promoters naturally found in those bacteria. Preferably, the promoter guides expression at a level that keeps the bacterial host cell viable, that is, maintaining a certain level of metabolic activity even if growth is not maintained.

[0027] The present invention includes combinations of the described aspects and preferred features, except where such combinations are clearly not permitted or explicitly avoided. Attached Figure Description

[0028] The embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying drawings, in which:

[0029] Figure 1This section illustrates the binding affinity of various probiotic strains to NPC cells. a) Quantification of the cancer-binding ability of probiotic strains using CNE-1 as a binding target. b) Lp localized on the surface of i) CNE-1, ii) CNE-2, iii) C666-1, iv) HK-1, and v) RPMI2650 cells, and absent on vi) HNC cells. Red (highlighted with white triangles): *Lactobacillus plantarum* WCFS1 (Lp) expressing mCherry. Blue: Mammal cell nuclei stained with Hoechst. Green: Cytoskeleton, cytoplasm, or microcilia of mammalian cells. Scale bar: 25 μm. c) Quantification of Lp-cell binding affinity. The y-axis represents the fold change in FITC fluorescence signal normalized relative to the HNC group. d) IF staining, showing the interaction between Lp and i) T24 bladder cancer cells, ii) A549 lung cancer cells, and iii) AGS gastric cancer cells. *Lactobacillus plantarum* WCFS1 (Lp) expressing mCherry (highlighted with white triangles). e) Inhibition of Lp-NPC binding by heparin salts in co-cultures of Lp and four NPC cell lines. n = 5 replicates. f) Schematic diagram showing possible competitive inhibition between heparan sulfate and heparin.

[0030] Figure 2 Identification of the OppA cancer-binding protein from the surface of *Lactobacillus plantarum* cells is shown. a) Flow cytometry analysis of the binding affinity of OppA protein to all NPC cell lines is shown. b) Increased Lp-NPC binding by adding Lp_0018 to a co-culture of *Lactobacillus plantarum* and NPC cell lines. c) α-sheet prediction model of Lp_0018 and simulation of its heparin docking complex. d) Schematic diagram showing Lp_0018 enhancing Lp-NPC binding and Lp_0018 SBD reducing Lp-NPC binding. e) Binding of Lp_0018 to the Lp surface. f) Reduction of Lp-NPC binding by adding Lp_0018 SBD.

[0031] Figure 3The images show the surface display of streptavidin and the loading of the prodrug on *Lactobacillus plantarum* WCFS1 (Lp). a) Expression cassette for Sav surface display in Lp. b) Schematic diagram of tetrameric Sav surface display. c) Western blot images showing the surface display of the Lp_1568-Sav fusion protein and the secreted Sav protein. d) Detection of the Lp_1568-Sav fusion protein on the Lp cell surface by i) flow cytometry and ii) IF. e) Detection of multimeric Sav on the Lp cell surface by flow cytometry and IF. f) Schematic diagram showing the synthetic route of TL-SN and the loading of TL-SN. g) Loading of TL-SN on Lp-Sav and EV strains.

[0032] Figure 4 The release of prodrug 1 (SN) from Lp-Sav is shown. a) and b) Schematic diagrams of the TL-SN activation and SN release mechanisms. c) and f) TL-SN activation and SN release over time in TL-SN-loaded Lp-Sav. d) and g) TL-SN activation kinetics over time obtained by spectral scanning. e) and h) Concentrations of TL-SN in the cell pellet and SN in the supernatant before and after activation.

[0033] Figure 5 Characterization of Lp-Sav loaded with prodrug in NPC cells in vitro. a) Accumulation of SN in C666-1 cells after TL-SN activation, incubation time 24 h. Red: β-actin stained by phalloidin. Blue: Cell nuclei stained by Hoechst. Green: SN (highlighted with white triangles). Scale bar, 25 μm. b) Viability of NPC cells after co-culturing with unloaded Lp-Sav at approximately 0.4 OD600 for 24 h. c) and d) IC50 of various treatments in NPC cells.

[0034] Figure 6The following diagram illustrates the treatment of a xenograft mouse model of NPC. a) Schematic diagram of the NPC xenograft mouse model used to i) evaluate treatment efficacy and ii) study Lp biodistribution. b) Table of treatment groups used in this study. c) Visualization of bioluminescent Lp in mouse organs at 24, 48, and 72 hours post-intravenous injection (tumor radiation is outlined in white). d) Bacterial density of Lp-CB in various mouse organs (including liver, spleen, tumor, cecum, heart, kidney, lung, and stomach) at 24, 48, and 72 hours post-injection. *P < 0.05; **P < 0.01; ***P < 0.001. e) Bacterial density of Lp-CB in blood at 1, 3, and 6 hours post-injection. n = 3 mice (p values ​​are as follows: 1 h vs. 3 h = 0.0261, 1 h vs. 6 h = 0.0172, 3 h vs. 6 h = 0.00029). f) Bacterial density of Lp-CB and LrD in the tumor 24 hours after injection. (p = 0.0417) g) Tumor-liver ratio of Lp-CB and LrD 24 hours after injection. (p = 0.0280)

[0035] Figure 7 The treatment of xenograft mouse NPC models is shown. a) Changes in body weight of mice in different groups during treatment. b) Tumor progression in mice in different treatment groups. Arrows indicate injections. c) Endpoint tumor burden in mice in different treatment groups.

[0036] Figure 8 This is a schematic diagram illustrating the delivery of a prodrug to cancer cells via an engineered Lp. a) Lp recognizes cancer cells by binding to heparan sulfate. b) and c) Specific binding of Lp to cancer cells. d) The biotinylated prodrug loaded on Lp and the release of the drug near the cancer cells via Lp.

[0037] Figure 9 SEM images of Lp cells co-cultured with CNE-1 monolayers are shown. Lp cells were found on the surface of CNE-1 cells.

[0038] Figure 10 The surface display of streptavidin in Lp is shown using various anchoring domains. a) Promoter library in Lp characterized by gusA assay. b) Replicon 256-based plasmid for genetic manipulation in Lp. c) Comparison of expression intensity in pTRK-892 and pHSSC256 plasmids. d) Expression cassette for streptavidin surface display. e) and f) Flow cytometry analysis and IF staining show that streptavidin was successfully displayed via fusion with the transmembrane protein Lp_1568. Scale bar, 25 µm.

[0039] Figure 11This image shows multiple sequence alignments of seven heparin-binding OppA proteins. Highly conserved residues are indicated by red uppercase letters; weakly conserved residues are indicated by blue lowercase letters; other residues are indicated by black. Detailed Implementation

[0040] Various aspects and embodiments of the invention will now be discussed with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated herein by reference.

[0041] The inventors have discovered that bacteria naturally occurring in the tumor microbiome can be used to target and deliver anticancer drugs to cancer cells. Unbound by theory, various cancer tissues, including nasopharyngeal carcinoma, breast cancer, ovarian cancer, and colorectal cancer, serve as hosts for symbiotic microorganisms that form unique cancer microbiomes. These microorganisms have evolved intrinsic cancer homing properties, and their metabolites directly influence the carcinogenesis, progression, and treatment of host tumor cells. This disclosure provides a drug delivery platform that utilizes bacteria-cancer interactions naturally occurring in the tumor microbiome to achieve targeted delivery of anticancer drugs to cancer cells.

[0042] Lactobacillus strains are naturally present in the microbiomes of many tumors and surrounding healthy tissues, such as in healthy and cancerous nasopharyngeal tissues. The inventors have identified a symbiotic *Lactobacillus plantarum* (Lp) strain (*Lactobacillus plantarum* WCFS1) with a surprising affinity for various cancer cells. Unbound by theory, this Lp strain contains an OppA adhesin protein on its surface with a strong affinity for heparan sulfate, which is highly expressed on many cancer cells. The presence of several of these adhesins on *Lactobacillus plantarum* makes this species particularly advantageous for targeting and colonizing cancerous tissues compared to other *Lactobacillus* bacteria or symbiotic bacteria. Heterologous expression of OppA adhesins or coating *Lactobacillus plantarum* with exogenous adhesins (e.g., recombinantly expressed and purified OppA protein) can further enhance cancer cell binding. For example, the inventors have found that incubating the Lp strain with OppA protein significantly increases the strain's binding ability to nasopharyngeal carcinoma (NPC) cells.

[0043] Lactobacillus species are symbiotic strains recognized as Generally Recognized As Safe (GRAS) by the US FDA and are suitable for human and many animal consumption. While other bacteria, such as strains from Clostridium, Escherichia, Listeria, and Salmonella, have been used for drug delivery, these bacteria are opportunistic pathogens that can potentially cause infection. In contrast, Lactobacillus plantarum (Lp) is cleared from non-cancerous tissues more quickly than strains like Salmonella Typhimurium, Listeria Monotrophus, Pseudomonas aeruginosa, and Escherichia coli, thus reducing the risk of post-application bacterial infection. Lactobacillus species are also naturally present in many mucous membranes, allowing for mucosal administration as an alternative to targeted delivery, thus avoiding the side effects of systemic administration of payload drugs.

[0044] The inventors have modified *Lactobacillus plantarum* (Lp) for drug delivery by expressing a capture domain on the bacterial surface capable of binding appropriately labeled drugs. Depending on the cancer to be treated, the range of drugs can be from small molecules to proteins (e.g., antibodies). Having a capture domain on the bacterial surface allows drugs to be easily loaded onto the bacteria, ensuring the drug remains accessible to target cells (e.g., cancer cells) and keeps the drug separate from the bacterial internal environment, thus preserving the drug's activity. The bacteria deliver the drug directly to the cancer or tumor site, reducing drug accumulation in healthy tissue. Cancer-targeted drug delivery utilizing *Lactobacillus plantarum*'s ability to attach to cancer cells can enhance the efficiency of chemotherapy and reduce the intensity of chemotherapy regimens.

[0045] To further target drug release to specific sites and reduce systemic drug toxicity, the inventors modified drugs into prodrugs with virtually no pharmacological activity until they were converted to their active form at the target site (e.g., tumor site), for example, in response to extracellular signals (cue) at the target site. The combination of targeted delivery using Lp bacteria and local drug activation using prodrug forms improved therapeutic specificity at the target site, thereby reducing systemic side effects and making chemotherapy more tolerable for patients.

[0046] Lactobacillus plantarum (Lp)-based bacterial drug delivery platforms can reduce the intensity of chemotherapy regimens for treating a variety of cancers. First, because Lp strains are naturally resident in the microbiota of many human tissues, they can be used via mucosal application to deliver chemotherapy to cancers in these tissues, reducing the systemic toxicity of conventional chemotherapy. Second, Lp-targeted drug delivery can increase the availability of chemotherapy drugs to cancer cells, reduce damage to healthy tissues, and improve treatment outcomes. Third, Lp exhibiting streptavidin or biotin-binding moieties on their surface allows for surface loading of various biotinylated chemotherapeutic drugs or prodrugs tailored to the unique characteristics and cellular microenvironment of specific cancers. The combination of Lp-mediated targeted delivery and local prodrug activation can enhance the efficiency of chemotherapy and allow for the use of lower-intensity chemotherapy regimens. Furthermore, Lp bacteria can be administered as adjuvant or neoadjuvant therapy to various mucosa to sensitize cancerous tissue to standard chemotherapy.

[0047] Therefore, this disclosure provides engineered Lactobacillus bacteria comprising a surface-displaying capture domain capable of binding to heterologous portions such as drugs or detectable markers; and methods for detecting or treating cancer using engineered bacteria. Methods for preparing said bacteria are also provided.

[0048] Methods of treating cancer

[0049] This article discloses a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of engineered Lactobacillus bacteria carrying a drug, wherein the drug binds to a capture domain displayed on the surface of the bacteria.

[0050] In some embodiments, the bacteria are derived from cancerous tissue or tissue that may develop cancer. The bacteria can be a component of the natural microbiome of the tissue or cancerous tissue. The tissue can be a mucosal tissue, such as the lining of the respiratory tract, gastrointestinal tract, or genitourinary tract. The bacteria can originate from a variety of tissues, examples of which include, but are not limited to, nasal, nasopharyngeal, larynx, pharynx, trachea, bronchi, oral cavity, esophagus, stomach, intestine, colon, rectum, bladder, urethra, vagina, ovarian, or breast tissue.

[0051] In one embodiment, the Lactobacillus bacterium is Lactobacillus plantarum (Lp). In another embodiment, the Lactobacillus bacterium is Lactobacillus plantarum WCFS1.

[0052] In some embodiments, the bacteria also include adhesins on their surface capable of attaching to cancer cells. Adhesins can attach to extracellular molecules on the surface of cancer cells. Adhesins can attach to peptides or polysaccharides. In some embodiments, the adhesins are capable of attaching to polysaccharides. In one embodiment, the polysaccharide is heparan sulfate. In one embodiment, the adhesin is an OppA peptide.

[0053] In some embodiments, the bacteria contain an OppA polypeptide on their surface. The OppA polypeptide can be any OppA polypeptide.

[0054] In one embodiment, the OppA peptide is an endogenous peptide expressed from the genome of a Lactobacillus bacterium. In another embodiment, the OppA peptide is a heterologous peptide expressed from a heterologous nucleic acid introduced into the bacteria. In yet another embodiment, the OppA peptide is an exogenous peptide not expressed by bacteria. The OppA peptide can be a purified or semi-purified formulation added to and coated on the surface of bacteria. The OppA peptide can be covalently or non-covalently attached to the surface of bacteria. The OppA peptide can be attached to the cell wall or cell membrane of bacteria. Bacteria may contain one or more OppA peptides.

[0055] In one embodiment, the OppA polypeptide comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequences shown in SEQ ID NO: 1-7. These OppA sequences bind to both *Lactobacillus* bacteria and heparan sulfate. In some embodiments, the OppA polypeptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequences shown in SEQ ID NO: 1-7. The bacteria may contain one or more OppA polypeptides. In a preferred embodiment, the bacteria contain two or more OppA polypeptides, such as two, three, four, five, six, or seven OppA polypeptides, said OppA polypeptides having an amino acid sequence having at least 70% sequence identity with the amino acid sequences shown in SEQ ID NO: 1-7. These OppA sequences bind to both *Lactobacillus* bacteria and heparan sulfate. In a preferred embodiment, the bacteria contain two or more OppA polypeptides, such as two, three, four, five, six or seven OppA polypeptides, wherein the OppA polypeptides contain an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 1-7.

[0056] In some embodiments, *Lactobacillus* bacteria express a substrate-binding domain (SBD) on their surface. The SBD is capable of binding heparan sulfate. This allows *Lactobacillus* to localize to cancer cells (e.g., NPC). In some embodiments, the SBD forms a portion of a polypeptide that can be presented on the surface of *Lactobacillus* bacteria. For example, the SBD can form a portion of a transmembrane protein on the surface of *Lactobacillus* bacteria, or it can be anchored to the surface membrane. In some embodiments, the OppA polypeptide contains an SBD. In some embodiments, the OppA polypeptide is composed of an SBD.

[0057] In some embodiments, the SBD comprises an amino acid sequence as defined at amino acid positions 73 to 453 of SEQ ID NO: 1. In some embodiments, the SBD comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence as defined at positions 73 to 453 of SEQ ID NO: 1.

[0058] In some embodiments, the SBD comprises an amino acid sequence as defined at amino acid positions 82 to 467 of SEQ ID NO: 2. In some embodiments, the SBD comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence as defined at positions 82 to 467 of SEQ ID NO: 2.

[0059] In some embodiments, the SBD comprises an amino acid sequence as defined at amino acid positions 87 to 474 of SEQ ID NO: 3. In some embodiments, the SBD comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence as defined at positions 87 to 474 of SEQ ID NO: 3.

[0060] In some embodiments, the SBD comprises an amino acid sequence as defined at amino acid positions 87 to 474 of SEQ ID NO: 4. In some embodiments, the SBD comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence as defined at positions 87 to 474 of SEQ ID NO: 4.

[0061] In some embodiments, the SBD comprises an amino acid sequence as defined at amino acid positions 79 to 468 of SEQ ID NO: 5. In some embodiments, the SBD comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence as defined at positions 79 to 468 of SEQ ID NO: 5.

[0062] In some embodiments, the SBD comprises the sequence defined at amino acid positions 76 to 467 of SEQ ID NO: 6. In some embodiments, the SBD comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence defined at positions 76 to 467 of SEQ ID NO: 6.

[0063] In some embodiments, the SBD comprises an amino acid sequence as defined at amino acid positions 82 to 463 of SEQ ID NO: 7. In some embodiments, the SBD comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence as defined at positions 82 to 463 of SEQ ID NO: 7.

[0064] Table 1. Exemplary OppA proteins (signal peptides are underlined; added Myc tags are shown in bold)

[0065]

[0066] The capture domain described herein can be an oligonucleotide, peptide, polypeptide, small molecule, or any combination thereof that specifically binds to a desired heterologous portion or binding partner on a heterologous portion to be carried on the bacterial surface. In a preferred embodiment, the capture domain is a polypeptide. The capture domain can be a protein or a protein fragment. The capture domain can be native to or a heterologous entity for Lactobacillus bacteria.

[0067] Lactobacillus bacteria are engineered to display capture domains on their surfaces. To display peptide capture domains, heterologous nucleic acid molecules encoding peptides can be introduced into the bacteria. In some embodiments, multiple heterologous nucleic acid molecules encoding multiple peptides can be introduced into the bacteria. In some embodiments, these peptides may encode the same capture domain. In other embodiments, these peptides may encode different capture domains.

[0068] The drug or heterologous portion can bind to the capture domain directly or via a binding partner on the drug or heterologous portion recognized by the capture domain. In some embodiments, the drug comprises a tag capable of binding to the capture domain on a bacterial surface. The tag can be covalently or nonvalently bound to the capture domain. The tag can be an oligonucleotide, peptide, polypeptide, small molecule, or any combination thereof.

[0069] In one implementation, the capture domain is a biotin-binding polypeptide, and the tag is biotin. The biotin-binding polypeptide can be endogenous or non-endogenous for Lactobacillus bacteria.

[0070] Non-limiting examples of biotin-binding polypeptides include avidin, streptavidin, neutral avidin, and their homologs and derivatives. A “homolog” is a protein that is functionally and / or structurally equivalent. Suitable homologs can be identified using methods and databases known in the art through comparative homology analysis based on amino acid sequences or protein domains. A “derivative” can be a protein variant engineered to have improved properties, such as improved biotin binding, greater thermal or pH stability, or improved or absent multimer formation.

[0071] In one embodiment, the biotin-binding polypeptide is streptavidin. In one embodiment, the streptavidin is polymeric streptavidin. In one embodiment, the polymeric streptavidin is tetrameric streptavidin.

[0072] In some embodiments, the capture domain includes an anchoring domain for anchoring to the bacterial surface. The anchoring domain may attach to the bacterial cell wall or cell membrane. In a preferred embodiment, the anchoring domain is a domain derived from an endogenous Lactobacillus protein.

[0073] In one embodiment, the anchoring domain is a lipoprotein anchoring domain. The lipoprotein anchoring domain may be an anchoring domain derived from the lp_1452 protein (Uniprot accession number Q88X05).

[0074] In one embodiment, the anchoring domain is a LysM anchoring domain. The LysM anchoring domain may be an anchoring domain derived from the lp_3014 protein (Uniprot accession number F9USE1).

[0075] In one embodiment, the anchoring domain is a bacterial transmembrane domain. The bacterial transmembrane domain may be a transmembrane domain derived from the lp_1568 protein (Uniprot accession number F9UNU7). In one embodiment, the bacterial transmembrane domain comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 8.

[0076] Lp_1568 Anchoring Structure Domain

[0077] (SEQ ID NO: 8)

[0078] Drugs can be peptides, nucleic acids, lipids, polysaccharides, small molecules, or radionuclides. In some embodiments, the drug comprises cytotoxic agents, non-limiting examples of which include radionuclides; chemotherapeutic agents; growth inhibitors; enzymes and fragments thereof, such as nucleases; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof; antimicrotubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase I inhibitors, topoisomerase II inhibitors, antimetabolites, hormones and hormone analogs, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, pro-apoptotic agents, LDH-A inhibitors, fatty acid biosynthesis inhibitors, cell cycle signaling inhibitors, HDAC inhibitors, proteasome inhibitors, and cancer metabolism inhibitors.

[0079] In some embodiments, the drug is a prodrug comprising a cleavable entity. When the prodrug contains a tag for binding to a capture domain on a bacterial surface, the cleavable entity can be a cleavable linker between the drug and the tag.

[0080] In some embodiments, the prodrug is activated near cancer cells or cancerous tissue through the cleavage of the cleavable entity. For example, the cleavable entity may be cleaved in response to extracellular signals near cancer cells or cancerous tissue. Extracellular signals may be cancer-specific signals. Non-limiting examples of extracellular signals include extracellular pH, extracellular redox conditions, extracellular oxygen levels, and extracellular metabolites, nucleic acids, peptides, or polypeptides near cancer cells or cancerous tissue.

[0081] In one embodiment, the cleavable entity is a redox-sensitive entity. A redox-sensitive entity may be sensitive to oxidants or oxidizing conditions or reducing agents or reducing conditions present in the vicinity of cancer cells or cancerous tissue. In one embodiment, the redox-sensitive entity is sensitive to oxidants or oxidizing conditions in the vicinity of cancer cells or cancerous tissue, such as reactive oxygen species (e.g., hydroperoxides, superoxides, hydroxyl radicals, or singlet oxygen).

[0082] In one embodiment, the cleavable entity is an enzyme-cleavable entity. The enzyme-cleavable entity may be an enzyme-cleavable linker. The enzyme-cleavable linker may be, for example, a peptide or peptide linker. The enzyme-cleavable entity may be cleaved by extracellular enzymes such as matrix metalloproteinases (MMPs) near cancer cells or cancerous tissue.

[0083] In some implementations, bacteria are administered via mucosa, around the tumor, or within the tumor. Administration can be via injection, spray, direct application (e.g., direct application to the mucosal surface or tumor tissue), or implantation (e.g., an implanted device or stent).

[0084] In one embodiment, bacteria are administered via a mucosa. The mucosa used herein can be any mucosa, such as the oral mucosa, buccal mucosa, nasal mucosa, lung mucosa, rectal mucosa, urethral mucosa, bladder mucosa, vaginal mucosa, and ocular mucosa. As used herein, mucosal administration encompasses delivery to one or more mucosa. Mucosal administration includes, but is not limited to, oral administration, intranasal administration, and bladder instillation. Oral mucosal administration includes buccal, sublingual, and gingival delivery routes.

[0085] In one implementation, the bacteria are applied around the tumor, i.e., in direct contact with or adjacent to the tumor. In another implementation, the bacteria are applied within the tumor, i.e., directly into the tumor mass. Technicians can determine the appropriate route of application based on the location of the cancer, the characteristics of the Lactobacillus bacteria (such as their ability to home to or adhere to specific tissues or mucous membranes), the drug to be delivered, and other factors.

[0086] In some implementations, the cancer is carcinoma. Carcinoma is a malignant tumor originating from epithelial cells (e.g., epithelial cells lining the inner surface of the body) and is generally associated with the mucosal layer, where the natural microbiome is ubiquitous. Examples of carcinoma include, but are not limited to, malignant melanoma, non-small cell lung cancer (e.g., squamous non-small cell lung cancer and non-squamous non-small cell lung cancer), small cell lung cancer, head and neck cancer (e.g., oral cancer, nasal cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, salivary gland cancer, and tongue cancer), renal cell carcinoma (e.g., clear cell renal cell carcinoma), breast cancer, ovarian cancer (e.g., serous ovarian cancer and clear cell adenocarcinoma of the ovary), uterine cancer (e.g., cervical cancer and endometrial cancer), anal cancer (e.g., anal canal cancer), and colorectal cancer (e.g., MSI-H and / or dMMR-positive colorectal cancer). Hepatocellular carcinoma, esophageal cancer, gastric cancer, esophagogastric junction cancer, pancreatic cancer, urothelial carcinoma (e.g., bladder cancer, upper urinary tract cancer, ureteral cancer, renal pelvis cancer, and urethral cancer), prostate cancer, fallopian tube cancer, primary peritoneal cancer, malignant pleural mesothelioma, gallbladder cancer, bile duct cancer, biliary tract cancer, skin cancer (e.g., uveal melanoma and Merkel cell carcinoma), testicular cancer (e.g., germ cell tumor), vaginal cancer, vulvar cancer, penile cancer, small bowel cancer, endocrine system cancers, thyroid cancer, parathyroid cancer, adrenal cancer, retinoblastoma of the eye, neuroendocrine tumors, squamous cell carcinoma, etc.

[0087] In some implementations, the cancer is cancer of the mucosal tissue. Non-limiting examples of mucosal cancer include head and neck cancers (e.g., oral cancer, nasal cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, salivary gland cancer, and tongue cancer), lung cancers (e.g., small cell lung cancer, non-small cell lung cancer), esophageal cancer, esophagogastric junction cancer, gastric cancer, colorectal cancer, pancreatic duct cancer, uterine cancers (e.g., cervical cancer and endometrial cancer), ovarian cancer, bladder cancer, vaginal cancer, vulvar cancer, and anal cancer.

[0088] In some embodiments, the cancer is nasopharyngeal carcinoma, oral cancer, lung cancer, bladder cancer, stomach cancer, colorectal cancer, skin cancer, breast cancer, or ovarian cancer. In one embodiment, the cancer is nasopharyngeal cancer, such as nasopharyngeal carcinoma.

[0089] This article discloses an engineered Lactobacillus bacterium carrying a cancer drug for treating cancer in a subject, wherein the drug binds to a capture domain displayed on the surface of the bacterium.

[0090] This article also discloses the use of engineered Lactobacillus bacteria carrying cancer drugs in the manufacture of agents for treating cancer in subjects, wherein the drugs bind to capture domains displayed on the surface of the bacteria.

[0091] This article discloses a method for delivering a drug to cancer cells, the method comprising contacting the cancer cells with a Lactobacillus bacterium carrying a cancer drug, wherein the cancer drug binds to a capture domain displayed on the surface of the bacteria. The method may be in vitro, ex vivo, or in vivo.

[0092] This article discloses a method for detecting cancer in a subject, the method comprising administering to the subject an effective amount of engineered Lactobacillus bacteria carrying a detectable marker, wherein the detectable marker binds to a capture domain displayed on the surface of the bacteria.

[0093] This article discloses a method for labeling cancer cells, the method comprising contacting the cancer cells with a Lactobacillus bacterium carrying a detectable label, wherein the detectable label binds to a capture domain displayed on the surface of the bacterium.

[0094] Detectable markers can be radioactive or optically detectable, such as fluorescent markers.

[0095] Engineered Lactobacillus bacteria

[0096] This article discloses an engineered Lactobacillus bacterium comprising (a) a heterologous nucleic acid molecule encoding a polypeptide containing a capture domain displayed on the surface of the bacterium; and (b) a heterologous portion that binds to the capture domain.

[0097] In one embodiment, the bacteria is Lactobacillus plantarum (Lp).

[0098] In some implementations, the heterogeneous portion includes tags capable of incorporating capture domains.

[0099] In some embodiments, the capture domain comprises a biotin-binding polypeptide, and the tag is biotin. In one embodiment, the biotin-binding polypeptide is streptavidin. In one embodiment, the streptavidin is multimeric streptavidin. In one embodiment, the multimeric streptavidin is tetrameric streptavidin.

[0100] In some embodiments, the heterologous portion is a drug. In one embodiment, the drug is a prodrug comprising a cleavable entity.

[0101] In some implementations, the heterogeneous portion includes a detectable marker.

[0102] In some embodiments, the bacteria also contain an OppA polypeptide on the surface of the bacteria. The bacteria may contain one or more OppA polypeptides on their surface. In one embodiment, the OppA polypeptide comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequences shown in SEQ ID NO:1-7.

[0103] This document discloses a composition comprising Lactobacillus bacteria as defined herein.

[0104] This article discloses a Lactobacillus bacterium or composition as defined herein, for use as a pharmaceutical agent.

[0105] This document discloses a method for preparing engineered Lactobacillus bacteria as defined herein, the method comprising: a) expressing in the bacteria a polypeptide containing a capture domain displayed on the surface of the bacteria; and b) contacting the bacteria with a heterologous portion capable of binding the capture domain.

[0106] The method may further include introducing a heterologous nucleic acid encoding a polypeptide into bacteria. The nucleic acid encoding the polypeptide may be operatively linked to a promoter, such as an inducible or constitutive promoter. The nucleic acid may be contained in a vector. Bacteria may be cultured under conditions suitable for polypeptide expression.

[0107] In one embodiment, the *Lactobacillus* bacteria are also engineered to produce and / or carry a drug for intranasal drug delivery to the olfactory region, brain, and CNS. In a preferred embodiment, the drug is a prodrug. In some embodiments, the bacteria are engineered to secrete the drug, preferably at an intranasal site (e.g., olfactory epithelium) after intranasal administration. The bacteria may comprise one or more expression constructs encoding a heterologous drug molecule (e.g., a recombinant peptide or polypeptide) or a component of a biosynthetic pathway required to synthesize the heterologous drug molecule. The expression construct may comprise one or more nucleotide sequences encoding a peptide or polypeptide, the one or more nucleotide sequences being operatively linked to one or more of the same or different promoters capable of directing the expression of the sequence in a *Lactobacillus* host. The promoters used according to the method of the invention are preferably constitutively expressed in the bacteria. The use of constitutive promoters avoids the need to provide inducers or other regulatory signals to cause expression to occur. The promoter may also be an inducible promoter, preferably a conditionally inducible promoter found at an intranasal site. The promoter may be homologous to the bacteria used, i.e., a promoter naturally found in the bacteria. Preferably, the promoter guides expression at a level that keeps the bacterial host cell viable, that is, maintaining a certain level of metabolic activity even if growth is not maintained.

[0108] definition

[0109] The terms “treatment,” “treat,” or “treating” are used herein to refer to a reduction in the severity of a disease or condition; a reduction in the duration of a disease; an improvement or elimination of one or more symptoms associated with a disease or condition; or to providing a beneficial effect to a subject suffering from a disease or condition. The term also covers the prevention of a disease or condition or its symptoms. As is known in the art, “prevention” refers to reducing or decreasing the occurrence or severity of a specific disease outcome. For example, delaying the progression of cancer in a subject.

[0110] As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). In many embodiments, the subject is a human. A subject can be a patient, which refers to a person presented to a healthcare provider for the diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." In some embodiments, the subject is a human. The subject may suffer from or be susceptible to a disease or disorder, but may or may not exhibit symptoms of said disease or disorder.

[0111] As used herein, “endogenous” refers to molecules that are naturally encoded and / or present in the host organism. For example, OppA nucleic acid or polypeptide, which is synthesized from the genome of Lactobacillus plantarum WCFS1, is endogenous to the host bacteria.

[0112] As used herein, “exogenous” means a molecule that is not naturally encoded and / or does not exist within a host organism. For example, an OppA nucleic acid or polypeptide that is not naturally encoded and / or does not exist in the host bacterium *Lactobacillus plantarum* WCFS1 is exogenous to said bacterium. As another example, an OppA nucleic acid or polypeptide synthesized outside *Lactobacillus plantarum* WCFS1 but introduced into the cytoplasm or surface of said host bacteria is exogenous to said host bacteria. Any polypeptide produced by the host organism from nucleic acids introduced into the host organism will be considered exogenous to said host organism.

[0113] As used herein, a "heterologous" sequence, gene, or polypeptide refers to any sequence, gene, or polypeptide that is naturally encoded and / or present in an organism that is not a host organism. Heterologous sequences, genes, or polypeptides may include synthetically engineered sequences, genes, or polypeptides.

[0114] As used herein, a "homologous" sequence, gene, or polypeptide refers to any sequence, gene, or polypeptide that is naturally encoded and / or present in a host organism.

[0115] As used herein, a "symbiotic" organism is any organism that resides on the surface or mucous membranes of a host organism without harming the health of the host organism. This includes, for example, *Lactobacillus plantarum*, a symbiotic bacterium in the microenvironment of human bladder tumors.

[0116] As used herein, “probiotics” refers to live organisms that provide health benefits when administered to a host organism. This includes, for example, engineered Lactobacillus plantarum as described herein, for the treatment of human bladder cancer.

[0117] As used herein, “and / or” means and covers any and all possible combinations of one or more related listed items, as well as the absence of combinations when explained in the alternative(or) section.

[0118] As used in this application, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly indicates otherwise. For example, the term “a pharmaceutical agent” includes multiple pharmaceutical agents, including mixtures thereof.

[0119] Throughout this specification and the statements that follow, unless the context otherwise requires, the words “comprise” and variations such as “comprises” and “comprising” shall be understood to imply inclusion of the stated whole or step or group of wholes or steps, but not to exclude any other whole or step or group of wholes or steps.

[0120] In this specification and the following statements, unless the context otherwise requires, the phrase “consistently of…” and its variations (such as “consistently of…”) should be understood to indicate that one or more of the listed elements are essential elements of the invention, i.e., necessary elements. This phrase allows for the presence of other unlisted elements, provided that such elements do not substantially affect the features of the invention, but excludes additional unspecified elements that would affect the fundamental and novel features of the defined method.

[0121] References to any prior publications (or information derived therefrom) or any known things in this specification are not, and should not be construed as, an acknowledgment or endorsement, or in any way imply that such prior publications (or information derived therefrom) or known things constitute part of the general knowledge in the technical field to which this specification pertains.

[0122] Those skilled in the art will recognize that, apart from those specifically described, the invention described herein is susceptible to variation and modification. It should be understood that the invention includes all such variations and modifications falling within its spirit and scope. The invention also includes all steps, features, compositions, and compounds individually or collectively mentioned or indicated in this specification, as well as all combinations of any one or any two or more of said steps or features.

[0123] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0124] Some embodiments of the invention will now be described with reference to the following examples, which are intended for illustrative purposes only and are not intended to limit the general scope of the above description.

[0125] ***

[0126] The features disclosed in the foregoing description, or the appended claims, or the drawings, expressed in their particular form or in terms of the means of performing the disclosed functions or the methods or processes for obtaining the disclosed results, may be used individually or in any combination of such features as appropriate to implement the invention in various forms.

[0127] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when this disclosure is given. Therefore, the exemplary embodiments of the invention described above are to be considered illustrative rather than restrictive. Various changes may be made to the described embodiments without departing from the spirit and scope of the invention.

[0128] To avoid any doubt, any theoretical explanations provided in this article are intended to improve the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0129] Any chapter headings used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0130] Throughout this specification, including the following claims, unless the context otherwise requires, the words “comprising” and “including”, as well as variations of “comprising,” “including,” and “including”, shall be understood to imply inclusion of the said whole or step or group of whole or steps, but not to exclude any other whole or step or other group of whole or steps.

[0131] It must be noted that, as used in the specification and appended claims, the singular forms “a / an” and “the” include plural indicators unless the context clearly specifies otherwise. A range may be expressed herein as from “about” one specific value and / or to “about” another specific value. When such a range is expressed, another embodiment includes from one specific value and / or to another specific value. Similarly, when a value is expressed as an approximation using the antecedent “about,” it will be understood that the specific value forms another embodiment. The term “about” in relation to numerical values ​​is optional and means, for example, + / - 10%.

[0132] Example

[0133] Example 1 - Tumor-seeking symbiotic bacteria for prodrug conjugation in targeted cancer therapy

[0134] To identify cancer-binding microorganisms, six symbiotic strains were investigated, including *Lactobacillus plantarum* WCFS1, *Lactobacillus casei* DSM 20011, *Lactobacillus acidophilus* DSM 20079, *Lactobacillus reuteri* DSM 20016, *Lactobacillus salivarius* DSM 20555, and *Escherichia coli* Nissle 1917. These strains were selected based on their prevalence within a high species abundance range in the nasopharynx and their applications in otolaryngology. The binding affinity of these strains to NPCs was tested using the human NPC cell line CNE-1 as a target. Figure 1 As shown in a, Lactobacillus plantarum WCFS1 (Lp) exhibited the highest binding affinity for CNE-1 cells compared to other bacteria and showed a positive correlation with confluence with CNE-1 cells.

[0135] The binding efficacy of Lp to both cancerous and non-cancer cells derived from human nasal cells was then tested to further evaluate its specificity for NPC. For visualization of the binding, Lp was engineered to express red fluorescent protein, and the engineered strain was co-cultured with four NPC cell lines (CNE-1, CNE-2, C666-1, and HK-1), the nasal squamous cell carcinoma line RPMI 2650, and the human healthy nasal cell line (HNC). Immunofluorescence staining (IF) of the Lp-NPC cocultures showed that the Lp strain bound to all cancer cells but not to HNC cells. Figure 1 b). Scanning electron microscopy (SEM) revealed that Lp was present on the surface of CNE-1 cells in the co-culture and was not internalized by the host cells. Figure 9 Furthermore, *Lp* was labeled with fluorescein isothiocyanate (FITC), and the binding efficacy of bacteria to different cells was quantitatively compared. The binding efficacy of *Lp* to various cancer cell lines was 6 to 20 times higher than that to *HNC*. Figure 1 c). Lp was also found to interact with bladder cancer, lung cancer, and gastric cancer cells ( Figure 1 d).

[0136] NPCs are known to overexpress heparan sulfate proteoglycans and expose heparan sulfate on their cell surface. Heparan sulfate has been reported as a binding target for Lactobacillus adhesins. Nasal respiratory epithelium lacks apical heparan sulfate, and ciliated HNCs mimicking nasal epithelium show negligible binding. For these reasons, we hypothesize that heparan sulfate is one of the binding targets in the Lp-NPC interaction.

[0137] For these reasons, we hypothesized that heparan sulfate is one of the binding targets in the Lp-NPC interaction. To test this hypothesis, we co-incubated cancer cells with heparan sulfate analogue heparin before co-culturing. The addition of heparin sodium (HS) significantly inhibited Lp-NPC binding ( Figure 1 e), which can be explained by HS competitively inhibiting the binding between Lp and heparan sulfate (e). Figure 1 f). At the highest concentration, HS reduced the binding of Lp to all four NPC cell lines by 35%–60% (f). Figure 1 e). In summary, these data indicate that Lp can bind to NPC with high affinity and that heparan sulfate on NPC may be a potential binding target for Lp.

[0138] Subsequently, we discovered a family of heparan sulfate proteins called oligopeptide-binding proteins (OppA) in Lp, which exhibited high binding affinity to all NPCs. Seven homologous copies of the OppA protein were identified within the Lp genome. To investigate their potential for targeting cancer cells, we recombinantly expressed and purified all seven OppA proteins: Lp_0018, Lp_0092, Lp_0200, Lp_0201, Lp_0783, Lp_1261, and Lp_3686. For ease of detection, we fused each OppA protein with a cMyc tag at its C-terminus. Flow cytometry analysis was performed to determine the binding affinity of each OppA protein to five different cancer cell types. The results showed that all OppA proteins bound to all five cancer cell types, with Lp_0018 exhibiting the highest binding affinity and being a universal binder for all cancer cells, while Lp_0092 exhibited the lowest binding affinity. Figure 2 a). Notably, the OppA protein exhibits varying binding affinity to different cancer cell lines, suggesting that the presence of all seven proteins in Lp provides the bacteria with a generally higher binding affinity to a range of cancer cells.

[0139] We then examined whether this enhanced binding was generalized and observable in other cell lines. When added to the co-culture, we found that Lp_0018 increased the binding bacteria in all NPC cells, and the increase was positively correlated with the concentration of Lp_0018. Figure 2 b). At a concentration of 10 μg / ml, Lp_0018 increased the number of bound bacteria on various NPC cells by 8 to 27 times ( Figure 2 b). This finding was unexpected and contradicted previous studies on the OppA protein in *Lactobacillus salivarius*. Interestingly, the addition of heparin salt to the co-culture reduced the enhanced binding of Lp_0018 ( Figure 2 (b) This indicates that heparin blocks the binding of Lp_0018 to heparan sulfate on cancer cells. To investigate the interaction between OppA and heparan sulfate, we predicted the 3D structures of all OppA proteins using AlphaFold42 and performed heparin docking on a simulated protein model using Cluspro43 and tetrameric heparin as ligands. Based on docking complex analysis, all proteins were predicted to have potential heparin-binding capacity and exhibited two common heparin-binding sites. Among the proteins, Lp_0783, Lp_0201, and Lp_1261 were predicted to have only one heparin-binding site, while Lp_0018, Lp_0092, Lp_0200, and Lp_3686 showed two heparin-binding sites. In the OppA proteins, Lp_0018 was predicted to have much lower heparin-binding energies at both sites, indicating a stronger interaction with the ligand heparin (b). Figure 2 c). The superimposed docking complex and sequence alignment clearly show that the contact map regions are highly conserved across different OppA proteins. However, compared to other OppA proteins, Lp_0018 has more arginine, lysine, and asparagine residues at both binding sites, which are known to have strong interactions with heparin and heparan sulfate. Figure 2 c), which explains the stronger heparin binding efficacy.

[0140] Both Lp_0200 and Lp_0018 exhibit high binding efficacy to CNE-1 cells. Figure 2 a(i)). It was also observed that both proteins adhered to the bacterial surface when incubated with Lp alone, but Lp_0018 exhibited significantly stronger adhesion to Lp than Lp_0200. This leads us to hypothesize that the OppA protein enhances Lp-NPC binding by simultaneously adhering to both Lp and NPC, and that the weaker bacterial binding ability of Lp_0200 may be due to the loss of its N-terminal sequence, limiting its ability to enhance Lp-CNE-1 binding (a). Figure 2d). To verify this, we removed the N-terminal sequence of Lp_0018 and recombined it to express its substrate-binding domain - Lp_0018 SBD. The loss of the N-terminal sequence in Lp_0018 SBD disrupted its binding ability with Lp ( Figure 2 e), and incubation with Lp_0018 SBD does not increase Lp-NPC binding in co-cultures of any cell line (e). Figure 2 f). This demonstrates the recombinant expression of Lp_0018.

[0141] It binds to both host bacteria and NPCs. Furthermore, in four cell lines, the addition of Lp_0018 SBD reduced Lp-NPC binding, decreasing by 25% in RPMI2650 cells to 65% in C666-1 cells. Figure 2 f). The absence of reduction in CNE-1 cells suggests the existence of mechanisms other than Lp_0018 in Lp-CNE-1 binding.

[0142] Therefore, we conclude that the OppA protein is the main effector of binding between Lp and NPC, and that heparan sulfate is the binding target of the OppA protein on NPC.

[0143] Lp was genetically modified to utilize its cancer-binding characteristics as a vector strain for prodrug delivery to cancer cells. Streptavidin (Sav) was chosen as the prodrug carrier protein due to the high binding affinity and specificity between tetrameric streptavidin and biotinylated molecules. Lp was engineered to display homotetrameric streptavidin (Lp-Sav) by combining it with a streptavidin secretion cassette. Figure 3 a and Figure 3 (As shown in b). Using Western blotting, flow cytometry analysis, and IF staining targeting the cMyc tag, Lp_1568-Sav was detected in both the control strain Lp-mSav and the Lp-Sav strain (as shown in b). Figure 3 c- Figure 3 (As shown in e). On the other hand, only the Lp-Sav strain displays HA-labeled streptavidin on its surface, possibly multimeric streptavidin, such as... Figure 3 This was demonstrated by e. This was further confirmed by Western blot analysis, which showed that excessive Sav protein was detected only in the culture supernatant of the Lp-Sav strain. This protein can interact with Lp_1568-Sav and polymerize into the tetramer streptavidin (…). Figure 3 c). In summary, Lp was successfully engineered to exhibit active tetrameric streptavidin protein on its bacterial surface, thereby allowing for surface loading of biotinylated molecules in the engineered strain.

[0144] Based on the chemotherapeutic agent SN-38 (referred to as SN), a biotinylated prodrug TL-SN was developed, which incorporates a thioacetate linker sensitive to reactive oxygen species (ROS) and glutathione (GSH). Figure 3 f). When excited at 363 nm, the prodrug produces a unique emission peak at 445 nm, which helps distinguish the TL-SN form of the prodrug from the natural SN form. The prodrug exhibits high binding affinity to Lp-Sav strains, with a maximum concentration reaching 8.9 μM (f). Figure 3 g).

[0145] The thioketal linker of TL-SN is sensitive to reactive oxygen species (ROS) and glutathione (GSH). Figure 4 a, Figure 4 b). TL-SN was incubated with either H2O2 or GSH as an activator to test the release of SN from Lp-Sav. Interestingly, TL-SN was more sensitive to H2O2 than GSH, and released SN at a higher reaction rate in H2O2. Figure 4 c. Figure 4 f). When excited at 363 nm, the TL-SN prodrug produces a unique emission peak at 445 nm, which helps distinguish the prodrug from the native SN, which emits at 560 nm. Fluorescence signal measurements revealed that byproducts of TL-SN decomposition produce an emission peak at 420 nm, which aids in background fluorescence readings of TL-SN at 445 nm. Figure 4 d、 Figure 4 g). However, we measured their concentrations based on the fluorescence signals of TL-SN and SN and found that after incubation with H2O2 or GSH for 5 or 10 hours, over 80% of the prodrug was converted to SN, indicating successful release of SN. Figure 4 e Figure 4 h).

[0146] In the absence of ROS or GSH stimulants, Lp-Sav strains loaded with prodrugs were co-cultured with C666-1 cells. Results showed that Lp-Sav strains loaded with TL-SN responded to the release of SN from intrinsic activators in C666-1 cells, leading to SN accumulation within C666-1 cells. Figure 5 a).

[0147] The anticancer efficacy of prodrug-loaded Lp-Sav strains was evaluated in vitro by co-culturing them with four types of NPC cells and measuring the 24-hour half-maximal inhibitory concentration (IC50). 50 To inhibit bacterial growth during co-culture, the antibacterial antibiotics erythromycin and chloramphenicol were added to the culture medium. It is noteworthy that 24 hours of co-culture did not affect the viability of NPC cells. Figure 5b). It was found that TL-SN had a similar IC to SN when treating most NPC cells. 50 Values, some of which showed higher IC50 values ​​to the prodrug. 50 value( Figure 5 c and Figure 5 d). In some cell lines, the IC50 of the prodrug is... 50 Value higher than SN ( Figure 5 c and Figure 5 d), this may be due to the delayed release of SN in the prodrug. Interestingly, when treating CNE-1, HK-1, and C666-1 cells, Lp-Sav cells loaded with TLSN resulted in IC50 compared to those loaded with SN. 50 A 5-10 fold reduction. This reduction can be attributed to potentially higher concentrations of SN released around cancer cells.

[0148] The limited reduction observed in CNE-2 cells can be attributed to the higher resistance of CNE-2 cells to SN. TL-SN exhibited similar IC50 values ​​to SN when treating most NPC cells, and almost twice the IC50 of SN when treating CNE-2 cells. However, the introduction of Lp-Sav improved the treatment efficiency of TL-SN in all NPC cells, indicating a synergistic effect between Lp-Sav and TL-SN. Figure 5 c and Figure 5 d). Therefore, these results indicate that Lp can deliver TL-SN to NPC cells, leading to SN accumulation near the cells and improved treatment outcomes.

[0149] To further evaluate the performance of Lp-Sav in animal models, a xenograft NPC mouse model was established in BALB / c nude mice by subcutaneous injection of C666-1 cells. Figure 6 a). After xenograft tumors were induced, Lp cells were administered intravenously to investigate the biodistribution of Lp in tumor-bearing mice. Lp cells were engineered to express bioluminescent proteins and injected via tail vein into tumor-bearing mice with relatively large tumors. To investigate biodistribution, mice were euthanized at 24, 48, and 72 hours post-injection, and organs were harvested for analysis. IVIS analysis revealed that tumors produced the highest bioluminescent signal in all organs, indicating that a large amount of Lp was localized in the tumor ( Figure 6 c). To quantify the amount of Lp in various organs, organ homogenates were prepared and bacterial colony-forming units (CFU) were measured. The highest bacterial colonization of Lp was found in the liver, spleen, and tumors. Figure 6 d). Furthermore, the amount of Lp in the tumor steadily increased over 72 hours, while bacterial cells in the liver, spleen, and other organs were mostly cleared within 48 hours. Figure 6(b) In non-tumor organs, *Lactobacillus* (Lp) is cleared much faster than other tumor-finding bacteria used in BMCT, including *Salmonella typhimurium* strains, *Listeria monocytogenes*, *Pseudomonas aeruginosa*, and *Escherichia coli*. Therefore, *Lactobacillus* strains such as *Lp* are readily available in the tumor microenvironment and a safer option for BMCT applications compared to many opportunistic pathogens that are not naturally occurring in the human microbiome.

[0150] The transient presence of Lp-CB in organs implies bacterial circulation in the mouse bloodstream. To assess the risk of sepsis, we determined the duration of Lp-CB presence in the blood by monitoring colony-forming units (CFU) of Lp-CB at 1, 3, and 6 h after intravenous injection. Figure 6 e). Notably, Lp-CB levels in mouse blood decreased rapidly and became undetectable six hours after injection. Figure 6 e).

[0151] To rule out non-specific localization of bacteria in tumors, the biodistribution of the control Lactobacillus strain Lactobacillus reuteri DSM 20016 (referred to as LrD) in tumor-bearing mice was also evaluated. Following intravenous injection, the density of Lp-CB in the tumor was measured 24 hours later. Figure 6 f) and the tumor-to-liver ratio of Lp-CB (f) Figure 6 The fg values ​​were more than 35-fold and 39-fold higher than those of LrD, respectively, indicating higher specificity and efficiency in the colonization of NPC tumors.

[0152] Subsequently, the efficacy of Lp-Sav-TL-SN in treating NPC in a xenograft mouse model was further evaluated. It was assumed that using the strategy of this invention would require a lower dose of the prodrug SN to achieve significant inhibition of tumor growth. Therefore, a dose-reduction treatment regimen was designed for animal experiments: treatment with a low dose of 50 μg / kg SN twice a week for two weeks, for a total of 200 μg / kg SN. Figure 6 a). In contrast, previous studies reported the use of

[0153] Irinotecan (an FDA-approved SN prodrug) is administered at higher total doses ranging from 150 mg / kg to 400 mg / kg over a period of less than four weeks.

[0154] To evaluate the efficacy of Lp-Sav-TL-SN, after the initial induction of subcutaneous C666-1 tumors (5-8 mm in size), one treatment group (Lp-Sav-TL-SN) along with four control groups (PBS, Lp-Sav, SN, and TL-SN) were administered intravenously to tumor-bearing mice. Figure 6 a and Figure 6b). For each group, mice were administered a total of four doses over a two-week span, and no significant weight loss or other side effects were observed in the mice. Figure 7 a). Based on tumor volume and tumor weight to body weight ratio, the tumors in the Lp-Sav-TL-SN group mice were 50% smaller than those in the SN group mice, 66% smaller than those in the TL-SN group mice, and 75% smaller than those in the PBS control group mice. Figure 7 b and Figure 7 c).

[0155] In summary, the Lp-Sav-TL-SN strain significantly inhibited the progression of NPC tumors. NPC recognition of the native OppA protein on the bacterial surface enables the bacteria to bind to the NPC surface, and the loaded TL-SN can release SN near NPC cells to inhibit cancer growth. In vitro experiments showed that the prodrug-loaded microbes significantly increased the potency of SN against NPC cell lines, up to 10-fold. In the NPC mouse xenograft model, prodrug delivery via engineered Lp resulted in 75% tumor growth inhibition, significantly increasing the efficacy of SN by 57%.

[0156] This invention demonstrates reduced potency of unloaded prodrug TLSN, which exhibits a higher IC50 value against NPC cells in vitro. Treatment with TL-SN in tumor-bearing mice did not result in a significant reduction in tumor size. In contrast, administration of Lp-Sav-TL-SN resulted in a significant reduction in IC50 and a significant reduction in tumor size compared to TL-SN and SN alone, signifying an immediate improvement in therapeutic efficacy. Furthermore, the amount of SN required to achieve significant tumor suppression with Lp-Sav-TL-SN is 750 to 2,000 times lower than that in previous studies (Guichard et al., 1998; Kawato et al., 1991; Slatter et al., 2000; Godavarthi et al., 2023). The strategy of this invention could be an effective method for treating cancer with reduced chemotherapy requirements and fewer associated side effects, making it a promising approach for dose reduction in chemotherapy regimens.

[0157] The engineered strain Lp-Sav was generated by reprogramming Lp to display tetrameric streptavidin protein on the bacterial surface. This modification allows for surface loading of biotinylated prodrugs, providing a universal platform for designing prodrugs with flexible compositions. This adaptability enables the selection of active drugs and release mechanisms tailored to the unique characteristics and sensitivities of individual cancers. First, the surface loading pattern serves to protect the prodrug from metabolism by complex intracellular biochemical processes within the bacteria. This protection expands the range of potential chemotherapeutic drugs that can be used. Second, the significant differences between bacterial metabolites and cancer metabolites make it possible to design connectives that respond to various signals within the tumor microenvironment, including hypoxia, acidosis, and high oxidative stress.

[0158] As described in this article, the combination of Lp-Sav-TL-SN constitutes a comprehensive therapy that achieves site-targeted delivery via Lp-Sav and site-specific conversion of TL-SN via an embedded thioacetate linker. This integrated system simplifies prodrug design and broadens the range of candidate active drugs, making it a promising and versatile approach in cancer therapy.

[0159] It should be understood that numerous further modifications and arrangements of the various aspects of the described implementation scheme are possible. Therefore, the described aspects are intended to cover all such changes, modifications, and variations that fall within the spirit and scope of the appended statement.

[0160] References

[0161] Numerous publications have been cited above to provide a more comprehensive description and disclosure of the invention and the current state of the art to which it pertains. Full citations of these references are provided below. The entirety of each of these references is incorporated herein by reference.

[0162] Fu, Z., Li, S., Han, S., Shi, C. & Zhang, Y. Antibody drug conjugate: the “biological missile” for targeted cancer therapy. Signal Transduct. Target. Ther. 7, 93 (2022).

[0163] Xu, S. Internalization, trafficking, intracellular processing and actions of antibody-drug conjugates. Pharm. Res. 32, 3577-3583 (2015).

[0164] Khera, E. & Thurber, GM Pharmacokinetic and immunological considerations for expanding the therapeutic window of nextgenerationantibody-drug conjugates. BioDrugs 32,465-480 (2018)

[0165] Mecklenburg, L. A brief introduction to antibody-drug conjugates fortoxicologic pathologists. Toxicol. Pathol. 46, 746-752 (2018).

[0166] Cheng, Y. et al. Nanomaterials-induced toxicity on cardiac myocytesand tissues, and emerging toxicity assessment techniques. Sci. Total Environ.800, 149584 (2021).

[0167] Tang, L.-L. et al. Global trends in incidence and mortality ofnasopharyngeal carcinoma. Cancer Lett 374, 22-30 (2016).

[0168] Zhang, Y., Rumgay, H., Li, M., Cao, S. & Chen, W. NasopharyngealCancer Incidence and Mortality in 185 Countries in 2020 and the ProjectedBurden in 2040: Population-Based Global Epidemiological Profiling. JMIRPublic Health Surveill 9, e49968 (2023).

[0169] McDowell, L., Corry, J., Ringash, J. & Rischin, D. Quality of life,toxicity and unmet needs in nasopharyngeal cancer survivors. Front. Oncol. 10(2020).

[0170] Wang, C. et al. Toxicities of chemoradiotherapy and radiotherapy innasopharyngeal carcinoma: an updated meta-analysis. J. Int. Med. Res. 47,2832-2847 (2019).

[0171] Guichard, S. et al. Comparison of the pharmacokinetics and efficacyof irinotecan after administration by the intravenous versus intraperitonealroute in mice. Cancer Chemother. Pharmacol. 42, 165-170 (1998).

[0172] Kawato, Y. et al. Antitumor activity of a camptothecin derivative,CPT-11, against human tumor xenografts in nude mice. Cancer Chemother.Pharmacol 28, 192-198 (1991).

[0173] Slatter, J. G. et al. Pharmacokinetics, metabolism, and excretion ofirinotecan (CPT-11) following I.V. infusion of [(14)C]CPT-11 in cancerpatients. Drug Metab. Dispos 28, 423-433 (2000).

[0174] Godavarthi, J. D. et al. The synergistic antitumor effect ofirinotecan and flavonoids on human colon cancer xenograft mice. J. Pharmacol.Exp. Ther. 385, 44 (2023).

[0175] For standard molecular biology techniques, see Sambrook, J., Russel,D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor,New York: Cold Spring Harbor Laboratory Press.

Claims

1. A method of treating a subject with cancer, the method comprising administering to the subject an effective amount of engineered Lactobacillus bacteria carrying a drug, wherein the drug binds to a capture domain displayed on the surface of the bacteria.

2. The method of claim 1, wherein the bacteria are derived from cancerous tissue or tissue capable of developing cancer.

3. The method according to claim 1 or 2, wherein the bacterium is Lactobacillus plantarum (Lp).

4. The method according to any one of claims 1 to 3, wherein the bacteria contain an OppA polypeptide on the surface of the bacteria.

5. The method according to claim 4, wherein the OppA polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequences shown in SEQ ID NO: 1-7.

6. The method according to any one of claims 1 to 5, wherein the drug comprises a tag capable of binding to a capture domain.

7. The method of claim 6, wherein the capturing domain is a biotin-binding polypeptide, and the tag is biotin.

8. The method according to claim 7, wherein the biotin-binding polypeptide is streptavidin.

9. The method according to any one of claims 1 to 8, wherein the capture domain comprises an anchoring domain for anchoring to the bacterial surface.

10. The method of claim 9, wherein the anchoring domain is a bacterial transmembrane domain.

11. The method of claim 10, wherein the bacterial transmembrane domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:

8.

12. The method according to any one of claims 1 to 11, wherein the drug is a prodrug comprising a cleavable entity.

13. The method of claim 12, wherein the prodrug is activated near cancer cells or cancerous tissue by cleavage of the cleavable entity.

14. The method according to claim 12 or 13, wherein the cleavable entity is a redox-sensitive entity.

15. The method according to any one of claims 1 to 14, wherein the bacteria are administered via a mucosa, around a tumor, or within a tumor.

16. The method according to any one of claims 1 to 15, wherein the cancer is cancer.

17. The method according to any one of claims 1 to 16, wherein the cancer is nasopharyngeal carcinoma, oral cancer, lung cancer, bladder cancer, gastric cancer, colorectal cancer, skin cancer, breast cancer, or ovarian cancer.

18. The method of claim 17, wherein the cancer is nasopharyngeal carcinoma.

19. An engineered Lactobacillus bacterium carrying a cancer drug for treating a subject's cancer, wherein the drug binds to a capture domain displayed on the surface of the bacterium.

20. Use of engineered Lactobacillus bacteria carrying cancer drugs in the manufacture of a medicament for treating cancer in a subject, wherein the drug binds to a capture domain displayed on the surface of the bacteria.

21. A method of delivering a drug to cancer cells, the method comprising contacting the cancer cells with a Lactobacillus bacterium carrying the drug, wherein the drug binds to a capture domain displayed on the surface of the bacteria.

22. A method for detecting cancer in a subject, the method comprising administering to the subject an effective amount of engineered Lactobacillus bacteria carrying a detectable marker, wherein the detectable marker binds to a capture domain displayed on the surface of the bacteria.

23. A method for labeling cancer cells, the method comprising contacting the cancer cells with a Lactobacillus bacterium carrying a detectable label, wherein the detectable label binds to a capture domain displayed on the surface of the bacterium.

24. An engineered Lactobacillus bacterium, said engineered Lactobacillus bacterium comprising: a) A heterologous nucleic acid molecule encoding a polypeptide containing a capture domain displayed on the surface of the bacteria; and b) The heterogeneous portion that is combined with the captured structural domain.

25. The engineered bacteria according to claim 24, wherein the bacteria is Lactobacillus plantarum (Lp).

26. The engineered bacteria of claim 24 or 25, wherein the heterologous portion comprises a tag capable of binding the capture domain.

27. The engineered bacteria of claim 26, wherein the capture domain comprises a biotin-binding polypeptide, and the tag is biotin.

28. The engineered bacteria of claim 27, wherein the biotin-binding polypeptide is streptavidin.

29. The engineered bacteria according to any one of claims 24 to 28, wherein the heterologous portion is a drug.

30. The engineered bacteria of claim 29, wherein the drug is a prodrug comprising a lysable entity.

31. The engineered bacteria according to any one of claims 24 to 30, wherein the heterologous portion comprises a detectable marker.

32. The engineered bacteria according to any one of claims 24 to 31, wherein the bacteria further comprises an OppA polypeptide on its surface.

33. A composition comprising Lactobacillus bacteria according to any one of claims 24 to 32.

34. The composition according to claim 33, wherein the composition comprises one or more Lactobacillus bacteria.

35. The Lactobacillus bacteria according to any one of claims 24 to 32 or the composition according to claim 33, used as a pharmaceutical agent.

36. A method for preparing engineered Lactobacillus bacteria according to any one of claims 24 to 32, the method comprising: a) Expressing in the bacteria a polypeptide containing a capture domain displayed on the surface of the bacteria; as well as b) Contact the bacteria with a heterologous portion capable of binding the capture domain.

37. A kit comprising bacteria according to any one of claims 24 to 32.

38. The kit of claim 37, wherein the kit comprises a first container containing a first composition according to any one of claims 33 or 34 and a second container containing a second composition according to any one of claims 33 or 34, wherein the drug carried by bacteria of the first composition comprises a different drug than the drug carried by bacteria of the second composition.

39. The kit of claim 38, further comprising a third container containing the third composition according to any one of claims 33 or 34, wherein the drug expressed and / or carried by the bacteria of the third composition comprises a drug different from the drug expressed and / or carried by the bacteria of the first composition and the second composition.

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