Treatment of Therapy-Induced Enteropathy

Abstract

The present invention provides lactic acid bacteria (LAB) engineered to express a therapeutic protein capable of promoting resolution of inflammation and / or wound healing for use in the treatment or prevention of therapeutic-induced enteropathy in human or animal subjects.

Description

[Technical Field]

[0001] The present invention relates generally to the treatment of enteropathy induced in a subject as a result of a treatment administered to the subject, for example in the treatment of cancer, and in particular to the administration of lactic acid bacteria recombinantly engineered to express a therapeutic protein capable of promoting resolution of inflammation and / or wound healing. [Background technology]

[0002] Various therapies administered to subjects for the treatment or management of a subject's clinical condition, such as cancer, among others, can have damaging or adverse side effects, particularly in the lower gastrointestinal (GI) tract. Such therapy-induced enteropathy can limit the application of the therapy and, in particular, can be a source of significant patient morbidity.

[0003] The introduction of immune checkpoint inhibitors (ICIs), which target, for example, cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed death receptor 1 (PD-1), and programmed death-ligand 1 (PD-L1), has improved the prognosis of many advanced cancers, including melanoma, urothelial and renal cell carcinoma, and non-small cell lung cancer. Although treatment with ICIs can facilitate effective tumor control in responders, many patients treated with ICIs develop immune-mediated adverse events, such as enterocolitis, hepatitis, arthritis, dermatitis, thyroiditis, and hypophysitis.

[0004] Furthermore, although combination ICIs are more effective in cancer control than monotherapy, this strategy carries a higher risk of immune-mediated adverse events, depending on the dose and duration of treatment. Immune-mediated enterocolitis, characterized by abdominal pain and diarrhea, is thought to occur in up to one-third of patients treated with ICIs, negatively impacting patients' quality of life and potentially limiting the continuation of ICI therapy. However, progression-free survival is still highest among patients who develop immune-related gastrointestinal AEs, e.g., as a surrogate for enhanced immune tumor-suppressive activity of T lymphocytes. Therefore, effective prevention or treatment of ICI-induced enterocolitis is essentially a critical component of long-term tumor management plans.

[0005] Current guidelines recommend treating ICI-induced enterocolitis based on symptom grade assessed using the Common Terminology Criteria for Adverse Events (CTCAE). For progressive, more severe disease, supportive measures, systemic corticosteroids, temporary or permanent discontinuation of ICI treatment, and the TNF inhibitor infliximab are sequentially recommended and reported. However, the potential adverse effects of TNF inhibitors on ICI efficacy have been a concern and reported. This current paradigm has several limitations. First, CTCAE grading is based on clinical symptoms, including stool frequency, bleeding, abdominal pain, and fever, rather than more objective markers of disease activity. Second, there are currently no validated instruments to describe and measure endoscopic and histological disease activity in treatment-induced enteropathy. Because clinical symptoms do not fully capture the severity of colitis for adequate assessment of the risk of rupture, biopsies are sometimes used for diagnosis and control. Third, there is limited evidence to inform therapeutic decisions, either for the prevention or treatment of ICI-induced enterocolitis. Clinically relevant questions regarding the optimal timing, sequence, and duration of treatment remain unresolved. Finally, the role of established systemic treatments for inflammatory bowel disease (including IBD, ulcerative colitis, and Crohn's disease), aside from infliximab, in the management of ICI-induced enterocolitis remains unclear.

[0006] In this regard, while ICI-induced enterocolitis shares certain histological features with acute colitis, such as that observed in IBD, the two entities appear to be distinctly immunologically and histopathologically distinct from each other. In particular, we observed increased fibrosis and epithelial ulcers in the colon with ICI-induced colitis, which does not necessarily correlate with the traditional clinical signs associated with colitis. While pathogenesis in ICI-induced colitis has been reported to be primarily driven by T cells, humoral (B cell) immunity has been shown to be more important in IBD (see, e.g., Yanai et al., Clin. Gastroenterol. Hepatol. 2017, 15:e8081; and Bertha et al., ACG Case Rep. J. 2017, 4th ed. 112). Furthermore, these different acute forms of colitis appear to differ clinically from each other, for example, in terms of severity and the potential for rapid progression of complications. Additionally, it is not yet clear whether the chronic colitis that has been observed in patients after ICI treatment resembles long-term IBD colitis disease (Hsieh et al., BMJ Case Rep. 2016, bcr-2016-216641).

[0007] Another commonly observed form of treatment-induced enteropathy is radiation-induced enteropathy. Intestinal radiation toxicity (radiation enteropathy) is generally classified as early (acute), occurring within 3 months of radiation therapy, or delayed (chronic), occurring more than 3 months after radiation therapy. While the incidence of severe (grade 3-4) delayed intestinal radiation toxicity has gradually decreased, largely due to improvements in radiation treatment planning and delivery techniques, it has been reported that approximately half of radiation therapy patients will experience some form of chronic GI dysfunction. Delayed radiation enteropathy is a chronic, often progressive disorder associated with significant long-term morbidity.

[0008] As mentioned above, the occurrence of ICI-induced enterocolitis appears to be associated with a better oncological response to ICI treatment. Recent data indicate that remission of ICI-induced enterocolitis after treatment with the TNF inhibitor infliximab may be associated with a poorer response to immune checkpoint inhibitors, which was indicated by further cancer progression. Based on this, the inventors speculated that local immunomodulatory treatment in the inflamed colon could provide the right balance between immunosuppression and immune activation without the systemic, global effects that dampen the immune system, which could negatively impact the anti-cancer effects of ICIs. This may represent an attractive approach to address the growing unmet medical need for additional or improved treatments for ICI-induced enteropathy, and for radiation- or other therapy-induced enteropathy, for that matter.

[0009] In WO2016 / 102660, the inventors describe the use of genetically modified lactic acid bacteria to express various proteins with wound healing activity, particularly CXCL12, CXCL17, and Ym1, to promote wound healing. The effects reported in this document, which are attributed to the proteins expressed by the bacteria, are thought to result from the immunomodulatory effects of these proteins on immune cells localized in the vicinity of the wound. The inventors therefore extended this research to the area of ​​treatment-induced enteropathy. Summary of the Invention

[0010] The inventors propose that lactic acid bacteria (LAB) modified in accordance with the disclosures herein to express certain proteins useful for promoting inflammation resolution and / or wound healing may be used in certain situations to treat therapy-induced enteropathy.

[0011] The above-mentioned differences between ICI-induced enterocolitis and IBD-related enterocolitis, and the need for local immunosuppression in ICI-induced enterocolitis, suggest that IBD treatment may not be effective or suitable in the setting of ICI induction. Nevertheless, the inventors still believe that the action of the inflammation-resolving and wound-healing proteins expressed by LAB on local B cells, follicular helper T cells, macrophages, and other immune cells in the intestinal areas rich in local immune cells and in the areas of damage induced by ICI or other treatments should be beneficial in repairing and treating this damage. The inventors tested this proposal and, using blinded evaluation with routine histopathology, showed that the administration of modified LAB to animal cancer models with colitis and / or treated with ICI treatment has a beneficial effect on reducing disease activity, crypt damage, and epithelial defects (erosion and ulceration of crypt epithelium) in the colon. Furthermore, the effect of the modified LAB was also demonstrated in reducing colon shortening induced by ICI treatment. Surprisingly, the beneficial effect of treatment with the modified LAB was observed on the disease manifestations during ICI treatment, and thus was not merely an effect of preventing disease onset. Rather, it was shown that the manifestations of damage in the GI tract could be treated, supporting the idea that the LAB could be administered after treatment-induced damage. Importantly, the beneficial effect of the modified LAB was not observed with the corresponding unmodified LAB (i.e., wild-type bacteria not modified to express wound-healing proteins).

[0012] Thus, in a broad and first aspect, the inventors provide herein engineered lactic acid bacteria (LAB) for use in the treatment or prevention of therapy-induced enteropathy in a subject, wherein said lactic acid bacteria have been engineered to express a protein that promotes resolution of inflammation and / or wound healing.

[0013] In particular, the engineered LAB are for use in the treatment of therapy-induced enteropathy.

[0014] In one particular embodiment, said treatment-induced enteropathy is tumor treatment-induced enteropathy, more particularly enteropathy induced by ICI treatment or radiation therapy.

[0015] The subject may be a human or animal subject.

[0016] In particular, the protein is a mammalian protein, more particularly an immunomodulatory protein. In one embodiment, the protein regulates the proliferation and / or activity of immune cells, particularly macrophage cells or their precursors. In one embodiment, the protein is a cytokine or chemokine. In one embodiment, the protein is a CXC protein.

[0017] In a more particular embodiment, said protein is selected from CXCL12, CXCL17 and Ym1, in particular from CXCL12 or CXCL17, hi another embodiment, said protein is TGF-β.

[0018] The LAB may be any genus, species, or strain of LAB, but as described below, the LAB is particularly of the genus Lactobacillus. In one embodiment, the LAB is Limosilactobacillus reuteri, formerly known as Lactobacillus reuteri.

[0019] In another, but related, aspect, provided herein is a pharmaceutical composition comprising the lactic acid bacteria (LAB) engineered herein for use in treating or preventing therapy-induced enteropathy in a subject, wherein the bacteria have been engineered to express a protein that promotes resolution of inflammation and / or wound healing.

[0020] Yet another aspect provides the use of engineered lactic acid bacteria (LAB) for the manufacture of a medicament for use in treating or preventing therapy-induced enteropathy in a subject, wherein the bacteria have been engineered to express a protein that promotes resolution of inflammation and / or wound healing.

[0021] Yet another aspect provides a method for treating or preventing therapy-induced enteropathy in a subject, the method comprising administering to a subject who has received or is receiving a therapy that induces enteropathy, engineered lactic acid bacteria (LAB) that have been engineered to express a protein that promotes wound healing. DETAILED DESCRIPTION OF THE INVENTION

[0022] The medical uses and methods herein are directed to the treatment or prevention, in particular the treatment, of therapy-induced enteropathy.

[0023] The treatment that induces enteropathy is not limited to, and can be any treatment that, when administered to human or animal subjects, causes or leads to enteropathy in the subject.However, in particular, said treatment is a treatment for cancer, or in other words, tumor treatment.Therefore, in one embodiment, the subject suffers from or has been diagnosed with cancer (i.e., is a cancer patient).

[0024] The cancer may be any cancer, without limitation. In particular, the cancer may be a cancer suitable for or susceptible to treatment with an immune checkpoint inhibitor (ICI). The cancer may be, for example, melanoma, urothelial or renal cell carcinoma, or lung cancer, such as non-small cell lung cancer, but these are merely representative examples. The cancer may be characterized by a solid tumor. In one embodiment, the cancer may be an advanced cancer. The nature of the cancer is not critical to the proposed use. Thus, the cancer may be cancer of any organ or tissue of the body. However, in one embodiment, the cancer does not include (or is not) colorectal cancer.

[0025] The enteropathy-inducing treatment can be any therapeutic treatment, including a pharmacological agent or pharmaceutical, including immunotherapy (e.g., immunotherapeutics), or radiation therapy. Thus, the treatment can include administering to the subject radiation, or a small molecule pharmaceutical, e.g., a chemotherapeutic agent, or a biomolecule, e.g., a protein, e.g., an antibody, or an antibody-derived or antibody-based protein.

[0026] In one embodiment, the treatment-induced enteropathy is immune checkpoint inhibitor (ICI)-induced enteropathy or radiation-induced enteropathy (which term is synonymous with "radiation enteropathy"). In a more particular embodiment, the radiation-induced enteropathy is delayed radiation-induced enteropathy.

[0027] The term "enteropathy" is used broadly herein to include any damage, injury, or inflammation to the intestine, or in other words, the GI tract, particularly the lower GI tract, especially the small intestine and large intestine. The term can include any histological changes to the GI tract compared to before treatment or to a healthy individual not receiving the enteropathy-inducing treatment. Specifically, these can be histopathological changes.

[0028] Histological features of treatment-induced enteropathy include crypt inflammation, intraepithelial neutrophilic lymphocytes, glandular destruction, erosion of mucosal surfaces, such as mucosal ulcers, crypt abscesses, apoptosis, and necrosis. Other symptoms include diffuse erythema, edema, loss of vascularity, increased hemodynamic instability, severe congestion, ischemic bowel, and perforation. Generally speaking, inflammatory changes are observed in lesions in the intestine, which may vary in size. Therefore, enteropathy to be treated herein can be defined as inflammation or inflammatory changes in the GI tract, or specifically in the intestine. Inflammatory changes may occur before, or may be present before, or after, overt inflammation is detected. Such changes may result in shortening of the colon or an increase in colon weight. As described above, the severity of treatment-induced enteropathy can be graded using the Common Terminology Criteria for Adverse Events (CTCAE) (grades 1 to 4, with more severe grades). Generally speaking, the enteropathy can manifest as colitis, i.e., inflammation of the colon. However, the term enteropathy also encompasses lower-grade damage or inflammatory changes, including damage or inflammatory changes that have not yet manifested as overt colitis by traditional clinical assessment. In a specific embodiment, the treatment-induced enteropathy to be treated herein may be characterized by crypt damage, epithelial defects, and / or colon shortening, or weight gain.

[0029] Furthermore, in some cases, treatment-induced enteropathy may alternatively or additionally be characterized by intestinal fibrosis. As mentioned above, the inventors have recently observed that fibrosis and edema can be observed in cases of ICI-induced enteropathy in mouse models of disease where more traditional clinical signs of colitis or overt colitis cannot be measured or are not yet observed. The observation of colon shortening, which is a reduction of more than 20% observed in these studies of ICI-induced colitis, is comparable to the most severe grade of colitis in other models, for example, chemically induced colitis using dextran sulfate sodium (DSS), which produces clinical signs measured mainly by weight loss, diarrhea, and bloody stool.

[0030] Markers commonly used to determine or assess the disease activity of inflammatory bowel disease (IBD) or colitis may be used to assess treatment-induced enteropathy. Thus, treatment-induced enteropathy can also be detected and / or diagnosed, at least in part, by detecting inflammatory markers. Inflammatory markers include, for example, proinflammatory cytokines, calprotectin, and / or the presence or activity of immune cells, including, for example, T cells, in the intestine.

[0031] Clinical signs of enteropathy include abdominal pain, increased daily bowel movements, diarrhea, rectal bleeding, and mucus in the stool. These clinical symptoms can interfere with active daily activities. As the severity increases, in stage 4, symptoms can include hemodynamic instability, severe congestion, ischemic bowel, perforation, megacolon, and sepsis. Ultimately, this can lead to death.

[0032] Treatment-induced enteropathy may be diagnosed based on one or more clinical symptoms, including diarrhea, as well as serum inflammatory markers and electrolyte measurements. Significant abnormalities indicative of treatment-induced enteropathy include anemia, elevated C-reactive protein, and low serum albumin levels. This diagnosis may be confirmed by endoscopy or rectoscopy and / or biopsy. Interestingly, mouse models have demonstrated that ICI-induced enteropathy does not necessarily exhibit common clinical features seen in other types of enteropathy and IBD, such as weight loss, diarrhea, and bloody stools, but instead exhibits clinical features such as increased fibrosis and epithelial ulcers, particularly in the colon (Examples 3 and 4). Therefore, increased fibrosis may also be evaluated as a clinical marker of treatment-induced enteropathy. Fibrosis can lead to colon shortening, and colon shortening may be observed as an indicator of colonic fibrosis.

[0033] Common clinical features of radiation-induced enteropathy include intestinal transit abnormalities, malabsorption, and dysmotility. Severe cases may progress to intestinal obstruction, fistula formation, or even frank intestinal perforation. Thus, the medical uses herein include the treatment of overt or overt clinical disease or preclinical conditions. That is, the enteropathy to be treated includes overt or overt disease, as evidenced by clinical symptoms such as diarrhea or symptoms of colitis, as well as preclinical enteropathy, in which clinical symptoms are not yet present but damage to the intestine has occurred, as determined by histological features or changes, including, for example, fibrosis and epithelial ulcers, especially in the colon, as described above. Thus, the enteropathy can be diagnosed by overt symptoms and / or by examination of the GI tract (e.g., by histological examination, e.g., by biopsy, or by endoscopy).

[0034] The medical uses herein include the treatment of established diseases, for example, in which the engineered LAB is administered after treatment-induced enteropathy is identified or diagnosed in a subject, which is a particularly advantageous embodiment of the treatment disclosed herein. In particular, the treatment herein includes the treatment of treatment-induced colitis after colitis has become apparent. However, the LAB can also be used in prevention, to delay or prevent the onset of treatment-induced enteropathy. In such cases, the LAB can be administered to a subject at the start of treatment, before enteropathy has developed or before enteropathy has been diagnosed, for example, before obvious or apparent colitis exists. For example, the LAB can be administered together with or immediately after treatment. In such cases, the LAB can be used to prevent the onset of colitis.

[0035] In one embodiment, the engineered LAB may be used in the treatment of subjects undergoing said treatment who exhibit increased or risk of fibrosis and / or epithelial ulceration in the intestine, particularly in the colon (i.e., as a result of said treatment).

[0036] In another embodiment, engineered LAB can be used to inhibit the development of fibrosis and / or epithelial ulcer in the intestine, particularly the colon, of the subject undergoing treatment.Treatment can be any treatment as discussed herein, and may also be referred to as enteropathy-induced treatment.The term " inhibiting " used herein includes any action of limiting fibrosis and / or epithelial ulcer, for example, reducing, preventing, and / or delaying fibrosis and / or epithelial ulcer.Therefore, the subject can be the subject at risk of developing treatment-induced enteropathy.

[0037] In another embodiment, the subject exhibits or is classified as having treatment-induced colitis at or above stage 2 by CTCAE.

[0038] In another embodiment, engineered LAB may be used to return or maintain the severity of treatment-induced colitis to Grade 2 or less by CTCAE.

[0039] In one embodiment, the treatment-induced enteropathy is steroid-resistant, particularly steroid-resistant ICI-induced enteropathy. However, in another embodiment, the subject is steroid-naive. In yet another embodiment, the subject is steroid-naive and exhibits treatment-induced colitis of grade 1-2 by CTCAE.

[0040] In another embodiment, the engineered LAB are administered or used (or are for use) in combination with a steroid.

[0041] ICI therapy can be therapy with any immune checkpoint inhibitor. An immune checkpoint inhibitor is broadly defined as any agent that inhibits the activity or function of a checkpoint protein. It can also be an agent that binds to a checkpoint protein or to a receptor of a checkpoint protein. Thus, a checkpoint inhibitor can be a binding agent for a checkpoint protein or its receptor. The binding agent can be an antibody, or can be based on or derived from an antibody. The antibody can be a natural antibody or a synthetic antibody, or a fragment or derivative thereof. Currently, the three major immune checkpoints targeted by ICI are PDL-1, PD-L1 (PD-L1 is Programmed Death Ligand 1, and PD-1 is the receptor for PD-L1), and CTLA-4 (cytotoxic T-lymphocyte antigen 4), but the medical uses and methods herein are not limited thereto and include targeting of any immune checkpoint.

[0042] Other checkpoint proteins include the costimulatory checkpoint protein CD-137 (4-1BB); the CD4-associated inhibitory receptor, lymphocyte-activation gene 3 (LAG-3, CD223), which is coexpressed with PD-1 on tolerant T cells; the B7 superfamily proteins B7-H3 and B7-H4; the T cell protein TIM3; and phosphatidylserine (PS), a phospholipid on normal cells that translocates to the outer membrane surface during apoptosis and suppresses otherwise excessive immune activation during the processing and clearance of decaying cellular material.

[0043] Examples of checkpoint inhibitors include: tremelimumab (CP-675,206), a human IgG2 monoclonal antibody with high affinity for CTLA-4; ipilimumab (MDX-010), a human IgG1 monoclonal antibody against CTLA-4; and nivolumab (BMS-936558), a human monoclonal anti-PD-1 antibody that essentially lacks detectable antibody-dependent cellular cytotoxicity (ADCC). IgG4 antibodies); MK-3475 (formerly lambrolizumab) (a humanized IgG4 anti-PD-1 antibody containing a mutation at C228P designed to prevent Fc-mediated ADCC); urelumab (BMS-663513) (a fully human IgG4 monoclonal anti-CD137 antibody); anti-LAG-3 monoclonal antibody (BMS-986016); and bavituximab (chimeric 3G4) (a chimeric IgG3 antibody against PS); MPDL3280A (RG7446) (a human IgG1-kappa anti-PD-L1 monoclonal antibody); and MEDI4736 (another IgG1-kappa PD-L1 inhibitor).

[0044] Another alternative approach is to use a B7-DC-Fc fusion protein to competitively block the PD-1 receptor, and therefore such a fusion protein can also be used.

[0045] Thus, in one embodiment, the immune checkpoint inhibitor is an antibody against PDL-1, PD-1, CTLA4, TIM3, CD137, CD223, PS or KIR on NK cells, or a B7-DC-Fc fusion protein.

[0046] For use herein, LAB are engineered to express wound-healing and / or inflammation-resolving proteins. One or more wound-healing and / or inflammation-resolving proteins may be expressed. The term "engineered" means that the LAB has been modified, specifically genetically modified, to express the protein. Thus, the term "engineered" is synonymous with and can be used interchangeably with "modified" or "genetically modified." In other words, engineered LAB are LAB into which one or more nucleic acid molecules containing a nucleotide sequence encoding the protein have been introduced. Thus, the introduced nucleic acid molecule encodes a protein that is heterologous to the LAB (i.e., not naturally expressed). The term "nucleotide sequence" is used herein synonymously and interchangeably with "gene" or "gene sequence" to refer to a sequence encoding the protein. In particular, the use of the term "gene" herein does not imply or require the presence of any promoter sequence or other expression control sequence associated with the coding sequence. Thus, the term "gene" does not imply or require the presence of a native promoter or other control sequence of a native gene, but simply refers to a coding sequence encoding a given protein.

[0047] The nucleic acid molecule may have been introduced into the LAB in or as part of a self-replicating element, such as a plasmid or another vector as described below, or may be integrated into the chromosome of the recipient or host LAB. Thus, the nucleotide sequence encoding the protein may be present in the engineered LAB integrated into the host genome, or may be present independently of the host genome in a vector present in the engineered LAB.

[0048] A wound-healing protein may be any protein that acts to promote wound healing. Similarly, an inflammation-resolving protein may be any protein that acts to promote or aid in the resolution of inflammation. In other words, an inflammation-resolving protein may also be called an "anti-inflammatory" protein. In particular, the wound-healing and / or inflammation-resolving protein is a mammalian protein. The wound-healing and / or inflammation-resolving protein may advantageously be an immunomodulatory protein, a protein that acts to regulate the activity of immune cells. Thus, in one embodiment, the wound-healing and / or inflammation-resolving protein may be defined as an immunoactive protein, in particular an immunoactive protein that is locally active on immune cells that are locally present in the vicinity of the wound, in this case in the vicinity of the enteropathy, i.e., in the intestine of the subject. In a particular embodiment, the protein is an anti-inflammatory protein or a protein with anti-inflammatory activity. In particular, the protein may act to stimulate the growth and / or activity of immune cells, in particular immune cells present in the intestine, specifically macrophage cells. The protein may act to promote or enhance the anti-inflammatory activity of immune cells (e.g., macrophages). The protein may stimulate proliferation of localized macrophages and / or other immune cells or induce a phenotypic shift to an anti-inflammatory phenotype. Alternatively or additionally, the protein may have anti-fibrotic activity, i.e., the protein may be an anti-fibrotic protein.

[0049] Wound healing and / or inflammation-resolving proteins may act to reverse (i.e., improve or reduce) one or more of the clinical signs or symptoms or histological characteristics of treatment-induced enteropathy as described above. In particular, the proteins may be observed to reverse disease progression, and thus, for example, the proteins may act to reduce crypt damage and epithelial defects or cell apoptosis in the intestine, to reduce inflammatory markers, to reduce colon shortening, etc., as determined histologically, or to reduce any marker of disease activity. The proteins may also act to inhibit (i.e., prevent or reduce) the development of fibrosis in the GI tract, or specifically in the intestine.

[0050] The protein may be a cytokine, such as an interleukin, or a chemokine, or a growth factor. The protein may be a CXC protein. In one embodiment, the protein is selected from one or more of CXCL12, CXCL17, Ym1, TGF-β, IL-22, IL-27, IL-4, IL-10, IL-12, IL-8, or SP1. In a more specific embodiment, the protein is selected from one or more of CXCL12, CXCL17, Ym1, or TGF-β.

[0051] CXCL12 (also known as SDF-1; SEQ ID NO: 3 and SEQ ID NO: 6) is constitutively expressed in tissues and acts through the receptor CXCR4 found on leukocytes and endothelial cells, where it induces multiple cellular effects. CXCL12 is found at elevated levels in differentiated macrophages during tissue remodeling.

[0052] CXCL17 (SEQ ID NO: 9 and SEQ ID NO: 12), originally classified as a chemokine, has effects on tissue macrophage phenotype similar to those of CXCL12. Similar to CXCL12, CXCL17 is co-regulated with VEGF-A as measured in cell culture media. CXCL17 is found primarily in mucosal tissues and has been reported to exhibit direct antibacterial activity against pathogenic bacteria found on the skin, but has no effect on the survival of Lactobacillus casei. In addition to its antibacterial activity, CXCL17 has been reported to have antimicrobial and antifibrotic effects, as well as chemotactic properties. More recently, the classification of CXCL17 as a chemokine has been questioned, but this is not relevant to its proposed use herein.

[0053] A further protein of interest is the chitinase-like protein Ym1 (SEQ ID NO: 15 and SEQ ID NO: 18). Chitin is a common polysaccharide in bacterial biofilms. Ym1 disrupts biofilm production, induces macrophage functions important for tissue remodeling and wound healing, and is macrophage-specific because it is not internalized by either vascular or epithelial cells.

[0054] Another protein of interest is TGF-β. TGF-β produces three different isoforms, TGF-β1, TGF-β2, and TGF-β3, all of which are included herein. TGF-β is a multifunctional cytokine that is secreted by many cell types, including macrophages, and plays a role in controlling inflammatory processes in the intestine and elsewhere.

[0055] Specifically, the protein may be mouse CXCL12, particularly mouse CXCL12-1α (SEQ ID NO: 3); human CXCL12, particularly human CXCL12-1α (SEQ ID NO: 6); mouse CXCL17 (SEQ ID NO: 9); human CXCL17 (SEQ ID NO: 12); mouse Ym1 (SEQ ID NO: 15); human Ym1 (SEQ ID NO: 18), and human TGF-β (SEQ ID NO: 27).

[0056] In one embodiment, the protein is selected from mouse CXCL12-1α having the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 2, or an amino acid sequence having at least 80% sequence identity thereto; human CXCL12-1α having the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 5, or an amino acid sequence having at least 80% sequence identity thereto; mouse CXCL17 having the amino acid sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 8, or an amino acid sequence having at least 80% sequence identity thereto; human CXCL17 having the amino acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 11, or an amino acid sequence having at least 80% sequence identity thereto; mouse Ym1 having the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 14, or an amino acid sequence having at least 80% sequence identity thereto; human Ym1 having the amino acid sequence set forth in SEQ ID NO: 18 or SEQ ID NO: 17, or an amino acid sequence having at least 80% sequence identity thereto; and human TGF-β having the amino acid sequence set forth in SEQ ID NO: 27, or an amino acid sequence having at least 80% sequence identity thereto.

[0057] A nucleic acid molecule comprising a nucleotide sequence encoding a protein as defined or described above may be prepared. The nucleic acid molecule may be provided in the form of a recombinant construct comprising a coding nucleotide sequence(s) operably linked to one or more expression control sequences, for example, a promoter, optionally with one or more additional control sequences. Each coding sequence may be under the control of a separate expression control sequence, although for convenience, all coding sequences to be introduced may be under the control of the same expression control sequence(s). A construct comprising a nucleic acid molecule comprising one or more coding sequences and one or more expression control sequences may also be referred to herein as an expression construct.

[0058] The nucleic acid molecule or recombinant construct may be contained in a vector, for example, for cloning purposes or for expression, for example, in an expression vector. As used herein, the term "vector" refers to any genetic element that can serve as a vehicle for gene transfer, expression, or replication of a foreign nucleic acid sequence in a host strain. A vector may exist as a single nucleic acid molecule or as two or more separate nucleic acid molecules. When present in a host LAB, the vector may be a single-copy vector or a multi-copy vector.

[0059] Particular vectors for use herein are expression vectors, in which one or more genes can be inserted into the vector molecule in the appropriate orientation and adjacent to the expression control elements present in the expression vector molecule, so that expression of one or more proteins is induced when the vector molecule is present in a host LAB.

[0060] Construction of suitable expression vectors and other recombinant or genetic modification techniques for carrying out the methods herein are known in the art (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY) (2012), and Ausubel et al., Short Protocols in Molecular Biology, Current Protocols John Wiley and Sons (New Jersey) (2002)).

[0061] The vector may be a plasmid, cosmid, phagemid or other phage vector, viral vector, episome, artificial chromosome, such as a bacterial artificial chromosome (BAC) or P1 artificial chromosome (PAC), or other polynucleotide construct, and may include, for example, one or more selectable marker genes and appropriate expression control sequences.

[0062] Generally, regulatory control sequences are operably linked to a coding nucleic acid sequence and include constitutive, regulatable, and inducible promoters, transcription enhancers, transcription terminators, etc., as known in the art. As noted above, coding nucleic acid sequences may be operably linked to one common expression control sequence or may be linked to different expression control sequences.

[0063] In a particular embodiment, the nucleotide sequence(s) are provided in a plasmid. Specifically, the plasmid is for use in expressing a protein in lactic acid bacteria, and is therefore provided or configured for such use (e.g., the plasmid is designed, selected, configured, or modified for a specific or particular use in lactic acid bacteria). Thus, in one embodiment, the plasmid is for specific expression in lactic acid bacteria compared to bacteria or microorganisms in general. The plasmid may be configured for expression in lactic acid bacteria, for example, by its regulatory elements (control sequences) and / or coding sequences being selected or modified for expression in lactic acid bacteria.

[0064] Thus, the plasmid may contain one or more regulatory (i.e., expression control) sequences that enable or are specific for expression in lactic acid bacteria. Thus, the plasmid may contain expression control sequences derived from or suitable for or specific for expression in lactic acid bacteria. Suitable expression control sequences include, for example, translational control elements (e.g., start and stop codons, ribosome binding sites) and transcriptional control elements (e.g., promoter-operator regions, termination sequences) linked in the corresponding reading frame with the nucleotide sequence(s) encoding the protein(s) to be expressed. The regulatory sequence(s) are operably linked to the nucleotide sequence encoding the protein(s) such that they facilitate or regulate the expression of the protein.

[0065] The plasmid can be introduced into the lactic acid bacterial cells by any suitable transformation technique, which are well described in the literature. The bacterial cells may be cultured or otherwise maintained under conditions that allow expression of the protein from the plasmid, which may include conditions in the GI tract of the subject.

[0066] In one embodiment, the promoter in the plasmid regulating the expression of the protein is a promoter that allows expression in or is specific for expression in lactic acid bacteria. Thus, the plasmid may comprise a nucleotide sequence(s) encoding the protein(s) under the control of (or operably linked to) a promoter that allows the protein(s) to be expressed in lactic acid bacteria. In a particularly preferred embodiment, the plasmid comprises a lactic acid bacteria promoter. That is, the promoter regulating the expression of the protein(s) is a promoter derived from lactic acid bacteria, or specifically a promoter obtained from or derived from a gene expressed in lactic acid bacteria.

[0067] In some embodiments, the plasmid may also contain, in addition to the lactic acid bacteria promoter, other regulatory elements or sequences obtained or derived from lactic acid bacteria to regulate expression of the protein(s). Thus, for example, such other lactic acid bacteria expression regulatory elements or sequences may include enhancers, terminators, and / or translation regulatory elements or sequences, as described above. In some embodiments, the plasmid may also contain regulatory elements or sequences, such as operator sequences, that regulate or control expression from the promoter, or one or more regulatory genes, as described below.

[0068] Alternatively or additionally, the plasmid may be configured (or modified, etc.) for use in lactic acid bacteria, with the nucleotide sequence encoding said protein(s) being codon-optimized for expression in lactic acid bacteria.

[0069] In a preferred embodiment, the promoter for the expression of the protein is a regulated (controllable) or inducible promoter. Thus, by providing the bacterium with a regulatory molecule or inducer that activates or turns on (induces) the promoter, or by contacting the bacterium with the regulatory molecule or inducer, the expression of the protein can be regulated or controlled (e.g., initiated at a desired or appropriate time). This is advantageous in the context of delivering the protein to the site of enteropathy.

[0070] An expression system for expressing the protein in lactic acid bacteria may comprise (i) a plasmid comprising a nucleotide sequence encoding the protein under the control of an inducible promoter capable of expressing the protein in lactic acid bacteria; and (ii) an inducer (or control molecule) for the promoter. The expression system may conveniently be provided in the form of a kit comprising the above components (i) and (ii).

[0071] Thus, in certain embodiments, (i) a lactic acid bacterium comprising a nucleotide sequence encoding a protein under the control of an inducible promoter capable of expressing said protein in the lactic acid bacterium; and (ii) an inducer (or regulatory molecule) for said promoter A pharmaceutical product (e.g., a kit or combination product) comprising: The pharmaceutical products are provided as combined preparations for separate, sequential, or simultaneous use in treating or preventing therapy-induced enteropathy in a subject.

[0072] Generally speaking, in any embodiment herein, the subject may be any human subject, or an animal subject, including, for example, a domestic animal, a livestock animal, a laboratory animal, a sport animal, or a zoo animal. The animal is particularly a mammal, but also includes other animals, such as birds. Thus, the animal may be a primate, a rodent (e.g., a mouse or rat), or a horse, dog, or cat. Most preferably, the subject is a human.

[0073] Lactic acid bacteria (LAB), or the order Lactobacillales, are a clade of Gram-positive, low-GC, acid-tolerant, generally non-spore-forming, non-respiring bacteria, either rod-shaped (bacilli) or cocci (cocci), that share common metabolic and physiological characteristics. These bacteria produce lactic acid as the primary metabolic end product of carbohydrate fermentation and are characterized by a high tolerance to acidity (low pH range). These characteristics of LAB enable them to outcompete other bacteria in natural fermentation because they can tolerate the high acidity resulting from organic acid production (e.g., lactic acid). Thus, LAB play an important role in food fermentation because acidification inhibits the growth of spoilage agents. Some LAB strains also produce proteinaceous bacteriocins, which further inhibit the growth of spoilage and pathogenic microorganisms. LAB have Generally Recognized as Safe (GRAS) status and are the most important group of microorganisms used in the food industry.

[0074] The core genera comprising the lactic acid bacteria group are Lactobacillus*, Leuconostoc, Pediococcus, Lactococcus, Enterococcus, Weissella, and Streptococcus, and the more peripheral genera Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Sporolactobacillus, Tetragenococcus, and Vagococcus. The methods herein may use any lactic acid bacteria from these genera, but in particular bacteria from the genus Lactobacillus or Lactococcus, more particularly lactic acid bacteria from the genus Lactobacillus. (*The genera Lactobacillus, Pediococcus, Weissella, and Leuconostoc were recently merged and further divided into 25 genera. For simplicity, the older taxonomic classification will be used herein. Reference: Zheng, J., Wittouck, S., Salvetti, E., Franz, CMAP, Harris, HMB, Mattarelli, P. et al. (2020). A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, amended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. International Journal of Systematic and Evolutionary Microbiology, 70(4), 2782-2858. http: / / doi.org / 10.1099 / ijsem.0.004107).

[0075] In one embodiment, the LAB is of the genus Lactobacillus.This includes all species of the genus Lactobacillus, and a wide range of species are known, and many species have been deposited and are publicly available from culture depositories.Specifically, the Lactobacillus is Lactobacillus reuteri.This species has recently been reclassified as rimosyllactobacillus reuteri.Therefore, the term "Lactobacillus" used herein includes rimosyllactobacillus (i.e., in one embodiment, the LAB is of the genus Lactobacillus or rimosyllactobacillus).

[0076] Lactobacillus reuteri may be any strain, and numerous strains are known and described in the art. Many strains of Lactobacillus reuteri are publicly available from culture collections, such as Lactobacillus reuteri DSM20016 or Lactobacillus reuteri ATCC PTA 6475. Particular mention may be made of the Lactobacillus reuteri R2LC strain.

[0077] Lactobacillus reuteri R2LC has been reported in the literature and is available upon request from Professor Siv Ahnre, Lund University, Sweden (see Ahnre et al., Nutrients 2011, 3, 104-117). Lactobacillus reuteri strain R2LC has been deposited in the Culture Collection of the University of Gothenburg (CCUG) under provisional accession number R2LC20211221 in December 2021. Lactobacillus reuteri strain R2LC has been deposited under the terms of the Budapest Treaty at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Inhoffenstrasse 7 B, D-38124 Braunschweig, Germany) on August 26, 2022, under the accession number DSM 34372.

[0078] As described above, the LAB may contain or express one or more of the proteins. Thus, the LAB may contain or express a combination of any two or more of the proteins listed above, such as a combination of CXCL12, CXCL17, Ym1, and / or TGF-β proteins (e.g., two or more of CXCL12, CXCL17, Ym1, or TGF-β). Alternatively, the LAB may contain or express two or more of CXCL12, CXCL17, Ym1, and / or TGF-β proteins, or even two or more of the other proteins listed or mentioned above (e.g., both mouse CXCL12 and human CXCL12). When multiple proteins are expressed, the proteins may be expressed from the nucleotide sequence encoding the proteins under the control of a single promoter, or multiple promoters may be used. For example, each protein may be expressed from a separate promoter, and these promoters may be the same or different. Techniques for expressing two or more proteins together from the same plasmid are known in the art, such as translationally coupled techniques, and means for achieving this are known and available in the art. For example, P2A and T2A sequences may be used to simultaneously express multiple transgenes under a single promoter.

[0079] The protein may be a native or naturally occurring protein (i.e., the nucleotide sequence may encode a protein having an amino acid sequence as found in nature) and may be derived from any species that produces such proteins. Generally, the protein will be a mammalian protein, with human and murine proteins being preferred, as noted above. However, the native nucleotide sequence or protein sequence may be modified, e.g., by the addition, insertion, deletion, and / or substitution of one or more amino acids, so long as the function or activity of the protein is not substantially or significantly altered, e.g., the activity of the protein is substantially maintained. The protein may also be a fragment or truncated variant of the native protein. For example, a sequence-modified variant protein may exhibit at least 80, 85, 90, or 95% of the activity of the parent protein from which the variant protein is derived. This may be assessed by tests known in the art for the activity of the protein. For example, activity can be measured in a system of receptor phosphorylation or calcium flux after binding in cultured cells treated with the protein, in a model of cell recruitment to the infected protein, or in a system of cell chemotaxis in vitro or in vivo. Chemotaxis-based in vitro assays are described in Nesmelova et al., JBC Papers in Press, June 12, 2008, DOI 10.1074 / jbc.M803308200, or Massena et al., Blood. October 22, 2015; 126(17):2016-26. doi:10.1182 / blood-2015-03-631572. Epub 8 / 18 / 2015. Hatse et al., Cytometry A. October 2004; 61(2):178-88, describes further in vitro chemokine activity tests that may be used. The terms "CXCL12," "CXCL17," "Ym1," or "TGF-β," or any of the other protein names or acronyms mentioned above, include not only the native proteins but also functionally equivalent variants or derivatives thereof.Thus, the protein may be a synthetic or sequence-altered variant, or may contain one or more other modifications, such as post-translational modifications.

[0080] As mentioned above, the encoded proteins may have the amino acid sequences shown above for the native human or mouse proteins, i.e., SEQ ID NO: 3 and SEQ ID NO: 6 for mouse CXCL12 and human CXCL12, respectively, SEQ ID NO: 9 and SEQ ID NO: 12 for mouse CXCL17 and human CXCL17, respectively, and SEQ ID NO: 15 and SEQ ID NO: 18 for mouse Ym1 and human Ym1, respectively, or SEQ ID NO: 27 for human TGF-β, or an amino acid sequence having at least 80% sequence identity to any of the above sequences. Advantageously, as further pointed out above, the nucleotide sequences encoding these native proteins may be codon-optimized for expression in lactic acid bacteria. This may result in a modified amino acid sequence of the encoded protein. For example, the codon-optimized sequence may encode a sequence, such as a secretory sequence, that is suitable (or more suitable) for lactic acid bacteria. Thus, the "optimized" protein encoded by the codon-optimized nucleotide sequence may contain an altered or replaced leader or signal sequence (e.g., a secretory sequence) compared to the native protein. In a preferred embodiment, the mature or truncated form of the protein encoded by the codon-optimized sequence is identical to the native protein. The protein encoded by the codon-optimized nucleotide sequence may have the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, or SEQ ID NO:17. Thus, the protein encoded by the plasmid may have the amino acid sequence set forth in SEQ ID NO:2 and SEQ ID NO:5 for mouse CXCL12 and human CXCL12, respectively, the amino acid sequence set forth in SEQ ID NO:8 and SEQ ID NO:11 for mouse CXCL17 and human CXCL17, respectively, and the amino acid sequence set forth in SEQ ID NO:14 and SEQ ID NO:17 for mouse Ym1 and human Ym1, respectively, or an amino acid sequence with at least 80% sequence identity to any of the aforementioned sequences.

[0081] In other embodiments, the encoded protein(s) may have an amino acid sequence having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing amino acid sequences.

[0082] Sequence identity can be easily determined by methods and software known and readily available in the art. Thus, sequence identity may be assessed by any convenient method. However, to determine the degree of sequence identity between sequences, computer programs that perform multiple alignment of sequences, such as Clustal W (Thompson et al., 1994) Nucleic Acids Res., 22:4673-4680, are useful.

[0083] Programs for comparing and aligning pairs of sequences, such as ALIGN (Myers and Miller (1988) CABIOS, 4:11-1), FASTA (Pearson and DJ Lipman 1988 PNAS, 85:2444-2448; and Pearson (1990), Methods Enzymol., 183:63-7), BLAST, and gapped BLAST (Altschul et al. (1997). Nucleic Acids Res., 25:3389-3402), are also useful for this purpose and may be used with default settings. In addition, the European Bioinformatics Institute's Dali server provides structure-based alignments of protein sequences. Multiple sequence alignments and percent identity calculations may be determined using standard BLAST parameters (e.g., using sequences from all available organisms, matrix Blosum 62, gap cost: existence 11, extension 1). Alternatively, the following programs and parameters may be used: Program: Align Plus 4, version 4.10 (Sci Ed Central Clone Manager Professional Suite). DNA comparison: global comparison, standard linear scoring matrix, mismatch penalty = 2, opening gap penalty = 4, extension gap penalty = 1. Amino acid comparison: global comparison, BLOSUM62 scoring matrix.

[0084] Variants of naturally occurring polypeptide sequences as defined herein can be made artificially, for example by using standard molecular biology techniques known in the art, for example standard mutagenesis techniques such as site-directed or random mutagenesis (e.g. using gene shuffling or error-prone PCR).

[0085] Derivatives of proteins as defined herein may also be encoded, by which is meant proteins as defined above or variants thereof in which amino acids have been chemically modified, for example by glycosylation.

[0086] When a protein contains amino acid substitutions relative to the sequence of a native protein, the substitutions can preferably be conservative substitutions. The term "conservative amino acid substitution" refers to any amino acid substitution in which an amino acid is replaced (substituted) with an amino acid having similar physicochemical properties, i.e., an amino acid of the same class / group. For example, small residues such as glycine (G), alanine (A), serine (S), or threonine (T); hydrophobic or aliphatic residues such as leucine (L), isoleucine (I); valine (V), or methionine (M); hydrophilic residues such as asparagine (N) and glutamine (Q); acidic residues such as aspartic acid (D) and glutamic acid (E); positively charged (basic) residues such as arginine (R), lysine (K), or histidine (H); or aromatic residues such as phenylalanine (F), tyrosine (Y), and tryptophan (W) can be interchangeably substituted without substantially changing the function or activity of the protein.

[0087] As mentioned above, it is advantageous to use an inducible promoter for the expression of the protein. "Inducible" refers to any promoter whose function (i.e., activity, or action in enabling or causing transcription of the coding nucleotide sequence) can be controlled or regulated. Thus, the term "inducible" is synonymous with "controllable" (or "controlled") and can be used interchangeably. Thus, there is no constitutive expression of the protein. Therefore, the expression of the protein can be induced or turned on (or specifically, can be turned on and off). Specifically, the expression can be induced or turned on for a finite or specific period. This may be because the expression stops after a certain period and / or the bacterial cell dies.

[0088] In some embodiments, there may be no expression (transcription) from a promoter until the promoter is induced (alternatively, said to be activated). However, as in any biological system, in the absence of promoter activation or induction, the lack of activity may not be complete, and there may be some basal promoter activity. However, in a preferred embodiment, the basal expression of the uninduced promoter is low, minimal, or insignificant, or more preferably, insignificant or negligible. Thus, expression from an inducible promoter is advantageously, measurably, or demonstrably increased when the promoter is induced compared to when the promoter is not induced.

[0089] Inducible promoters are known in the art, including inducible promoters for use in lactic acid bacteria, and any suitable inducible promoter suitable for expression in lactic acid bacteria may be used.

[0090] An inducible promoter may be induced (or activated) in the presence of an inducer or activator molecule. The inducer or activator molecule may act directly or indirectly on the promoter and may be added to induce the promoter, or more generally, to cause or facilitate the induction or activation of the promoter and allow protein expression. Alternatively, the inducible promoter may be induced (or activated) by a change in bacterial conditions containing the plasmid, for example, by introducing a change in conditions, such as starvation or depletion of a specific nutrient, into the lactic acid bacteria. The inducer of the promoter may be encoded by a regulatory gene, and in one embodiment, the inducer may itself be induced or activated. Thus, the term "inducer" is used broadly herein to include any regulatory molecule, or for that matter, any permissive condition, that can activate or turn on an inducible promoter or that can allow or cause the induction of an inducible promoter. Thus, induction of an inducible promoter may involve the introduction of a regulatory molecule (e.g., contacting the lactic acid bacteria containing the plasmid with a regulatory molecule) or the introduction of conditions permissive for promoter induction (activation). In some embodiments, the inducer may be an activation peptide, which may act directly or indirectly to induce the promoter, e.g., as described below.

[0091] As mentioned above, promoters obtained from or derived from lactic acid bacteria are particularly suitable. These may be natural promoters or modified or mutant promoters. For example, suitable promoters can be identified by growing lactic acid bacteria in a wound and determining which genes are expressed or upregulated in the bacteria in the wound. Then, promoters from such genes can be identified. Alternatively, numerous different promoters and expression systems for use in or with lactic acid bacteria have been identified and reported or are available in the art, including expression plasmids or expression systems containing such promoters for use in LAB. Any such known plasmid or expression system may be used as the basis for the recombinant plasmid of the present invention.

[0092] Various inducible expression systems for use in LAB, such as Lactobacillus, are known in the art. One example is the autoinduction system based on the manganese starvation-inducible promoter from the manganese transporter of L. plantarum NC8, as described by Bohmer et al., FEMS Microbiol Lett 342 (2013) 37-44. This system does not require the addition of an external inducer for recombinant protein production.

[0093] Duong et al., Microbial Biotechnology (2010) 4(3), 357-367, describe expression vectors for use in Lactobacillus based on the broad pWVO1 replicon and containing promoters from operons involved in fructooligosaccharide (FOS), lactose, or trehalose metabolism or transport, or in glycolysis. Such promoters can be induced by their specific carbohydrates and repressed by glucose.

[0094] Specifically, the inducible expression system may comprise an inducible promoter involved in the production of LAB proteins, particularly bacteriocins. The activity of such promoters may be regulated by a cognate control system based on the bacteriocin regulon, e.g., by a two-component control (signal transduction) system that responds to an exogenously added activator peptide (i.e., a peptide-type inducer / control molecule) and genes encoding a histidine protein kinase and response regulator required to activate the promoter upon induction by the activator peptide.

[0095] In one embodiment, the expression system may be based on the nisin-regulated expression (NICE) system, which is based on the combination of the nisA promoter and the nisRK regulatory gene. This system is based on the promoter and regulatory gene from the nisin gene cluster of Lactococcus lactis and has been used to develop regulated gene expression systems for lactococci, lactobacilli, and other Gram-positive bacteria (briefly reviewed in Sorvig, FEMS Microbiol Lett. 2003;229(1):119-126, and Sorvig et al., Microbiology. 2005 June;151(Pt 7):2439-49).

[0096] While NICE systems are efficient and well-regulated in lactococci, they can exhibit significant basal activity, which can be circumvented by chromosomal integration of the histidine kinase and response regulator genes and restricting the expression system to specifically designed host strains.

[0097] In another embodiment, the expression system may be based on the genes and promoters involved in the production of the class II bacteriocin sakacin A (sap gene) via the sakacin A regulon, or sakacin P (spp gene) via the sakacin P regulon. Such vectors are known as pSIP vectors and contain promoter elements from either the sakacin A or sakacin P structural gene with an engineered NcoI site for translational fusion cloning. A variety of such vectors containing different promoters and / or different replicons from the regulons are described in the above-mentioned Sorvig et al. paper, and any of these vectors could be used as the basis for the recombinant plasmids of the present invention.

[0098] In an exemplary embodiment, the promoter is a P gene from the sakacin A or P regulon with its associated or cognate regulatory gene. sapA , P sppA , or P orfX It may also be a promoter.

[0099] In one particularly preferred embodiment, the plasmid comprises the pSH71 replicon, the P orfX The plasmid contains a promoter and cognate regulatory genes and is based on the vector pSIP411 described in Sorvig et al., 2005. Plasmid pSIP411 is referred to herein as pLAB112. The inducer for use in such embodiments is preferably an activation peptide based on the peptide SppIP, for example, an activation peptide having the sequence of SEQ ID NO: 19 or an amino acid sequence having at least 80% (or more specifically, at least 85%, 90%, or 95%) sequence identity thereto. In one particular embodiment, the plasmid is derived from a plasmid designated pLAB112, which has the nucleotide sequence set forth in SEQ ID NO: 20 (the corresponding amino acid translation is set forth in SEQ ID NO: 34).

[0100] The use of an inducible promoter (or inducible expression system) can provide the advantage of more regulated, particularly long-term, expression of the protein in the intestinal environment, i.e., when the bacterium is administered to a subject. For better efficacy in treating or preventing therapy-induced enteropathy, it is advantageous to have the protein expressed by the bacterium at the site of enteropathy for a period of time, for example, at least 40, 45, 50, 55, or 60 minutes, particularly at least 1 hour or longer. Thus, the protein may be expressed for a limited, defined, or extended period of time. Results reported in WO 2016 / 102660 indicate that the protein can be expressed at the wound surface for a period of approximately 1 hour. In some embodiments, the bacterium may be optimized to allow expression of the protein for 2, 3, or 4 hours or longer.

[0101] Thus, continuous expression and delivery of proteins is desirable, and this can be achieved by using engineered LAB. "Continuous" or "long-term" means that there is expression, and thus delivery, of the protein over a period of time, for example, over a period of at least one hour (or so, as described above). In particular, this can allow the protein to be available for an extended period of time compared to when the protein is administered directly (i.e., as a protein product, rather than by expression by the bacteria).

[0102] As mentioned above, the nucleotide sequences encoding said protein(s) may be codon-optimized for expression in LAB. Thus, in a preferred embodiment, the nucleotide sequences encoding said proteins may be selected from the codon-optimized nucleotide sequences set forth in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 16, which encode mouse CXCL12, human CXCL12, mouse CXCL17, human CXCL17, mouse Ym1, and human Ym1, respectively, or nucleotide sequences having at least 80% sequence identity thereto.

[0103] Thus, in exemplary embodiments, the recombinant plasmid may be selected from the group consisting of the following named plasmids: mLrCK1, comprising the nucleotide sequence set forth in SEQ ID NO:1 (with the corresponding amino acid translation set forth in SEQ ID NO:28); mLrCK1.4, comprising the nucleotide sequence set forth in SEQ ID NO:1; mLrCK1.7, comprising the nucleotide sequence set forth in SEQ ID NO:1; hLrCK1, comprising the nucleotide sequence set forth in SEQ ID NO:4 (with the corresponding amino acid translation set forth in SEQ ID NO:29); mLrCK2, comprising the nucleotide sequence set forth in SEQ ID NO:7 (with the corresponding amino acid translation set forth in SEQ ID NO:30); hLrCK2, comprising the nucleotide sequence set forth in SEQ ID NO:10 (with the corresponding amino acid translation set forth in SEQ ID NO:31); hLrMP1, comprising the nucleotide sequence set forth in SEQ ID NO:13 (with the corresponding amino acid translation set forth in SEQ ID NO:32); and mLrMP2, comprising the nucleotide sequence set forth in SEQ ID NO:16 (with the corresponding amino acid translation set forth in SEQ ID NO:33).

[0104] In some embodiments, the protein is encoded by a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of the following codon-optimized inserts: mLrCK1 (i.e., to the nucleotide sequence of SEQ ID NO: 1), mLrCK1.4 (i.e., to the nucleotide sequence of SEQ ID NO: 1), mLrCK1.7 (i.e., to the nucleotide sequence of SEQ ID NO: 1), hLrCK1 (i.e., to the nucleotide sequence of SEQ ID NO: 4), mLrCK2 (i.e., to the nucleotide sequence of SEQ ID NO: 7), hLrCK2 (i.e., to the nucleotide sequence of SEQ ID NO: 10), hLrMP1 (i.e., to the nucleotide sequence of SEQ ID NO: 13), and mLrMP2 (i.e., to the nucleotide sequence of SEQ ID NO: 16).

[0105] Sequence identity of nucleotide molecules can be determined by methods and software known and widely available in the art, for example, by FASTA searching using the GCG package with windows of 6 nucleotides, default values ​​and a variable pamfactor, and gap creation penalty settings of 12.0 and gap extension penalty settings of 4.0.

[0106] Such sequence identity with respect to a nucleotide sequence may be functionally equivalent to the nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:13, or SEQ ID NO:16. Such nucleotide sequences can be considered functionally equivalent if they encode a polypeptide that would be considered a functional equivalent to the CXCL12, CXCL17, or Ym1 protein, respectively. Particular functional equivalents are those that encode specific proteins as described above.

[0107] The bacteria may be administered in any convenient or desired manner, for example orally, or by direct administration to the GI tract, for example rectally, or by direct injection or infusion, or application or introduction of a pharmaceutical composition or dressing or device containing the bacteria. In one particular embodiment, the bacteria are administered orally.

[0108] The bacteria may be formulated or prepared in any convenient or desired way for administration by any of the above routes, using such means according to procedures well known and routine in the art. Thus, products for use herein may be formulated and provided as or in nutritional supplements or foods, e.g., functional foods, as well as pharmaceutical compositions, medical devices, dressings, and the like.

[0109] Oral dosage forms include powders, tablets, capsules, liquids, etc. Additionally, the bacteria may be provided on (e.g., coated on) a medical device such as an implant (e.g., a prosthetic implant), a tube, a conduit, or a catheter.

[0110] The bacteria may be provided in any convenient or desired form, for example as an active or growing culture, or in lyophilized or freeze-dried form.

[0111] Generally speaking, in the administration herein, the LAB can be conveniently provided as an enteric preparation, i.e., a preparation designed to protect the LAB from digestion in the stomach and release the LAB in the intestine.The preparation can therefore be gastric juice resistant.The preparation can also be designed to release the LAB at neutral pH.The formulation of enteric preparations and compositions is known in the art and described in the literature, for example, using coating agents and / or excipients.In particular, the composition containing the LAB can be provided with an enteric coating.Such coating agents are commercially available, for example, the enteric coating agent available from Evonik Health Care under the trade name Eudragit.

[0112] In one embodiment, the LAB may be provided as or in tablets, capsules, or microparticles that are provided with an enteric coating.

[0113] The composition comprising the LAB may also contain an inducer (if an inducible promoter is used), which may be provided as part of the product (e.g., incorporated or contained in a tablet or capsule) or separately, for example as part of a kit or combination product, as defined above.

[0114] When the bacteria and inducer are co-formulated in a product, they may be provided in a format in which the bacteria are separated from the inducer and are brought together (or contacted) during use. For example, the bacteria and inducer may be in separate compartments that are brought together (e.g., contacted or mixed), or may be separated by a barrier (e.g., a membrane or other partition) that can be broken or destroyed or opened during use.

[0115] Alternatively, the inducer may be formulated separately and provided separately (e.g., in a kit also containing the bacteria or in a product containing the bacteria), and the inducer and the bacteria (or product containing the bacteria) may be brought together (e.g., contacted) during use. This may be before, during, or after administration to a subject. For example, a product containing the bacteria may be administered first, and then the inducer may be added to or given to the bacteria. In another embodiment, the bacteria and inducer may be pre-mixed prior to administration, e.g., shortly before or immediately before administration, or during administration.

[0116] If the bacteria are provided in lyophilized or freeze-dried form, it may be desirable to reconstitute or resuspend them prior to administration, e.g., prior to or during use, however, for use herein it is advantageous to provide them in lyophilized or freeze-dried form for reconstitution by body fluids upon release in the GI tract.

[0117] Conveniently, for use herein, the LAB may be formulated in an enteric capsule containing freeze-dried bacteria and freeze-dried inducer.

[0118] Alternatively, the tablet may contain at least two separate compartments, one containing the freeze-dried bacteria and the other containing the inducer and optionally a liquid, and the tablet is crushed before ingestion to rupture the inner membrane separating the two compartments and mix the contents.

[0119] The engineered LAB can be used as proposed herein to treat therapy-induced enteropathy in an advantageous manner. It is believed that improved therapeutic effects can be obtained compared to existing treatments, particularly steroid treatments. Moreover, because therapy-induced enteropathy can become resistant or refractory to steroid treatment, this proposal opens up opportunities to treat previously intractable patient populations, such as those diagnosed with steroid-resistant ICI-induced colitis.

[0120] A further advantage offered by the proposed treatment is that it frees the subject from steroid therapy.

[0121] Representative embodiments are further described in light of the following non-limiting examples and figures. [Brief explanation of the drawings]

[0122] [Figure 1] Figure 1 shows the effect of ILP100 on PD-1 inhibitor-induced colitis in mice. Hematoxylin and eosin stained mouse colon tissue sections (A), crypt damage scores (B), and a graph of the average crypt damage score (C). Mice were subcutaneously inoculated with breast cancer tumor cells. Treatment began when tumors reached a certain size. Group A: No treatment for 3 weeks. Group B: Anti-PD-1 antibody + isotype control monoclonal antibody (mAb) for 3 weeks. Group C: Anti-PD-1 antibody isotype control mAb for 3 weeks + ILP100 treatment for 1 week (week 3). One-way ANOVA multiple comparisons followed by Holm-Sidak post-hoc test. Mice / sample per group: n=4. [Figure 2]Figure 1 shows the effect of ILP100 on PD-1-induced epithelial loss in mice. The graph shows epithelial loss measured at termination for mice treated with isotype control, anti-PD-1, or anti-PD-1 followed by R2LC_CXCL12. One-way ANOVA multiple comparisons followed by Holm-Sidak post-hoc test. n = 4-6 mice / sample per group. [Figure 3] 1 shows the effect of ILP100 on PD-1 and PDL-1-induced colon shortening in mice. The graph shows colon length (cm) in mice treated with an isotype control antibody and vehicle; a PD-1 inhibitor and vehicle; a PD-1 inhibitor and ILP100; a PD-L1 inhibitor and vehicle; or a PD-L1 inhibitor and ILP100. [Figure 4] 1 shows the effect of ILP100 on the development of PD-1 inhibitor-induced colonic erosion / ulceration in mice. The graph shows the development of colonic erosion / ulceration measured at termination for mice treated with isotype control, anti-PD-1, or anti-PD-1 followed by R2LC_CXCL12. [Figure 5] Figure 5 shows the effects of WT_R2LC and R2LC_hCXCL12 on colitis induced by the combination of DSS and a PD-1 inhibitor in mice. The graphs show the disease activity index (A), body weight loss (B), intestinal bleeding score (C), stool consistency score (D), colon length (E), colon weight (mg) (F), and colon weight-to-length ratio (G) in mice treated with either vehicle (DSS + ICI), WT_R2LC (DSS + ICI + WT_R2LC), or R2LC_hCXCL12 (DSS + ICI + R2LC_hCXCL12). The untreated (UT) group in Figures 5E-5G was included as a reference and was not included in statistical tests. [Figure 6]Figure 6 shows the effects of ILP100, WT_R2LC, anti-TNF, and anti-α4β7 on DSS-induced colitis in mice. Figures 6A, 6B, and 6E show the disease activity index (A), colon length (cm) (B), and incidence of colonic erosion / ulceration measured at the end of the study in healthy mice and mice treated with either DSS alone (DSS), ILP100 (DSS + R2LC_CXCL12), or WT_R2LC (DSS + WT_R2LC). Figures 6C and D show the disease activity index (C) and colon length (cm) (D) in healthy mice and mice treated with either DSS alone (DSS), ILP100 (DSS + R2LC_CXCL12), anti-TNF (DSS + anti-TNF), or anti-α4β7 (DSS + anti-α4β7). Mice / sample per group: n=22–32 for A and C, n=4 for B and D. [Example]

[0123] Example 1 (Preparation of Lactobacillus reuteri expressing CXCL12 (ILP100)) Lactobacillus reuteri strain R2LC was genetically modified with a plasmid containing a nucleotide sequence encoding human CXCL12 as described in Example 1 of WO2016 / 102660. This modified strain is designated herein as ILP100.

[0124] Briefly, the sequence for hCXCL12 was codon-optimized for expression in LAB, and the resulting sequence (SEQ ID NO: 4) was introduced into plasmid pLAB112 (SEQ ID NO: 20), which corresponds to plasmid pSIP411 as described in Sorvig et al., Construction of vectors for inducible gene expression in Lactobacillus sakei and L plantarum. FEMS Microbiol Lett. 2003;229(1):119-126.

[0125] The resulting plasmid, designated hLrCK1 (pLAB112 containing the optimized hCXCL12-1α insert), was transformed into L. reuteri strain R2LC, and positive clones for this construct were collected. The hLrCK1 plasmid obtained from the positive colony was verified by sequence analysis. This positive clone was designated strain ILP100.

[0126] Example 2 ILP100 treatment reduces crypt damage in mice with PD-1 inhibitor-induced colitis. The effect of ILP100 on PD-1 inhibitor-induced colitis in mice was examined in three experimental groups: A, B, and C (Figure 1). On day 0, female BALB / c mice were orthotopically inoculated with 7,000 4T1 cells. Tumor size in this study was monitored twice weekly. Animals were treated with either A) an isotype control mAb once weekly for 3 weeks, B) an anti-PD-1 antibody once weekly for 3 weeks, or C) an anti-PD-1 antibody once weekly for 3 weeks, and orally administered ILP100 treatment by gavage for 7 days prior to termination. At termination, blood smears and Swiss rolls of colon and small intestine from each animal were prepared and fixed in 4% PFA.

[0127] Swiss roll specimens of colon and small intestinal tissue were embedded in paraffin, sectioned, and stained with hematoxylin. Slides were scanned using a microscope (Figure 1A). Image files were blinded and keyed by individuals not involved in image analysis or interpretation of the results. Images were analyzed using histopathological methods, including assessment of crypt damage. Each image is displayed in y × y mm. 2 The tissues were divided equally into squares and scored from 0 to 3 (Figure 1B). The mean value for each tissue was calculated. The analysis was then unblinded. The mean and SEM for the group were calculated, and the results are shown in Figure 1C.

[0128] Figure 1C shows that Group B had a higher mean crypt damage score than Group A, indicating that 3 weeks of PD-1 treatment induced significant damage to the intestine. Group C had a significantly lower mean crypt damage score compared to Group B, indicating that ILP100 treatment of PD-1-induced colitis in Group C mice was effective.

[0129] Example 3 ILP100 treatment reduces epithelial defect damage in mice with PD-1 inhibitor-induced colitis. The effect of ILP100 treatment on epithelial defects in female Balb / C mice treated with a PD-1 inhibitor was examined.

[0130] The effect of ILP100 on PD-1 inhibitor-induced colitis in mice was examined in three experimental groups (Figure 2). On day 0, mice were inoculated intraperitoneally with 7,000 4T1 cells. Tumor size in this study was monitored twice weekly. Animals were treated with an isotype control mAb once per week for 3 weeks, an anti-PD-1 antibody once per week for 3 weeks, or an anti-PD-1 antibody once per week for 3 weeks followed by daily ILP100 treatment (administered orally by gavage) for 7 days before termination.

[0131] At the end of the experiment, colons were harvested and rolled into Swiss rolls. The tissues were paraffin-embedded, sectioned, and stained with hematoxylin and eosin. The length of the areas with erosion and ulceration was measured in each section. Erosion was defined as an area without epithelium but with intact crypt structure, and ulceration was defined as an area without epithelium and with no remaining crypt structure. Because the areas of ulceration were few and very small, these two parameters were merged to define epithelial loss. Figure 2 shows that mice treated with a PD-1 inhibitor in combination with ILP100 exhibited less epithelial loss than mice treated with vehicle. Figure 4 shows that mice treated with a PD-1 inhibitor in combination with ILP100 exhibited reduced incidence of erosion / ulceration in the colon.

[0132] Tumor size in mice was not affected by ILP100 treatment compared to anti-PD1 antibody (PD1 inhibitor).

[0133] Example 4 ILP100 treatment reduces colon shortening in mice with colitis induced by PD-1 and PD-L1 inhibitors. We also examined the effect of ILP100 treatment on colon length, a measure of fibrosis development, in mice treated with PD-1 and PD-L1 inhibitors. Figure 3 shows that mice treated with the isotype control and vehicle had the longest colon lengths at the end of the experiment. In contrast, mice treated with PD-1 and vehicle or PD-L1 inhibitors and vehicle had significantly shorter colon lengths. However, mice treated with ILP100 in combination with PD-1 or PD-L1 inhibitors showed preserved colon length and a trend toward reduced colon shortening.

[0134] Furthermore, we observed that anti-PD1-treated mice did not exhibit classical symptoms of colitis as measured by disease activity indices, including weight loss, stool consistency, and bloody stool. However, the mice developed severe fibrosis (20% colon shortening) and a clear increase in erosions and ulcers, which were reversed by ILP100 treatment even at short treatment times.

[0135] Example 5 (Manufacture and coating of capsules containing SppIP and ILP100) SppIP was dissolved in a solution containing 15% glucose. To control the content, this peptide solution was dispensed into well plates and freeze-dried in a form similar to tablets. The SppIP freeze-dried tablets were then placed into capsules together with freeze-dried ILP100, and the capsules were then coated with a coating polymer (Eudragit L 30 D-55) by drum coating.

[0136] Example 6 ILP100 treatment reduces colitis symptoms in mice with colitis induced by a combination of DSS and PD-1 inhibitors The effects of ILP100 (R2LC hCXCL12) and wild-type R2LC treatment on disease activity index (DAI), body weight loss, intestinal bleeding, stool consistency, colon length, colon weight, and colon weight-to-length ratio were examined in mice treated with a combination of DSS and a PD-1 inhibitor (anti-CTLA-4) to induce colitis. ILP100 administration during overt inflammation significantly improved colitis symptoms assessed by DAI compared with mice treated with either vehicle or wild-type R2LC (Figure 5A). Furthermore, clinical symptoms of colitis (Figure 5B-G) were improved in mice treated with ILP100 compared with mice treated with either vehicle or wild-type R2LC. Colon weight may be related to edema, a component of inflammation, and can be used as a marker of inflammation (heavier weight indicates more severe inflammation). Colon length is an indicator of fibrosis (shorter colon length indicates greater fibrosis and inflammation), therefore the weight / length ratio is a good indicator of induced colitis.

[0137] Example 7 ILP100 reverses DSS-induced colitis and improves symptoms with efficacy better than or comparable to established treatments for ulcerative colitis. Mice were treated with 3% DSS for 6 days to induce colitis. On day 6, the animals were then treated with vehicle (HO), WT_R2LC, or ILP100 (R2LC hCXCL12) by oral gavage three times daily for 6 days, or with anti-TNF or anti-α4β7 by intraperitoneal injection once daily. At termination, colons were fixed and processed as described in Example 2.

[0138] The effects of ILP100 and wild-type R2LC treatment were examined in mice with DSS-induced colitis (Figures 6A and 6B). Administration of ILP100 during overt inflammation significantly improved colitis symptoms, as assessed by DAI, compared with vehicle-treated and wild-type R2LC-treated mice (Figure 6A). Furthermore, ILP100-treated mice had significantly longer colons than vehicle-treated or wild-type R2LC-treated mice.

[0139] The effects of ILP100 treatment on DSS-induced colitis were compared with current standard of care treatments for IBD (anti-TNF and anti-α4β7; Figures 6C and 6D). ILP100 administration significantly improved colitis symptoms, as assessed by DAI, compared with current standard of care treatments, with better efficacy (Figure 6C). Furthermore, ILP100-treated mice had colon lengths comparable to those of anti-TNF-treated and anti-α4β7-treated mice (Figure 6D). ILP100(R2LC_hCXCL12)-treated mice had the lowest DAI (not significant) compared with anti-TNF-treated and anti-α4β7-treated mice throughout the study, and these animals were the only animals to show a significant reduction in colon shortening compared with controls. A more rapid reduction in intestinal bleeding was observed in ILP100(R2LC_hCXCL12)-treated animals compared with the other treatments.

[0140] Considering that the action of the engineered LAB (ILP100(R2LC CXCL12)) occurs at the local level, the significant positive effect of the engineered bacterium was surprising. array Sequence Listing Overview

[0141] [Table 1]

[0142] TIFF2025504333000002.tif216170

Claims

1. An engineered lactic acid bacteria (LAB) for use in treating therapy-induced enteropathy in a subject, the LAB having been modified to express a mammalian protein that promotes resolution of inflammation and / or wound healing.

2. The engineered LAB for use according to claim 1, wherein the protein that promotes the resolution of inflammation and / or wound healing is an immunomodulatory protein that regulates the activity of immune cells.

3. 3. The engineered LAB for use according to claim 1 or claim 2, capable of expressing a protein selected from the group consisting of CXCL12, CXCL17 and Ym1.

4. The engineered LAB for use according to any one of claims 1 to 3, wherein the treatment-induced enteropathy is tumor treatment-induced enteropathy.

5. The engineered LAB for use according to any one of claims 1 to 4, wherein the treatment-induced enteropathy is immune checkpoint inhibitor-induced (ICI-induced) enteropathy or radiation-induced enteropathy.

6. The protein is (i) murine CXCL12-1α having the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:2, or an amino acid sequence having at least 80% sequence identity thereto; (ii) human CXCL12-1α having the amino acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:5, or an amino acid sequence having at least 80% sequence identity thereto; (iii) a mouse CXCL17 having an amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:8, or an amino acid sequence having at least 80% sequence identity thereto; (iv) human CXCL17 having the amino acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 11, or an amino acid sequence having at least 80% sequence identity thereto; (v) mouse Ym1 having an amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 14, or an amino acid sequence having at least 80% sequence identity thereto; and (vi) a human Ym1 having an amino acid sequence as set forth in SEQ ID NO: 18 or SEQ ID NO: 17 or having at least 80% sequence identity thereto; 7. The engineered LAB for use according to any one of claims 1 to 6, selected from:

7. 7. The engineered LAB for use according to any one of claims 1 to 6, which is transformed with a plasmid expressing said protein.

8. 8. The engineered LAB for use according to claim 7, wherein the plasmid comprises one or more nucleotide sequences encoding one or more of the proteins under the control of an inducible promoter.

9. The engineered LAB for use according to claim 7 or claim 8, wherein the plasmid comprises an inducible promoter and regulatory elements derived from the nisin regulon, the sakacin A regulon, or the sakacin P regulon of lactic acid bacteria.

10. The engineered LAB for use according to any one of claims 7 to 9, wherein the inducible promoter is the PorfX promoter from the Sakacin P regulon.

11. The engineered LAB for use according to any one of claims 7 to 10, wherein said plasmid is derived from a plasmid named pSIP411.

12. 12. The engineered LAB for use according to any one of claims 1 to 11, wherein the LAB has been engineered by introducing a nucleotide sequence encoding the protein, the nucleotide sequence being codon-optimized for expression in lactic acid bacteria.

13. The engineered LAB for use according to any one of claims 1 to 12, wherein the LAB comprises one or more nucleotide sequences selected from the group consisting of a nucleotide sequence comprising the sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, or SEQ ID NO:16, and a nucleotide sequence having at least 80% sequence identity to any of the sequences.

14. The engineered LAB for use according to any one of claims 1 to 13, wherein the LAB is of the genus Lactobacillus.

15. 15. The engineered LAB for use according to any one of claims 1 to 14, wherein the LAB is a strain of Lactobacillus reuteri.

16. 16. The engineered LAB for use according to claim 15, wherein the LAB is Lactobacillus reuteri strain R2LC.

17. The engineered LAB for use according to any one of claims 1 to 16, wherein the LAB is provided as an orally administrable pharmaceutical composition, the LAB being included in the pharmaceutical composition together with one or more pharma- ceutically acceptable excipients.

18. 18. The engineered LAB for use according to any one of claims 1 to 17, wherein the LAB is freeze-dried.

19. 19. The engineered LAB for use according to claim 17 or claim 18, wherein the LAB is provided in the form of an enteric coated capsule.

20. 20. The engineered LAB for use according to any one of claims 17 to 19, wherein the LAB expresses the protein under the control of an inducible promoter, and the pharmaceutical composition or capsule further comprises an inducer for the promoter.

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