Chloride-inducible prokaryotic expression system

Abstract

The present invention is directed to recombinant bacteria, recombinant plasmids, pharmaceutical compositions and kits, and the use of reconstituted media containing chloride ions to reconstitute recombinant bacteria.

Description

[Technical Field]

[0001] The present invention is directed to recombinant bacteria, recombinant plasmids, pharmaceutical compositions and kits, and the use of reconstituted media containing chloride ions to reconstitute recombinant bacteria. [Background technology]

[0002] Biopharmaceuticals or biotherapeutics relate to a wide range of medical biological products produced by means of biological processes involving, for example, recombinant DNA technology.

[0003] Biopharmaceuticals or biotherapeutics include, for example, polypeptides that are identical or nearly identical to the polypeptide of the subject to be treated, such as the hematopoietic stimulating protein erythropoietin, biosynthetic human insulin and its analogs, or monoclonal antibodies that can be made specifically to inhibit or block any given target, such as a specific cell type, a specific polypeptide, or an endogenous antigen, and RNA molecules that are designed to modulate gene expression or translation by neutralizing the target mRNA molecule.

[0004] Biopharmaceuticals will have a profound impact on many medical areas of medicine, adding major therapeutic options for the treatment of many diseases, including some for which no effective therapy was available and others for which previously existing therapies were clearly inadequate.

[0005] However, the advent of biopharmaceuticals also raises complex regulatory issues and significant pharmacoeconomic concerns, as the costs of biotherapeutics are dramatically higher than those of conventional drugs. This factor is particularly important because many biologics are used for the rest of a person's life to treat chronic diseases or to treat otherwise untreatable cancers.

[0006] Furthermore, biopharmaceuticals often have the disadvantage of limited stability against degradation, for example by proteases or nucleases, especially after administration to a subject. Partial or complete degradation of the biopharmaceutical molecules leads to a significantly reduced half-life of the biopharmaceutical after administration to a subject.

[0007] In order to provide a sufficient amount of the respective biopharmaceutical to the subject, alternative modes of application are often required instead of direct application of the biopharmaceutical to the subject.

[0008] For example, biopharmaceuticals such as polypeptides can be provided either by autologous or heterologous cells secreting the respective polypeptide, or by transferring the gene encoding the desired polypeptide into the target tissue by gene transfer methods, e.g., using viral vectors.

[0009] A commercially available example is a dermal substitute containing human embryonic cells, which secrete various growth factors after application to the diseased wound area.

[0010] The dermal substitute Dermagraft is composed of fibroblasts, extracellular matrix, and a bioabsorbable substrate. Dermagraf is manufactured from human fibroblasts derived from donor neonatal foreskin tissue. During the manufacturing process, the human fibroblasts are seeded onto a bioabsorbable polygalactin substrate.

[0011] The commercially available dermal substitute Apligraf contains two cell types derived from neonatal foreskin: live human keratinocytes and fibroblasts embedded in a wound type 1 collagen matrix.

[0012] The previously mentioned disadvantage of dermal substitutes is the considerable cost resulting from the manufacturing process. Furthermore, each cell used in the dermal substitute must be tested for evidence of infection with human viruses, such as immunodeficiency virus types 1 and 2, hepatitis B virus, hepatitis C virus, syphilis, human T-lymphotropic virus types 1 and 2, and Epstein-Barr virus.

[0013] Another commercially available example is boretigene neparvovec-rzyl (LUXTURNA), an adeno-associated viral vector-based gene therapy suspension for subretinal injection designed to deliver normal copies of the gene encoding bioactive human retinal pigment epithelial 65 kDa protein (RPE65) to cells in the retina of individuals with reduced or absent levels of RPE65.

[0014] The disadvantages of viral products or any other viral vector-based gene therapy products are the high manufacturing costs and limitations to scale-up production.

[0015] For example, WO9714806A2 describes the delivery of biologically active polypeptides to a subject by non-invasive bacteria.

[0016] WO9611277A1 is directed to the use of microorganisms as vehicles for the delivery of therapeutic compounds to a subject.

[0017] WO2011160062A2 provides a method for treating inflammatory bowel disease, comprising administering to a subject a recombinant microorganism capable of producing a therapeutically effective amount of interleukin-27 (IL-27) or a variant or fragment thereof in situ in the intestinal mucosa.

[0018] US2013209407A is directed to commensal strains of E. coli, which are capable of colonizing the urogenital and / or gastrointestinal mucosa and which are capable of blocking the infectious and / or disease-causing activity of pathogens by secreting heterologous antimicrobial polypeptides.

[0019] Several expression systems for heterologous polypeptides are known in bacteria, using either inducible or constitutive promoters.

[0020] A constitutive promoter is preferably active in the cell under all circumstances and allows preferably high-level production of the desired heterologous polypeptide; however, this may also lead to a significantly increased metabolic burden on the bacteria used and therefore a reduced viability and / or growth rate. This may again have a direct impact on the bacterial yield during fermentation of the production process. The higher the metabolic burden, the lower the yield may be during the fermentation process.

[0021] In contrast, in response to a specific inducer, the regulated, preferably inducible, promoter becomes active or the promoter activity is enhanced. By applying the inducer to the recombinant bacterium expressing the desired heterologous polypeptide under the control of the respective regulated, preferably inducible promoter, the promoter becomes active and the nucleic acid of interest encoding the respective heterologous polypeptide is expressed. Preferably, this is an advantage during the production of the recombinant bacterium, in which higher yields of recombinant bacteria can be achieved because the metabolic burden is minimized.

[0022] A widely used controlled gene expression system is, for example, the nisin-controlled gene expression system (NICE) of Lactococcus lactis.

[0023] Nisin, well known to those skilled in the art, is a 34-amino acid lantibiotic polypeptide with a broad host spectrum produced by several L. lactis strains. Nisin is widely used in foods as a preservative. Initially, nisin is synthesized as a precursor by ribosomes. After subsequent enzymatic modification, the modified molecule translocates through the cytoplasmic membrane and is processed to its mature form.

[0024] When the gene of interest is placed behind the inducible promoter PnisA of the nisin system, expression of the nucleic acid sequence of interest can be induced by the addition of nisin.

[0025] Nisin-controlled gene expression systems favorably allow for high protein yields, however, they are dependent on exogenous inducers. When used for administration to subjects, the addition of nisin is costly because it requires the provision of pharmaceutical-grade nisin, which poses additional regulatory issues.

[0026] Because nisin is a polypeptide, it is also prone to degradation by proteases, and therefore, if continuous expression of the nucleic acid sequence of interest is intended, nisin must preferably be supplied repeatedly after administration to a subject of a recombinant bacterium expressing the nucleic acid sequence of interest under the control of a nisin promoter.

[0027] The requirement for the addition of nisin for continuous expression of the nucleic acid sequence of interest further limits the applicable administration routes of each recombinant bacterium.

[0028] For example, systemic administration of recombinant bacteria expressing nucleic acid sequences of interest under the control of a nisin promoter would, in addition, potentially require the administration of significant amounts of nisin to a subject to achieve a concentration of nisin in the subject that induces expression of each nucleic acid sequence of interest. Furthermore, the toxicity profile of nisin and the need for availability of pharmaceutical-grade nisin must be taken into consideration. Summary of the Invention [Problem to be solved by the invention]

[0029] It is an object of the present invention to provide a bacterial expression system that allows for the expression of at least one nucleic acid sequence encoding at least one heterologous factor or complex thereof, independently a heterologous polypeptide, wherein the heterologous polypeptide comprises at least one eukaryotic polypeptide, at least one fragment thereof, or a combination thereof, without negatively impacting the viability of the respective recombinant bacterium used prior to administration to a subject.

[0030] The bacterial expression system should provide for inducible expression of each heterologous factor, at least upon administration to a subject.

[0031] The bacterial expression system should also provide for easy administration of at least one beneficial agent or complex thereof, which is independently a heterologous polypeptide, to a subject, preferably over a prolonged period of time, without the need to supply additional inducers, wherein the heterologous polypeptide comprises at least one eukaryotic polypeptide, at least one fragment thereof, or a combination thereof.

[0032] Furthermore, bacterial expression systems should provide controlled amounts of each beneficial factor, which can be used in medicine. [Means for solving the problem]

[0033] The object of the present invention is to a) at least one nucleic acid sequence operably coupled to a chloride-inducible promoter in a prokaryotic system and encoding at least one heterologous factor, which heterologous factor is independently a heterologous polypeptide or complex thereof; b) at least one prokaryotic regulator gene that controls the activity of a chloride-inducible promoter; The problem is solved by providing a recombinant bacterium according to claim 1, comprising: The heterologous polypeptide comprises at least one eukaryotic polypeptide, at least one fragment thereof, or a combination thereof.

[0034] Preferably, the recombinant bacteria of the present invention are for medical use.

[0035] Preferred embodiments of the recombinant bacterium are disclosed in any one of the dependent claims 3 to 13, 15 or 17 to 23.

[0036] The object of the present invention is further to provide a) at least one nucleic acid sequence operably coupled to a chloride-inducible promoter in a prokaryotic system and encoding at least one heterologous factor, which heterologous factor is independently a heterologous polypeptide or complex thereof; b) at least one prokaryotic regulator gene that controls the activity of a chloride-inducible promoter; This is solved by providing a recombinant nucleic acid according to claim 2, which comprises The heterologous polypeptide comprises a eukaryotic polypeptide, at least a fragment thereof, or a combination thereof.

[0037] Preferably, the recombinant nucleic acids, preferably plasmids, of the invention are used in a method for producing a recombinant bacterium of the invention.

[0038] Preferred embodiments of the recombinant nucleic acid are disclosed in any one of the dependent claims 4 to 11 or 14 to 16.

[0039] The object of the present invention is further solved by providing a pharmaceutical composition according to claim 24 comprising a recombinant bacterium according to any one of claims 1, 3 to 13, 15 or 17 to 21 and at least one pharmaceutically acceptable excipient.

[0040] Preferably, the pharmaceutical composition of the present invention is for use in medicine, preferably for the treatment of chronic inflammatory wounds or degenerative conditions, or for the treatment of tumors, preferably malignant tumors.

[0041] Preferred embodiments of the pharmaceutical composition are disclosed in either one of the dependent claims 27 or 28.

[0042] The object of the present invention is further to provide a) a recombinant bacterium according to any one of claims 1, 3 to 13, 15, or 17 to 21, capable of expressing at least one heterologous factor under the control of a chloride-inducible promoter in a prokaryotic system; b) at least one inducer comprising chloride ions; This is solved by a kit according to claim 25 for medical use, comprising:

[0043] Preferred embodiments of the kit are disclosed in any one of the dependent claims 28 to 30.

[0044] The object of the present invention is further to provide a) a recombinant bacterium according to any one of claims 1, 3 to 13, 15, or 17 to 21, capable of expressing at least one heterologous factor under the control of a chloride-inducible promoter in a prokaryotic system; This is solved by a medical device according to claim 26, comprising:

[0045] A preferred embodiment of the kit is disclosed in dependent claim 28.

[0046] The object of the present invention is further solved by the use of a reconstituted medium according to claim 31 containing chloride ions for reconstituting a recombinant bacterium according to any one of claims 1, 3 to 13, 15 or 17 to 23.

[0047] The inventors have found that recombinant bacteria comprising the above-referenced nucleic acid sequences may be used in medicine, preferably in the treatment of chronic inflammatory wounds or degenerative conditions, or in the treatment of tumors, preferably malignant tumors, to provide suitable prophylactic and / or therapeutic factors to subjects in need thereof without the additional administration of exogenous inducers.

[0048] The inventors have further discovered that the extracellular tissue environment or bodily fluids of a subject provide a sufficiently high concentration of chloride ions to initiate and / or maintain expression of at least one nucleic acid sequence operably coupled to a chloride-inducible promoter in a prokaryotic system and encoding at least one heterologous factor.

[0049] Suitable body fluids are, for example, extracellular fluids, such as interstitial fluid, intravascular fluids, such as blood, plasma, and serum, cerebrospinal fluid, peritoneal fluid, urine, tears, and lymphatic fluid.

[0050] Furthermore, the intracellular fluid of some mammalian phagocytes, such as neutrophils and monocytes, preferably has a sufficiently high resting intracellular chloride ion concentration to initiate and / or maintain expression of at least one nucleic acid sequence operably coupled to a chloride-inducible promoter in a prokaryotic system and encoding at least one heterologous factor.

[0051] However, in the intracellular fluid of other mammalian cells, the intracellular chloride ion concentration is preferably insufficient for protein production, and therefore, when the recombinant bacteria of the invention are to enter the cytoplasm of these cells, the intracellular chloride concentration will, as a safety feature, prevent the initiation and / or maintenance of expression of at least one nucleic acid sequence operably coupled to a chloride-inducible promoter in a prokaryotic system and encoding at least one heterologous factor.

[0052] These findings make it possible to provide recombinant bacteria that can be engineered to produce at least one heterologous factor, preferably based on non-pathogenic lactic acid bacteria, and that can be designed as a single pharmaceutical entity.

[0053] According to the present invention, the term "heterologous factor" means a factor, preferably a polypeptide, or a complex thereof, which is not naturally present in or expressed by the bacterium used.

[0054] When referring generally to a "heterologous factor(s)," or when referring to a specific "heterologous factor(s)," such as, for example, FGF-2, IL-4, CSF-1, etc., it is intended that the term also encompasses functional analog(s).

[0055] According to the present invention, the term "functional analog" of a factor refers to an agent that binds to the same receptor(s) and preferably activates the same second messengers in target cells as the respective factor.

[0056] Preferably, when each heterologous factor is a polypeptide or a complex thereof, a "functional analog" of at least one heterologous factor has at least 50%, preferably at least 80%, more preferably at least 90%, more preferably at least 93%, more preferably at least 95%, and more preferably at least 97% amino acid sequence identity.

[0057] Preferably, when each heterologous factor is a ribonucleic acid, a "functional analog" of at least one heterologous factor has at least 80%, more preferably at least 90%, more preferably at least 93%, more preferably at least 95%, and more preferably at least 97% sequence identity with the ribonucleic acid sequence.

[0058] A "functional analog" may also be referred to as a biosimilar.

[0059] Upon induction with chloride ions, the recombinant bacterium of the present invention comprising at least one nucleic acid sequence referred to above is capable of producing at least one heterologous factor by transcribing, and preferably translating, the at least one nucleic acid sequence.

[0060] In the presence of chloride ions, the recombinant bacteria of the present invention are preferably capable of delivering at least one heterologous factor as defined in claim 1 to a subject, e.g., to diseased tissue, thereby mediating a beneficial effect and / or enabling healing of the subject. Preferably, the recombinant bacteria of the present invention release the at least one heterologous factor after administration to a subject.

[0061] In the presence of chloride ions, the recombinant bacteria of the present invention preferably further provide a constant release of at least one heterologous factor, more preferably after administration to a subject in need thereof.

[0062] Thereby, significantly improved, safer, and more cost-effective treatment options are available for subjects suffering from medical conditions, such as, preferably, chronic inflammatory wounds or degenerative conditions, or tumors, preferably malignant tumors.

[0063] Preferably, the at least one heterologous factor exerts at least one bioactive function after release from the bacteria to support healing of the subject and / or prevent deterioration of the medical condition.

[0064] Furthermore, the at least one heterologous factor may have a prophylactic and / or therapeutic effect after release from the bacteria, for example by exerting paracrine and / or endocrine activity that impacts local or systemic metabolism, and / or by regulating the activity of cells of the body, and / or by impacting the survival, growth, and differentiation of various cells of the body, and / or by impacting immune control or induction of an acute phase inflammatory response to injury and / or infection.

[0065] The recombinant bacterium of the present invention: a) at least one nucleic acid sequence operably coupled to a chloride-inducible promoter in a prokaryotic system and encoding at least one heterologous factor, which heterologous factor is independently a heterologous polypeptide or complex thereof; b) at least one prokaryotic regulator gene that controls the activity of a chloride-inducible promoter; Including, The heterologous polypeptide comprises at least one eukaryotic polypeptide, at least one fragment thereof, or a combination thereof.

[0066] The term "prokaryotic promoter" is known to those skilled in the art and refers to a nucleic acid sequence that controls the initiation of transcription of a particular gene, preferably by providing a binding site for RNA polymerase and / or at least one transcription factor that recruits RNA polymerase.

[0067] Prokaryotic promoters are preferably located near the start of transcription of a gene, more preferably on the same strand of the gene to be transcribed and upstream, towards the 5' region of the same strand.

[0068] By the term "operably coupled to" is meant that transcription of at least one nucleic acid sequence encoding at least one heterologous factor is preferably initiated and controlled by a respective prokaryotic promoter.

[0069] Preferably, the prokaryotic promoter used according to the present invention to initiate and control transcription of at least one nucleic acid sequence encoding at least one heterologous factor is a prokaryotic promoter that is inducible by chloride ions.

[0070] More preferably, the activity of the chloride-inducible promoter in a prokaryotic system is dependent on the concentration of chloride ions in the environment of the recombinant bacterium, such as in reconstituted medium or body fluids after application to a subject.

[0071] The term "activity" of a chloride-inducible promoter in a prokaryotic system preferably refers to the amount of at least one heterologous factor expressed from at least one nucleic acid sequence operably coupled to the promoter.

[0072] Preferably, the term "activity" of a chloride-inducible promoter in a prokaryotic system refers to the amount of protein(s) resulting from transcription of at least one nucleic acid sequence encoding at least one heterologous factor.

[0073] Preferably, the at least one heterologous element is subsequently produced from the transcribed mRNA, in particular by translation of the mRNA.

[0074] Preferably, the activity of the promoter used according to the invention to initiate and control the transcription of at least one nucleic acid sequence encoding at least one heterologous factor is further controlled by at least one prokaryotic regulatory factor gene.

[0075] The term "regulatory element gene" is known to those skilled in the art and refers to a nucleic acid sequence, preferably a gene, involved in controlling the expression of one or more other genes. Preferably, the regulatory sequence encoding the regulatory element gene is preferably located 5' to the transcription start site of the gene to be controlled. The regulatory element may also be located 3' to the transcription start site or at a remote site on the chromosome.

[0076] The regulator gene may be located preferably on the operon, adjacent to it, or distant from it, more preferably within the same bacterial cell.

[0077] In a preferred embodiment, the regulator gene encodes a regulator protein such as a repressor protein or an activator protein, and more preferably, expression of the regulator protein is initiated and controlled by a respective prokaryotic promoter selected from at least one of a constitutive promoter or a regulated, preferably inducible, promoter.

[0078] More preferably, expression of the regulator protein is controlled by a constitutive prokaryotic promoter, thereby providing the recombinant bacterium of the present invention with a sufficient amount of regulator protein intracellularly to control the activity of the chloride-inducible promoter.

[0079] The repressor protein preferably binds to the operator or promoter and prevents RNA polymerase from transcribing RNA. At least one inducer preferably can cause the repressor protein to change shape or otherwise become unable to bind DNA, allowing RNA polymerase to initiate and / or continue transcription.

[0080] An activator protein such as GadR preferably binds to a site on a DNA molecule, preferably near a promoter to be controlled by a regulator protein, and allows transcription and / or enhances the rate of transcription.

[0081] According to the present invention, at least one nucleic acid sequence operably coupled to a prokaryotic chloride-inducible promoter and encoding at least one heterologous factor, and at least one prokaryotic regulator gene controlling the activity of the chloride-inducible promoter, are each located within the same bacterial cell, preferably within each bacterial cell, of the recombinant bacterium of the present invention.

[0082] Preferably, at least one nucleic acid sequence operably coupled to the prokaryotic chloride-inducible promoter and encoding at least one heterologous factor and at least one prokaryotic regulator gene controlling the activity of the chloride-inducible promoter are each independently located on the chromosome and / or at least one plasmid within the same bacterial cell of the recombinant bacterium.

[0083] For example, at least one nucleic acid sequence operably coupled to a prokaryotic chloride-inducible promoter and encoding at least one heterologous factor is located on at least one chromosome and / or plasmid in at least one bacterial cell of a recombinant bacterium of the invention, and independently, at least one prokaryotic regulatory factor gene is preferably located on at least one chromosome and / or plasmid in the same bacterial cell of the recombinant bacterium.

[0084] Preferably, at least one nucleic acid sequence operably coupled to a prokaryotic chloride-inducible promoter and encoding at least one heterologous factor, and at least one prokaryotic regulatory factor gene controlling the activity of the chloride-inducible promoter, are each located on the same recombinant nucleic acid molecule, which is at least one of a chromosome and / or a plasmid.

[0085] More preferably, at least one nucleic acid sequence operably coupled to a prokaryotic chloride-inducible promoter and encoding at least one heterologous factor, and at least one prokaryotic regulatory factor gene controlling the activity of the chloride-inducible promoter, are both located on a recombinant nucleic acid of the invention, more preferably on a recombinant plasmid.

[0086] The recombinant nucleic acid of the present invention, preferably a recombinant plasmid, a) at least one nucleic acid sequence operably coupled to a chloride-inducible promoter in a prokaryotic system and encoding at least one heterologous factor, which heterologous factor is independently a heterologous polypeptide or complex thereof; b) at least one prokaryotic regulator gene that controls the activity of a chloride-inducible promoter; Including, The heterologous polypeptide comprises a eukaryotic polypeptide, at least a fragment thereof, or a combination thereof.

[0087] The term "plasmid" is known to those skilled in the art and refers to a preferably circular, more preferably double-stranded DNA molecule within a bacterial cell that is physically separate from chromosomal DNA and capable of independent replication.

[0088] Thus, the recombinant nucleic acid of the present invention is preferably a circular, more preferably a double-stranded DNA molecule, a) at least one nucleic acid sequence operably coupled to a chloride-inducible promoter in a prokaryotic system and encoding at least one heterologous factor, which heterologous factor is independently a heterologous polypeptide or complex thereof; b) at least one prokaryotic regulator gene that controls the activity of a chloride-inducible promoter; Including, The heterologous polypeptide comprises a eukaryotic polypeptide, at least a fragment thereof, or a combination thereof.

[0089] A recombinant bacterium of the invention preferably comprises at least one copy of a recombinant nucleic acid, preferably a recombinant plasmid, of the invention.

[0090] At least one nucleic acid sequence operably coupled to a chloride-inducible promoter in a prokaryotic system encodes at least one heterologous factor.

[0091] Preferably, at least one nucleic acid sequence each encodes one or more factor(s) that are not naturally occurring in or expressed by the bacterium used.

[0092] More preferably, at least one nucleic acid sequence encodes one, two, three, four, or more factors, each of which is not naturally occurring in or expressed by the bacterium used, and which are independently heterologous polypeptides or complexes thereof, preferably heterologous polypeptides or complexes thereof.

[0093] The term "complex" refers to a protein complex of two or more polypeptide chains, which may be referred to as "subunits," preferably associated or linked by at least one non-covalent protein-protein interaction, such as a hydrogen bond, an ionic interaction, van der Waals forces, and / or a hydrophobic bond, and / or by at least one covalent protein-protein bond that is not a peptide bond, such as a disulfide bond.

[0094] The subunits of a multimeric protein complex can be identical, as in a homomultimeric protein complex, or different, as in a heteromultimeric protein complex.

[0095] Preferably, the complex of heterologous polypeptides is formed before and / or after release of at least one heterologous polypeptide from the recombinant bacterium of the invention.

[0096] For example, the recombinant bacteria of the invention express one heterologous polypeptide that, after release from the bacterium, forms a homomultimeric protein complex having two or more identical subunits.

[0097] Alternatively, the recombinant bacteria of the invention express two or more heterologous polypeptides that are distinct from one another and that form a heteromultimeric protein complex having two or more distinct subunits after release from the bacteria.

[0098] Preferably, the at least one heterologous factor has a therapeutic and / or preventative effect in a subject, preferably after administration of a recombinant bacterium of the invention to a subject.

[0099] In a further preferred embodiment of the present invention, the at least one heterologous factor is a heterologous polypeptide or complex thereof comprising or consisting of at least one eukaryotic polypeptide, at least one fragment thereof, or a combination thereof, respectively.

[0100] More preferably, the term "fragment of a eukaryotic polypeptide" refers to a biologically active fragment of a eukaryotic polypeptide.

[0101] Preferably, the recombinant bacteria of the invention express at least one heterologous polypeptide or complex thereof after application to a subject, and more preferably, the recombinant bacteria of the invention release, preferably secrete, the at least one heterologous polypeptide or complex thereof into the surrounding environment, e.g., the site of application and / or body fluids of the subject.

[0102] The at least one heterologous polypeptide or complex thereof may itself have a biological effect on at least one cell of the subject, for example by stimulating or inhibiting cell growth, proliferation, and / or cell differentiation, by inducing or suppressing apoptosis, by activating or inhibiting the immune system, by regulating metabolism, by controlling cell migration, and / or by regulating the production and / or release of endogenous factors of the subject, thereby mediating a therapeutic and / or preventive effect in the subject, preferably after application of the recombinant bacterium of the present invention to the subject, preferably after release from the recombinant bacterium of the present invention, and more preferably after secretion.

[0103] The therapeutic and / or preventive effect in a subject may also be mediated by binding of at least one heterologous polypeptide or a complex thereof to at least one endogenous or exogenous target molecule, such as a bacterial antigen, a viral antigen, a tumor antigen, and / or an endogenous polypeptide, present in the subject.

[0104] For example, the at least one heterologous polypeptide or conjugate thereof is an antibody and / or at least one biologically active fragment thereof, which is released from the bacteria after application of the recombinant bacteria of the present invention to a subject and thereafter binds to at least one target molecule, preferably a foreign antigen, such as a part of a bacterial cell or virus, or to an endogenous antigen or polypeptide, which would otherwise mediate a harmful effect, such as an overactive immune response.

[0105] By binding to at least one target molecule present in the subject, the at least one heterologous polypeptide or complex thereof preferably reduces the amount of the molecule in the subject and / or reduces and / or prevents the biological activity of the molecule in the subject, for example by reducing or inhibiting the enzymatic activity of the molecule and / or by reducing or inhibiting the binding of the molecule to an endogenous receptor.

[0106] Preferably, the at least one heterologous factor is a heterologous polypeptide, preferably at least one fragment thereof having at least 5, preferably at least 7 amino acids linked by peptide bonds, and / or a complex thereof.

[0107] Examples of suitable polypeptides include polypeptides, precursors thereof, fragments thereof, and combinations thereof from eukaryotic species, preferably mammalian species, more preferably humans, that can act locally and / or systemically.

[0108] More preferably, the at least one heterologous polypeptide is selected from the group consisting of growth factors, cytokines, chemokines, enzymes, polypeptide hormones, neuropeptides, antibodies, cell surface receptors, soluble receptors, receptor ligands, intrabodies, which may also be called intracellular antibodies, cofactors, transcription factors, adhesion molecules, tumor antigens, precursors thereof, preferably biologically active fragments thereof, and combinations thereof from eukaryotic species, preferably mammalian species, more preferably human.

[0109] Suitable tumor antigens are known to those skilled in the art and are preferably disclosed in Cheever, MA et al. (2009) ("The Prioritization of Cancer Antigens: A National Cancer Institute Pilot Project for the Acceleration of Translational Research", Clin. Cancer Res. 15(17), pages 5323 to 5337; DOI: 10.1158 / 1078-0432.CCR-09-0737).

[0110] It will be appreciated that functional analogs of the above-mentioned or below-mentioned polypeptides or biosimilars thereof may also be used within the scope of the claimed invention.

[0111] Growth factors are preferably polypeptides capable of stimulating cell growth, proliferation, healing, and / or cell differentiation.

[0112] Preferably, the growth factor is selected from the group consisting of fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta), nerve growth factor (NGF), activin, functional analogs thereof, biosimilars thereof, and mixtures thereof.

[0113] Fibroblast growth factors are a family of growth factors involved in angiogenesis, wound healing, and various endocrine signaling pathways. 22 members of the FGF family, FGF-1 to FGF-14 and FGF-16 to FGF-23, have been identified in humans and can be used in the present invention. FGF-1 to FGF-10 bind to fibroblast growth factor receptors (FGFRs).

[0114] In a preferred embodiment, the fibroblast growth factor is selected from the group consisting of FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, and mixtures thereof, more preferably FGF-1, FGF-2, FGF-7, FGF-10, and mixtures thereof, more preferably FGF-2, FGF-7, functional analogs thereof, biosimilars thereof, and mixtures thereof, more preferably FGF-2.

[0115] For example, FGF-1 and FGF-2 can stimulate angiogenesis and are mitogenic for several cell types present at sites of inflammatory skin lesions, including fibroblasts and keratinocytes. Furthermore, FGF-7 can stimulate wound re-epithelialization in a paracrine manner.

[0116] The nucleic acid sequence of human fibroblast growth factor 2 (hFGF-2) mRNA is available under NCBI accession number NM_002006.4. The amino acid sequences of each of the AUG isomers are available under NCBI accession number NP_001997.5 and UniProt accession number P09038 version 182.

[0117] The precursor includes a propeptide spanning amino acids 1 to 142 of the precursor and a mature human fibroblast growth factor 2 peptide spanning amino acids 143 to 288 of the precursor.

[0118] In a preferred embodiment, the Fibroblast Growth Factor 2 comprises one or at least one of the amino acid sequences of SEQ ID NOs: 5 to 7. The amino acid sequences of SEQ ID NOs: 5 to 7 are depicted in Figures 5a to 5c, respectively.

[0119] Insulin-like growth factors (IGFs) are proteins with high sequence similarity to insulin. Insulin-like growth factors include two proteins, IGF-1 and IGF-2, which may be used in the present invention.

[0120] The epidermal growth factor (EGF) family is a group of proteins with highly similar structural and functional characteristics, including the proteins epidermal growth factor (EGF), heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor-α (TGF-α), amphiregulin (AR), epiregulin (EPR), epigen (EPGN), betacellulin (BTC), neuregulin-1 (NRG1), neuregulin-2 (NRG2), neuregulin-3 (NRG3), and neuregulin-4 (NRG4). Rhin-4 (NRG4), preferably epidermal growth factor (EGF), heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor-α (TGF-α), amphiregulin (AR), epiregulin (EPR), epigen (EPGN), and betacellulin (BTC), more preferably epidermal growth factor (EGF), heparin-binding EGF-like growth factor (HB-EGF), and transforming growth factor-α (TGF-α), which may be used in the present invention.

[0121] Transforming growth factor-α (TGF-α), preferably human transforming growth factor-α (hTGF-α), can be produced by macrophages, brain cells, and keratinocytes. hTGF-α induces epithelial development. hTGF-α and hEGF bind to the same receptor, epidermal growth factor receptor (EGFR; ErbB-1; HER1 in humans). When TGF-α binds to EGFR, it can initiate multiple cell proliferation events, including wound healing.

[0122] Human transforming growth factor-α exists as at least five isoforms produced by alternative splicing.

[0123] The amino acid sequence of human transforming growth factor alpha isoform 1 precursor is available under NCBI accession number NP_003227.1. The respective nucleic acid sequence of the mRNA is available under NCBI accession number NM_003236.2.

[0124] The precursor of human transforming growth factor alpha isoform 1 includes a signal peptide spanning amino acids 1 to 23 of the precursor, a protransforming growth factor alpha isoform 1 spanning amino acids 24 to 160 of the precursor, and a mature transforming growth factor alpha peptide spanning amino acids 40 to 89 of the precursor.

[0125] The amino acid sequence of human transforming growth factor alpha isoform 2 precursor is available under NCBI accession number NP_001093161.1. The respective nucleic acid sequence of the mRNA is available under NCBI accession number NM_001099691.1.

[0126] The amino acid sequence of human transforming growth factor alpha isoform 3 precursor is available under NCBI accession number NP_001295087.1. The respective nucleic acid sequence of the mRNA is available under NCBI accession number NM_001308158.1.

[0127] The amino acid sequence of human transforming growth factor alpha isoform 4 precursor is available under NCBI accession number NP_001295088.1. The respective nucleic acid sequence of mRNA is available under NCBI accession number NM_001308159.1.

[0128] The amino acid sequence of human transforming growth factor alpha isoform 5 precursor is available under NCBI accession number AAF05090.1. The respective nucleic acid sequence of the mRNA is available under NCBI accession number AF149097.1.

[0129] Amphiregulin (AREG), preferably human amphiregulin (hAREG), is another ligand of EGF receptor.Human amphiregulin is an autocrine growth factor and mitogen for a wide range of target cells, including astrocytes, Schwann cells, and fibroblasts.Human amphiregulin promotes the growth of epithelial cells.

[0130] The amino acid sequence of the human amphiregulin precursor is available under NCBI accession number NP_001648.1. The respective nucleic acid sequence of the mRNA is available under NCBI accession number NM_001657.3.

[0131] Epiregulin (EPR), preferably human epiregulin (hEPR), is a ligand for the EGF receptor, which can stimulate cell proliferation and / or angiogenesis.

[0132] The amino acid sequence of the human epiregulin precursor is available under NCBI accession number NP_001423.1. The respective nucleic acid sequence of the mRNA is available under NCBI accession number NM_001432.1.

[0133] Epigen (EPGN), preferably human epigen (hEPGN), promotes epithelial cell growth. Human epigen exists as at least seven isoforms produced by alternative splicing.

[0134] The amino acid sequence of human epigen isoform 1 precursor is available under NCBI accession number NP_001257918.1. The respective nucleic acid sequence of mRNA is available under NCBI accession number NM_001270989.1.

[0135] The amino acid sequences of human epigen isoforms 1 to 7 precursors are also available at UniProt accession number Q6UW88 version 101.

[0136] Betacellulin (BTC), preferably human betacellulin (hBTC), is a growth factor that also binds to the epidermal growth factor receptor and is synthesized by a wide range of adult tissues and in many cultured cells, including smooth muscle cells and epithelial cells. The amino acid sequence of the human probetacellulin precursor is available under NCBI accession number NP_001720.1. The respective nucleic acid sequence of the mRNA is available under NCBI accession number NM_001729.1.

[0137] Insulin-like growth factor 1 (IGF-1) is also called somatomedin C. The nucleic acid sequence of human IGF-1 mRNA is available under NCBI accession number NM_000618.2. The respective amino acid sequence is available under NCBI accession number NP_000609.1 and UniProt accession number P05019 version 178.

[0138] The nucleic acid sequence of human insulin-like growth factor 2 (hIGF-2) mRNA is available under NCBI accession number NM_000612.4. The respective amino acid sequence of the human insulin-like growth factor 2 precursor is available under NCBI accession number NP_000603.1 and UniProt accession number P01344 version 192.

[0139] The vascular endothelial growth factor (VEGF) family is a group of growth factors, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PGF), which may be used in the present invention.

[0140] In a preferred embodiment, the vascular endothelial growth factor is vascular endothelial growth factor A (VEGF-A). VEGF-A can induce angiogenesis, vasculogenesis, and endothelial cell growth.

[0141] The nucleic acid sequence of human vascular endothelial growth factor A (hVEGF-A) mRNA is available under NCBI accession number NM_001025366.1. The respective amino acid sequence of human vascular endothelial growth factor A is available under NCBI accession number NP_001020537.2 and UniProt accession number P15692 version 197.

[0142] Platelet-derived growth factor (PDGF) regulates cell growth and division. Human platelet-derived growth factor (hPDGF) has four subunits, PDGF-A, PDGF-B, PDGF-C, and PDGF-D, which form either homo- or heterodimers of each subunit, and these can be used in the present invention.

[0143] Preferably, the platelet-derived growth factor is PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, PDGF-DD, or a mixture thereof.

[0144] More preferably, the platelet-derived growth factor is a dimeric protein composed of two PDGF-A subunits, a dimeric protein composed of two PDGF-B subunits, a dimeric protein composed of a PDGF-A subunit and a PDGF-B subunit, or a mixture thereof.

[0145] The nucleic acid sequence of human platelet-derived growth factor subunit A (hPDGF-A) mRNA is available under NCBI accession number NM_002607.4. The respective amino acid sequence is available under NCBI accession number NP_002598.4 and UniProt accession number P04085 version 159.

[0146] The nucleic acid sequence of human platelet-derived growth factor subunit B (hPDGF) mRNA is available under NCBI accession number NM_002608.1. The amino acid sequences of each of the human platelet-derived growth factor subunit precursors are available under NCBI accession number NP_002599.1 and UniProt accession number P01127 version 181.

[0147] Hepatocyte growth factor (HGF) is a growth factor secreted by mesenchymal cells that acts primarily on epithelial and endothelial cells, but also on hematopoietic progenitor cells, and can be used in the present invention.

[0148] The nucleic acid sequence of human hepatocyte growth factor (hHGF) mRNA is available under NCBI accession number NM_000601.3. The amino acid sequence of each of the human hepatocyte growth factor precursors is available under NCBI accession number NP_000592.3 and UniProt accession number P14210 version 186.

[0149] Transforming growth factor beta (TGF-β), preferably human transforming growth factor beta (hTGF-β), is a cytokine secreted by many cell types, including macrophages.

[0150] TGF-β exists in at least three isoforms, TGF-β1, TGF-β2, and TGF-β3, which may be used in the present invention.

[0151] Human transforming growth factor-β1 is a secreted protein that is cleaved into the latency-associated peptide (LAP) and the mature TGF-β1 peptide, which can form either TGF-β1 homodimers or heterodimers with other TGF-β family members.

[0152] The nucleic acid sequence of the mRNA of human transforming growth factor-β1 precursor can be obtained under NCBI accession number NM_000660.4. The respective amino acid sequence is available under NCBI accession number NP_000651.3 or UniProt accession number P01137 version 199.

[0153] Transforming growth factor beta 2 (TGF-β2), preferably human transforming growth factor beta 2 (hTGF-β2), is a multifunctional cytokine that regulates the proliferation, differentiation, adhesion, and migration of many cell types.

[0154] Alternatively, splice transcript variants of the human transforming growth factor β2 gene that encode two different isoforms have been identified.

[0155] The nucleic acid sequence of the mRNA of human transforming growth factor beta 2 isoform 1 precursor is available under NCBI accession number NM_001135599.3. The respective amino acid sequence is available under NCBI accession number NP_001129071.1.

[0156] The nucleic acid sequence of the mRNA for human transforming growth factor beta 2 isoform 2 precursor is available under NCBI accession number NM_003238.3. The respective amino acid sequence is available under NCBI accession number NP_003229.1. The amino acid sequence of transforming growth factor beta 2 is also available under UniProt accession number P61812 version 128.

[0157] Transforming growth factor beta 3 (TGF-β3), preferably human transforming growth factor beta 3 (hTGF-β3), is a secreted cytokine involved in embryonic development and cell differentiation.

[0158] The nucleic acid sequence of the mRNA for human transforming growth factor-β3 precursor protein is available under NCBI accession number NM_003239.3. The corresponding amino acid sequence is available under NCBI accession number NP_003230.1 and UniProt accession number P10600 version 170.

[0159] Activin is a disulfide-linked dimeric protein originally purified from gonadal fluids as a protein that stimulates pituitary follicle-stimulating hormone (FSH) release. Activin proteins have a wide range of biological activities, including roles in mesoderm induction, neural cell differentiation, bone remodeling, hematopoiesis, and reproductive physiology.

[0160] Activins are homodimers or heterodimers of various beta subunit isoforms, while inhibins are heterodimers of a unique alpha subunit and one of four beta subunits, beta A, beta B, beta C, and beta E.

[0161] Cytokines are preferably polypeptides that are involved in autocrine, paracrine, and endocrine signaling as immunomodulatory agents.

[0162] Preferably, the cytokine is selected from the group consisting of interferons, interleukins, lymphokines, tumor necrosis factors, colony stimulating factors, functional analogs thereof, biosimilars thereof, and mixtures thereof.

[0163] Preferably, the interferon, more preferably a human interferon, is selected from the group consisting of interferon alpha (IFN-α), interferon beta (IFN-β), interferon epsilon (IFN-ε), interferon kappa (IFN-κ), interferon gamma (IFN-γ), interferon omega (IFN-ω), interferon lambda (IFN-λ), functional analogs thereof, biosimilars thereof, and mixtures thereof.

[0164] Interferon alpha, preferably human interferon alpha, is preferably interferon alpha-1 (IFN-α1), interferon alpha-2 (IFN-α2), interferon alpha-4 (IFN-α4), interferon alpha-5 (IFN-α5), interferon alpha-6 (IFN-α6), interferon alpha-7 (IFN-α7), interferon alpha-8 (IFN-α8), interferon alpha-10 (IFN-α10), ...1 (IFN-α1), interferon alpha-1 (IFN-α1), interferon alpha-1 (IFN-α1), interferon alpha-2 (IFN-α2), interferon alpha-3 (IFN-α3), interferon alpha-4 (IFN-α4), interferon alpha-5 (IFN-α5), interferon alpha-6 (IFN-α6), interferon alpha-7 (IFN-α7), interferon alpha-8 (IFN-α8), interferon alpha-10 (IFN-α10), interferon alpha-1 (IFN-α1), interferon alpha-1 (IFN-α1), interferon alpha-1 (IFN-α1), interferon alpha-1 (IFN-α1), interferon alpha-1 (IFN-α1), interferon alpha-1 (IFN-α1), interferon alpha-1 (IFN-α1 Interferon alpha-13 (IFN-α13), interferon alpha-14 (IFN-α14), interferon alpha-16 (IFN-α16), interferon alpha-17 (IFN-α17), interferon alpha-21 (IFN-α21), functional analogs thereof, biosimilars thereof, and mixtures thereof, preferably interferon alpha-2 (IFN-α2), functional analogs thereof, biosimilars thereof, and mixtures thereof.

[0165] The nucleic acid sequence of human interferon alpha 1 (IFN-α1) mRNA is available under NCBI accession number NM_024013.2. The nucleic acid sequence of human interferon alpha 13 (IFN-α13) mRNA is available under NCBI accession number NM_006900.3.

[0166] Human interferon alpha-1 and alpha-13 have identical protein sequences. The respective amino acid sequences of the human interferon alpha-1 / 13 precursors are available under NCBI accession numbers NP_076918.1 and NP_008831.3 and UniProt accession number P01562 version 180.

[0167] The precursor of human interferon alpha-1 / 13 includes a signal peptide spanning amino acids 1 to 23 of the precursor, and mature interferon alpha-2 spanning amino acids 24 to 189 of the precursor.

[0168] The nucleic acid sequence of human interferon alpha 2 (IFN-α2) mRNA is available under NCBI accession number NM_000605.3. The respective amino acid sequence of the human interferon alpha-2 precursor is available under NCBI accession number NP_000596.2 and UniProt accession number P01563 version 176.

[0169] The precursor of human interferon alpha-2 includes a signal peptide spanning amino acids 1 to 23 of the precursor, and mature interferon alpha-2 spanning amino acids 24 to 188 of the precursor.

[0170] More preferably, the interferon alpha-2 comprises one or at least one of the amino acid sequences of SEQ ID NOs: 28 to 30, which are also depicted in Figures 14a to 14c, respectively.

[0171] The nucleic acid sequence of human interferon alpha 4 (IFN-α4) mRNA is available under NCBI accession number NM_021068.2. The respective amino acid sequence of the human interferon alpha-4 precursor is available under NCBI accession number NP_066546.1 and UniProt accession number P05014 version 168.

[0172] The precursor of human interferon alpha-4 includes a signal peptide spanning amino acids 1 to 23 of the precursor, and mature interferon alpha-4 spanning amino acids 24 to 189 of the precursor.

[0173] The nucleic acid sequence of human interferon alpha 5 (IFN-α5) mRNA is available under NCBI accession number NM_002169.2. The respective amino acid sequence of the human interferon alpha-5 precursor is available under NCBI accession number NP_002160.1 and UniProt accession number P01569 version 158.

[0174] The precursor of human interferon alpha-5 includes a signal peptide spanning amino acids 1 to 21 of the precursor, and mature interferon alpha-5 spanning amino acids 22 to 189 of the precursor.

[0175] The nucleic acid sequence of human interferon alpha 6 (IFN-α6) mRNA is available under NCBI accession number NM_021002.2. The respective amino acid sequence of the human interferon alpha-6 precursor is available under NCBI accession number NP_066282.1 and UniProt accession number P05013 version 158.

[0176] The precursor of human interferon alpha-6 includes a signal peptide spanning amino acids 1 to 20 of the precursor, and mature interferon alpha-6 spanning amino acids 21 to 189 of the precursor.

[0177] The nucleic acid sequence of human interferon alpha 7 (IFN-α7) mRNA is available under NCBI accession number NM_021057.2. The respective amino acid sequence of the human interferon alpha-7 precursor is available under NCBI accession number NP_066401.2 and UniProt accession number P01567 version 160.

[0178] The precursor of human interferon alpha-7 includes a signal peptide spanning amino acids 1 to 23 of the precursor, and mature interferon alpha-7 spanning amino acids 24 to 189 of the precursor.

[0179] The nucleic acid sequence of human interferon alpha 8 (IFN-α8) mRNA is available under NCBI accession number NM_002170.3. The respective amino acid sequence of the human interferon alpha-8 precursor is available under NCBI accession number NP_002161.2 and UniProt accession number P32881 version 157.

[0180] The precursor of human interferon alpha-8 includes a signal peptide spanning amino acids 1 to 23 of the precursor, and mature interferon alpha-8 spanning amino acids 24 to 189 of the precursor.

[0181] The nucleic acid sequence of human interferon alpha 10 (IFN-α10) mRNA is available under NCBI accession number NM_002171.2. The respective amino acid sequence of the human interferon alpha-10 precursor is available under NCBI accession number NP_002162.1 and UniProt accession number P01566 version 160.

[0182] The precursor of human interferon alpha-10 includes a signal peptide spanning amino acids 1 to 23 of the precursor, and mature interferon alpha-10 spanning amino acids 24 to 189 of the precursor.

[0183] The nucleic acid sequence of human interferon alpha 14 (IFN-α14) mRNA is available under NCBI accession number NM_002172.2. The respective amino acid sequence of the human interferon alpha-14 precursor is available under NCBI accession number NP_002163.2 and UniProt accession number P01570 version 172.

[0184] The precursor of human interferon alpha-14 includes a signal peptide spanning amino acids 1 to 23 of the precursor, and mature interferon alpha-14 spanning amino acids 24 to 189 of the precursor.

[0185] The nucleic acid sequence of human interferon alpha 16 (IFN-α16) mRNA is available under NCBI accession number NM_002173.3. The respective amino acid sequence of the human interferon alpha-16 precursor is available under NCBI accession number NP_002164.1 and UniProt accession number P05015 version 161.

[0186] The precursor of human interferon alpha-16 includes a signal peptide spanning amino acids 1 to 23 of the precursor, and mature interferon alpha-16 spanning amino acids 24 to 189 of the precursor.

[0187] The nucleic acid sequence of human interferon alpha 17 (IFN-α17) mRNA is available under NCBI accession number NM_021268.2. The respective amino acid sequence of the human interferon alpha-17 precursor is available under NCBI accession number NP_067091.1 and UniProt accession number P01571 version 162.

[0188] The precursor of human interferon alpha-17 includes a signal peptide spanning amino acids 1 to 23 of the precursor, and mature interferon alpha-17 spanning amino acids 24 to 189 of the precursor.

[0189] The nucleic acid sequence of human interferon alpha 21 (IFN-α21) mRNA is available under NCBI accession number NM_002175.2. The respective amino acid sequence of the human interferon alpha-21 precursor is available under NCBI accession number NP_002166.2 and UniProt accession number P01568 version 171.

[0190] The precursor of human interferon alpha-21 includes a signal peptide spanning amino acids 1 to 23 of the precursor, and mature interferon alpha-21 spanning amino acids 24 to 189 of the precursor.

[0191] The nucleic acid sequence of human interferon beta 1 (IFN-β1) mRNA is available under NCBI accession number NM_002176.3. The amino acid sequence of each of the human interferon beta precursors is available under NCBI accession number NP_002167.1 and UniProt accession number P01574 version 196.

[0192] The precursor of human interferon beta includes a signal peptide spanning amino acids 1 to 21 of the precursor, and mature interferon beta spanning amino acids 22 to 187 of the precursor.

[0193] The nucleic acid sequence of human interferon gamma (IFN-γ) mRNA is available under NCBI accession number NM_000619.2. The amino acid sequence of each of the human interferon gamma precursors is available under NCBI accession number NP_000610.2 and UniProt accession number P01579 version 205.

[0194] The precursor of human interferon gamma includes a signal peptide spanning amino acids 1 to 23 of the precursor, and mature interferon gamma spanning amino acids 24 to 161 of the precursor, and a propeptide spanning amino acids 162 to 166 of the precursor.

[0195] The nucleic acid sequence of human interferon kappa (IFN-κ) mRNA is available under NCBI accession number NM_020124.2. The respective amino acid sequence of the human interferon kappa precursor is available under NCBI accession number NP_064509.2 and UniProt accession number Q9P0W0 version 120.

[0196] The precursor of human interferon kappa includes a signal peptide spanning amino acids 1 to 27 of the precursor, and mature interferon gamma spanning amino acids 28 to 207 of the precursor.

[0197] The nucleic acid sequence of human interferon epsilon (IFN-ε) mRNA is available under NCBI accession number NM_176891.4. The respective amino acid sequence of the human interferon epsilon precursor is available under NCBI accession number NP_795372.1 and UniProt accession number Q86WN2 version 124.

[0198] The precursor of human interferon epsilon includes a signal peptide spanning amino acids 1 to 21 of the precursor, and mature interferon epsilon spanning amino acids 22 to 208 of the precursor.

[0199] The nucleic acid sequence of human interferon omega 1 (IFN-ω1) mRNA is available under NCBI accession number NM_002177.2. The amino acid sequences of the respective human interferon omega precursors are available under NCBI accession number NP_002168.1 and UniProt accession number P05000 version 165.

[0200] The precursor of human interferon omega includes a signal peptide spanning amino acids 1 to 21 of the precursor, and mature interferon omega spanning amino acids 22 to 195 of the precursor.

[0201] The human interferon lambda (IFN-λ) is preferably selected from the group consisting of interferon lambda-1 (IFN-λ1), interferon lambda-2 (IFN-λ2), interferon lambda-3 (IFN-λ3), interferon lambda-4 (IFN-λ4), functional analogs thereof, biosimilars thereof, and mixtures thereof.

[0202] The nucleic acid sequence of human interferon lambda 1 (IFN-λ1) mRNA is available under NCBI accession number NM_172140.1. The respective amino acid sequence of the human interferon lambda-1 precursor is available under NCBI accession number NP_742152.1 and UniProt accession number Q8IU54 version 125.

[0203] The precursor of human interferon lambda-1 includes a signal peptide spanning amino acids 1 to 19 of the precursor, and mature interferon lambda-1 spanning amino acids 20 to 200 of the precursor.

[0204] The nucleic acid sequence of human interferon lambda 2 (IFN-λ2) mRNA is available under NCBI accession number NM_172138.1. The respective amino acid sequence of the human interferon lambda-2 precursor is available under NCBI accession number NP_742150.1 and UniProt accession number Q8IZJ0 version 105.

[0205] The precursor of human interferon lambda-2 precursor includes a signal peptide spanning amino acids 1 to 25 of the precursor, and mature interferon lambda-2 spanning amino acids 26 to 200 of the precursor.

[0206] The nucleic acid sequence of human interferon lambda 3 (IFN-λ3) mRNA contains two transcript variants. The sequence of human interferon lambda 3 transcript variant 1 mRNA is available under NCBI accession number NM_001346937.1. The sequence of human interferon lambda 3 transcript variant 2 mRNA is available under NCBI accession number NM_172139.3.

[0207] The respective amino acid sequences of the human interferon lambda-3 isoform 1 precursor are available under NCBI accession number NP_001333866.1. The respective amino acid sequences of the human interferon lambda-3 isoform 2 precursor are available under NCBI accession number NP_742151.2 and UniProt accession number Q8IZI9 version 113.

[0208] The precursor of human interferon lambda-3 isoform 2 includes a signal peptide spanning amino acids 1 to 21 of the precursor, and mature interferon lambda-3 spanning amino acids 22 to 196 of the precursor.

[0209] The nucleic acid sequence of human interferon lambda 4 (IFN-λ4) mRNA is available under NCBI accession number NM_001276254.2. The respective amino acid sequence of the human interferon lambda-4 precursor is available under NCBI accession number NP_001263183.2 and UniProt accession number K9M1U5 version 24.

[0210] The precursor of human interferon lambda-4 includes a signal peptide spanning amino acids 1 to 21 of the precursor, and mature interferon lambda-4 spanning amino acids 22 to 179 of the precursor.

[0211] Preferably, the interleukin, more preferably a human interleukin, is interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-14 (IL-14), interleukin-15 (IL-15), interleukin-16 (IL-16), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-19 (IL-19), interleukin-20 (IL-20), interleukin-21 (IL-21), interleukin-22 (IL-22), interleukin-23 (IL-23), interleukin-24 (IL-24), interleukin-25 (IL-25), interleukin-26 (IL-26), interleukin-27 (IL-27), interleukin-28 (IL-28), interleukin-29 (IL-29), interleukin-30 (IL-29), interleukin-31 (IL-29), interleukin-32 (IL-29), interleukin-33 (IL-29), interleukin-34 (IL-29), interleukin-35 (IL-29), interleukin-36 (IL-29), interleukin-37 (IL-29), interleukin-38 (IL-29), interleukin-39 (IL-39), interleukin Interleukin-13 (IL-13), interleukin-14 (IL-14), interleukin-15 (IL-15), interleukin-16 (IL-16), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-19 (IL-19), interleukin-20 (IL-20), interleukin-21 (IL-21), interleukin-22 (IL-22), interleukin-23 (IL-23), interleukin-24 (IL-24), interleukin-25 (IL-25), interleukin -26 (IL-26), interleukin-27 (IL-27), interleukin-28 (IL-28), interleukin-29 (IL-29), interleukin-30 (IL-30), interleukin-31 (IL-31), interleukin-32 (IL-32), interleukin-33 (IL-33), interleukin-34 (IL-34), interleukin-36 alpha (IL-36α), interleukin-36 beta (IL-36β), interleukin-36 gamma (IL-36γ), interleukin-37 (IL-37) , cardiotrophin-like cytokine factor 1 (CLCF1), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin-M (OSM), interleukin receptor antagonist protein, interleukin binding protein, functional analogs thereof, biosimilars thereof, and mixtures thereof, preferably selected from the group consisting of interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-18 (IL-18), functional analogs thereof, biosimilars thereof, and mixtures thereof.

[0212] Preferably, the interleukin-1 (IL-1), more preferably human interleukin-1, is selected from the group consisting of interleukin-1 alpha (IL-1α), interleukin-1 beta (IL-1β), functional analogs thereof, biosimilars thereof, and mixtures thereof.

[0213] The nucleic acid sequence of the mRNA of the human interleukin-1 alpha (IL-1α) precursor is available under NCBI accession number NM_000575.4. The respective amino acid sequence of the precursor is available under NCBI accession number NP_000566.3.

[0214] The amino acid sequence of human interleukin-1 alpha precursor is also available at UniProt accession number P01583 version 1908. The amino acid sequence includes the propeptide spanning amino acids 1 to 112 of the interleukin-1 alpha precursor and mature interleukin-1 alpha spanning amino acids 113 to 271 of the precursor.

[0215] The nucleic acid sequence of the human interleukin-2 precursor mRNA is available under NCBI accession number NM_000586.3. The respective amino acid sequence is available under NCBI accession number NP_000577.2.

[0216] The amino acid sequence of the human interleukin-2 precursor is also available at UniProt accession number P60568 version 161. The amino acid sequence includes a signal peptide spanning amino acids 1 to 20 of the interleukin-2 precursor and mature interleukin-2 spanning amino acids 21 to 153 of the precursor.

[0217] The nucleic acid sequence of the human interleukin-3 precursor mRNA is available under NCBI accession number NM_000586.3. The respective amino acid sequence is available under NCBI accession number NP_000577.2.

[0218] The amino acid sequence of the human interleukin-3 precursor is also available at UniProt accession number P08700 version 177. The amino acid sequence includes a signal peptide spanning amino acids 1 to 19 of the interleukin-3 precursor and mature interleukin-3 spanning amino acids 20 to 152 of the precursor.

[0219] Interleukin-4 (IL-4), preferably human interleukin-4 (hIL-4), is a multifunctional cytokine. Interleukin-4 is a ligand for the interleukin-4 receptor.

[0220] The nucleic acid sequence of the mRNA of human interleukin-4 isoform 1 precursor is available under NCBI accession number NM_000589.3. The respective amino acid sequence of interleukin-4 isoform 1 precursor is available under NCBI accession number NP_000580.1.

[0221] The nucleic acid sequence of the mRNA of human interleukin-4 isoform 2 precursor is available under NCBI accession number NM_172348.2. The respective amino acid sequence of interleukin-4 isoform 2 precursor is available under NCBI accession number NP_758858.1.

[0222] The amino acid sequence of the human interleukin-4 precursor is also available under UniProt accession number P05112 version 178. The amino acid sequence includes the signal peptide spanning amino acids 1 to 24 of the interleukin-4 isoform 1 and isoform 2 precursors.

[0223] Preferably, the interleukin-4 comprises one or at least one of the amino acid sequences of SEQ ID NOs: 8 to 10, which are also depicted in Figures 6a to 6c, respectively.

[0224] The nucleic acid sequence of the human interleukin-5 precursor mRNA is available under NCBI accession number NM_000879.2. The respective amino acid sequence is available under NCBI accession number NP_000870.1.

[0225] The amino acid sequence of the human interleukin-5 precursor is also available at UniProt accession number P05113 version 189. The amino acid sequence includes a signal peptide spanning amino acids 1 to 19 of the interleukin-5 precursor and mature interleukin-5 spanning amino acids 20 to 134 of the precursor.

[0226] The nucleic acid sequence of the mRNA of transcript variant 1 of the human interleukin-6 precursor is available under NCBI accession number NM_000600.4. The respective amino acid sequence of the interleukin-6 isoform 1 precursor is available under NCBI accession number NP_000591.1.

[0227] The amino acid sequence of the human interleukin-6 precursor is also available under UniProt accession number P05231 version 212. The amino acid sequence includes a signal peptide spanning amino acids 1 to 29 of the interleukin-6 precursor and mature interleukin-6 spanning amino acids 30 to 212 of the precursor.

[0228] The nucleic acid sequence of the mRNA of transcript variant 1 of human interleukin-7 precursor is available under NCBI accession number NM_000880.3. The respective amino acid sequence of interleukin-7 isoform 1 precursor is available under NCBI accession number NP_000871.1.

[0229] The amino acid sequence of the human interleukin-7 precursor is also available at UniProt accession number P05231 version 212. The amino acid sequence of the human interleukin-7 isoform 1 precursor contains a signal peptide spanning amino acids 1 to 25 of the isoform 1 precursor and mature interleukin-7 spanning amino acids 26 to 177 of the isoform 1 precursor.

[0230] The nucleic acid sequence of the mRNA of transcript variant 1 of the human interleukin-8 precursor is available under NCBI accession number NM_000584.3. The respective amino acid sequence of the interleukin-8 isoform 1 precursor is available under NCBI accession number NP_000575.1.

[0231] The amino acid sequence of the human interleukin-8 precursor is also available under UniProt accession number P10145 version 210. The amino acid sequence of the human interleukin-8 isoform 1 precursor contains a signal peptide spanning amino acids 1 to 20 of the isoform 1 precursor. The 99 amino acid precursor peptide undergoes cleavage to generate several active IL8 isoforms.

[0232] Preferably, the mature human interleukin-8 comprises amino acids 31 to 99 of the interleukin-8 isoform 1 precursor.

[0233] Interleukin-9 (IL-9), preferably human interleukin-9 (hIL-9), is produced by various cells. The nucleic acid sequence of the mRNA of human interleukin-9 precursor can be found under NCBI accession number NM_000590.1. The respective amino acid sequences can be found under NCBI accession number NP_000581.1 and UniProt accession number P15248 version 153.

[0234] The amino acid sequence of the human interleukin-9 precursor includes a signal peptide spanning amino acids 1 to 18 of the human interleukin-9 precursor protein, and mature interleukin-9 spanning amino acids 19 to 144 of the precursor.

[0235] Interleukin-10 (IL-10), preferably human interleukin-10 (hIL-10), is a cytokine produced primarily by monocytes. The nucleic acid sequence of human interleukin-10 precursor mRNA can be found under NCBI accession number NM_000572.2. The respective amino acid sequences can be found under NCBI accession number NP_000563.1 and UniProt accession number P22301 version 156.

[0236] The amino acid sequence of the human interleukin-10 precursor includes a signal peptide spanning amino acids 1 to 18 of the human interleukin-10 precursor protein.

[0237] Interleukin-12 is a heterodimeric cytokine encoded by two separate genes, interleukin-12 subunit alpha (IL-12A), also called p35, and interleukin-12 subunit beta (IL-12B), also called p40. An active heterodimer, termed "p70," and a homodimer of p40 are formed after protein synthesis.

[0238] The nucleic acid sequence of the mRNA for human interleukin-12 subunit alpha precursor contains three transcript variants, which are available under NCBI accession numbers NM_000882.4 (variant 1), NM_001354582.1 (variant 2), and NM_001354583.1 (variant 3).

[0239] The amino acid sequences of each of the human interleukin-12 subunit alpha precursor isoforms 1, 2, and 3 are available under NCBI accession numbers NP_000873.2, NP_001341511.1, and NP_001341512.1, respectively, and UniProt accession number P29459 version 170.

[0240] The human interleukin-12 subunit alpha isoform 1 precursor contains a signal peptide spanning amino acids 1 to 56 of the precursor protein deposited at NCBI reference sequence: NP_000873.2.

[0241] Human IL12A variants 2 and 3 lack an alternative in-frame exon compared to variant 1. The resulting isoforms 2 and 3 have the same N- and C-termini but are shorter compared to isoform 1.

[0242] The nucleic acid sequence of the murine interleukin-12 subunit alpha precursor mRNA contains two transcript variants, which are available under NCBI accession numbers NM_001159424.2 (variant 1) and NM_008351.3 (variant 2).

[0243] The amino acid sequences of each of the murine interleukin-12 subunit alpha precursor isoforms 1 and 2 are available under NCBI accession numbers NP_001152896.1 and NP_032377.1, respectively, and UniProt accession number P43431 version 140.

[0244] The mouse interleukin-12 subunit alpha isoform 2 precursor contains a signal peptide spanning amino acids 1 to 22 of the precursor protein deposited at NCBI reference sequence: NP_032377.1.

[0245] Preferably, the interleukin-12 subunit alpha comprises the amino acid sequence of mature murine interleukin-12 subunit alpha isoform 2, which is depicted in Figure 13b and SEQ ID NO:25.

[0246] The nucleic acid sequence of the mRNA for human interleukin-12 subunit beta precursor is available under NCBI accession number NM_002187.2. The respective amino acid sequence is available under NCBI accession number NP_002178.2 and UniProt accession number P29460 version 210.

[0247] The precursor of human interleukin-12 subunit beta precursor contains a signal peptide spanning amino acids 1 to 22 of the amino acid sequence deposited in NCBI reference sequence NP_002178.2.

[0248] The nucleic acid sequence of the mRNA for mouse interleukin-12 subunit beta precursor is available under NCBI accession number NM_001303244.1. The respective amino acid sequence is available under NCBI accession number NP_001290173.1 and UniProt accession number P43432 version 163.

[0249] The precursor of mouse interleukin-12 subunit beta precursor contains a signal peptide spanning amino acids 1 to 22 of the amino acid sequence deposited in NCBI reference sequence NP_001290173.1.

[0250] Preferably, the interleukin-12 subunit beta comprises the amino acid sequence of mature murine interleukin-12 subunit beta, which is depicted in Figure 13a and SEQ ID NO:24.

[0251] More preferably, interleukin-12 is expressed as a recombinant fusion protein comprising the mature form of interleukin-12 subunit alpha and the mature form of interleukin-12 subunit beta, preferably linked by a linker sequence, more preferably comprising at least one amino acid.

[0252] More preferably, interleukin-12 is expressed as a recombinant fusion protein comprising one or at least one of the amino acid sequences of SEQ ID NOs: 26 to 27, which are also depicted in Figures 13c and 13d, respectively.

[0253] Interleukin-13 (IL-13), preferably human interleukin-13 (hIL-13), is an immunoregulatory cytokine. The nucleic acid sequence of the mRNA of human interleukin-13 precursor is available under NCBI accession number NM_002188.2. The respective amino acid sequences are available under NCBI accession number NP_002179.2 and UniProt accession number P35225 version 157.

[0254] The interleukin-13 precursor contains a signal peptide spanning amino acids 1 to 24 of the interleukin-13 precursor protein deposited under UniProt accession number P35225 version 157.

[0255] Interleukin-18 (IL-18) is a cytokine belonging to the IL-1 superfamily and is produced, for example, by macrophages.

[0256] The nucleic acid sequence of the human interleukin-18 (hIL-18) precursor mRNA contains two transcript variants, which are available under NCBI accession numbers NM_001562.4 (variant 1) and NM_001243211.1 (variant 2).

[0257] Transcript variant 1 represents the major and longer transcript and encodes the longer hIL-18 isoform 1.

[0258] Transcript variant 2, also known as Delta3pro-IL-18, lacks an in-frame coding exon compared to variant 1. The encoded hIL-18 isoform 2 is shorter than hIL-18 isoform 1 and may be resistant to proteolytic activation.

[0259] The amino acid sequences of each of the human interleukin-18 precursor isoforms 1 and 2 are available under NCBI accession numbers NP_001553.1 and NP_001230140.1, respectively, and UniProt accession number Q14116 version 1174.

[0260] The human interleukin-18 isoform 1 precursor contains a propeptide, which spans amino acids 1 to 36 of the precursor protein deposited under NCBI reference sequence: NP_001553.1, and is processed in vivo by caspase 1 (CASP1) or caspase 4 (CASP4) to yield the active form.

[0261] Preferably, the interleukin-18 comprises one or at least one of the amino acid sequences of SEQ ID NOs: 16 to 18, which are also depicted in Figures 10a to 10c, respectively.

[0262] Interleukin-34 (IL-34) is a cytokine that also promotes the differentiation and survival of monocytes and macrophages. Due to alternative splicing, human interleukin-34 exists as two isoforms, which can be used in the present invention.

[0263] The nucleic acid sequence of the mRNA for human interleukin-34 isoform 1 precursor is available under NCBI accession number NM_001172772.1. The corresponding amino acid sequence is available under NCBI accession number NP_001166243.1.

[0264] The nucleic acid sequence of human interleukin-34 isoform 2 precursor mRNA is available under NCBI accession number NM_001172771.1. The corresponding amino acid sequence is available under NCBI accession number NP_001166242.1.

[0265] The respective amino acid sequences are also available under UniProt accession number Q6ZMJ4 version 80. The human interleukin-34 precursor includes a signal peptide spanning amino acids 1 to 20 of the respective amino acid sequence of the precursor protein.

[0266] The interleukin receptor antagonist protein is preferably selected from interleukin-1 receptor antagonist protein (IL-1RA), interleukin-36 receptor antagonist protein (IL-36RA), and mixtures thereof.

[0267] A suitable interleukin-binding protein is preferably interleukin-18 binding protein (IL-18BP).

[0268] Colony stimulating factors are preferably polypeptides capable of stimulating proliferation, healing, and / or cell differentiation.

[0269] Preferably, the colony-stimulating factor is selected from the group consisting of colony-stimulating factor 1 (CSF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), erythropoietin, thrombopoietin, functional analogs thereof, biosimilars thereof, and mixtures thereof, preferably colony-stimulating factor 1 (CSF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), erythropoietin, functional analogs thereof, biosimilars thereof, and mixtures thereof.

[0270] Colony-stimulating factor-1 (CSF-1), also known as macrophage colony-stimulating factor (M-CSF), is a cytokine that controls the production, differentiation, and function of macrophages.

[0271] Due to alternative splicing, human CSF-1 exists in different isoforms, which may be used in the present invention.

[0272] The nucleic acid sequence of the mRNA for human CSF-1 isoform 1, also called macrophage colony-stimulating factor 1 isoform A precursor, is available under NCBI accession number NM_000757.5. The corresponding amino acid sequence is available under NCBI accession number NP_000748.3.

[0273] The nucleic acid sequence of the mRNA for human CSF-1 isoform 2 precursor, also called human macrophage colony-stimulating factor 1 isoform B precursor, is available under NCBI accession number NM_172210.2. The corresponding amino acid sequence is available under NCBI accession number NP_757349.1.

[0274] The nucleic acid sequence of the mRNA for human CSF-1 isoform 3 precursor, also called macrophage colony-stimulating factor 1 isoform C precursor, is available under NCBI accession number NM_172211.3. The corresponding amino acid sequence is available under NCBI accession number NP_757350.1.

[0275] The respective amino acid sequences are also available in UniProt accession number P09603 version 158. Isoform 1 was chosen as the canonical UniProt sequence.

[0276] The protein sequence of each of human CSF-1 precursor isoforms 1 to 3 includes an N-terminal signal peptide spanning amino acid number 1 to amino acid number 32 of the respective amino acid sequence.

[0277] The active form of human CSF-1 can be found extracellularly as a disulfide-linked homodimer, which is produced by proteolytic cleavage of the membrane-bound precursor, resulting in the loss of the N-terminal signal peptide.

[0278] Preferably, colony stimulating factor 1 comprises one or at least one of the amino acid sequences of SEQ ID NOs: 11 to 13, which are also depicted in Figures 7a to 7c, respectively.

[0279] Granulocyte-macrophage colony-stimulating factor (GM-CSF) is also known as colony-stimulating factor 2 (CSF-2).

[0280] The nucleic acid sequence of the mRNA of the human granulocyte-macrophage colony-stimulating factor precursor is available under NCBI accession number NM_000758.3. The respective amino acid sequence of the GM-CSF precursor is available under NCBI accession number NP_000749.2.

[0281] The amino acid sequence of the human GM-CSF precursor is also available at UniProt accession number P04141 version 180. The amino acid sequence of the human GM-CSF precursor includes a signal peptide spanning amino acids 1 to 17 of the precursor and mature GM-CSF spanning amino acids 18 to 144 of the GM-CSF precursor.

[0282] Preferably, the granulocyte-macrophage colony-stimulating factor (GM-CSF) comprises one or at least one of the amino acid sequences of SEQ ID NOs: 19 to 21, which are also depicted in Figures 11a to 11c, respectively.

[0283] Granulocyte colony-stimulating factor (G-CSF or GCSF) is also known as colony-stimulating factor 3 (CSF3).

[0284] In humans, two distinct G-CSF polypeptides, designated isoform 1 and isoform 2, are synthesized from the same gene by differential splicing of mRNA. The two polypeptides differ only by the presence or absence of three amino acids. Expression studies indicate that both preferably have G-CSF activity.

[0285] The nucleic acid sequence of the mRNA of human G-CSF isoform 1 precursor, also called G-CSF isoform a precursor, is available under NCBI accession number NM_000759.3. The corresponding amino acid sequence is available under NCBI accession number NP_000750.1.

[0286] The nucleic acid sequence of the mRNA of human G-CSF isoform 2 precursor, also called G-CSF isoform b precursor, is available under NCBI accession number NM_172219.2. The corresponding amino acid sequence is available under NCBI accession number NP_757373.1.

[0287] The respective amino acid sequence of human G-CSF is also available in UniProt accession number P09919 version 193. Isoform 1 was chosen as the canonical UniProt sequence.

[0288] The protein sequences of each of human G-CSF precursor isoforms 1 and 2 include an N-terminal signal peptide spanning amino acid number 1 to amino acid number 29 of the respective amino acid sequences.

[0289] Erythropoietin (EPO) stimulates the production of red blood cells (erythropoiesis) in the bone marrow.

[0290] The nucleic acid sequence of the mRNA of the human erythropoietin precursor is available under NCBI accession number NM_000799.3. The amino acid sequence of each of the erythropoietin precursors is available under NCBI accession number NP_000790.2.

[0291] The amino acid sequence of the human erythropoietin precursor is also available under UniProt accession number P01588 version 183. The amino acid sequence of the human erythropoietin precursor includes a signal peptide spanning amino acids 1 to 27 of the precursor, and mature erythropoietin spanning amino acids 28 to 193 of the precursor.

[0292] Chemokines are a group of chemotactic polypeptides that direct the recruitment of various leukocytes, preferably to sites of inflammation. Based on the homology of their respective polypeptide sequences and the number of amino acids between the first two cysteines, chemokines can be subdivided into C, CC, CXC, and CX3C subgroups.

[0293] Preferably, the chemokine is selected from the group consisting of CCL1 to CCL28, CXCL1 to CXCL17, XCL1, XCL2, CX3CL1, functional analogs thereof, biosimilars thereof, and mixtures thereof.

[0294] Examples of polypeptide hormones are known to those of skill in the art and include, for example, corticotropin-releasing hormone, islet amyloid polypeptide, gastric inhibitory polypeptide, ghrelin, glucagon-like peptide-1, growth hormone-releasing hormone, appetite-regulating hormone, insulin, leptin, motilin, thyrotropin-releasing hormone, urocortin, pancreatic polypeptide, somatostatin, natriuretic peptide, growth hormone, prolactin, calcitonin, luteinizing hormone, parathyroid hormone, thyroid-stimulating hormone, vasoactive intestinal peptide, functional analogs thereof, biosimilars thereof, and mixtures thereof.

[0295] Examples of neuropeptides are known to those of skill in the art and include, for example, agouti-related protein (AgRP), gastrin-releasing peptide (GRP), calcitonin gene-related peptide (CGRP), cocaine- and amphetamine-regulated transcript peptide (CART), dynorphin, endorphin, enterostatin, galanin peptide (GAL), galanin-like peptide (GALP), hypocretin / orexin, melanin-concentrating hormone (MCH), neuromedin, neuropeptide B, neuropeptide K, neuropeptide S, neuropeptide W, neuropeptide Y, neurotensin, oxytocin, prolactin-releasing peptide, proopiomelanocortin and derived melanocortins, apolipoprotein A-IV, oxyntomodulin, gastrin-releasing peptide, glucose-dependent insulinotropic polypeptide, orphanin FQ, enkephalins, functional analogs thereof, biosimilars thereof, and mixtures thereof.

[0296] Examples of enzymes are known to those skilled in the art and include, for example, eukaryotic thrombolytic enzymes, including tissue plasminogen activator, urokinase, or other enzymes such as Factor VII-activating protease.

[0297] The antibodies described herein can be full-size antibodies or functional fragments thereof, such as Fabs, fusion proteins, or multimeric proteins.

[0298] The term "functional" as used herein refers to an antibody fragment that can still perform its intended function, i.e., antigen binding. The term antibody as used herein includes, but is not limited to, conventional antibodies, chimeric antibodies, dAbs, bispecific antibodies, trispecific antibodies, multispecific antibodies, bivalent antibodies, trivalent antibodies, multivalent antibodies, VHHs, nanobodies, Fab, Fab', F(ab')2, scFv, Fv, dAbs, Fd, diabodies, triabodies, single-chain antibodies, single-domain antibodies, and single antibody variable domains.

[0299] In this context, the term "antibody" is used to describe an immunoglobulin, whether natural or partially or fully engineered. Because antibodies can be modified in several ways, the term "antibody" covers antibody fragments, derivatives, functional equivalents, and homologs of antibodies, as well as single-chain antibodies, bifunctional antibodies, bivalent antibodies, VHHs, nanobodies, Fab, Fab', F(ab')2, scFv, Fv, dAb, Fd, diabodies, triabodies, and camelid antibodies, and encompasses any polypeptide comprising an immunoglobulin binding domain, whether natural or fully or partially engineered. Thus, chimeric molecules comprising an immunoglobulin binding domain or equivalent fused to another polypeptide are encompassed. The term also covers any polypeptide or protein having a binding domain that is or is homologous to an antibody binding domain, e.g., an antibody mimic.

[0300] Examples of antibodies are immunoglobulin isotypes and their isotype subclasses, including IgG (IgG1, IgG2a, IgG2b, IgG3, IgG4), IgA, IgD, IgM, and IgE. Therefore, those skilled in the art will understand that the present invention also relates to antibody fragments comprising an antigen-binding domain, such as VHHs, nanobodies, Fabs, scFvs, Fvs, dAbs, Fds, diabodies, and triabodies. In certain embodiments, the present invention relates to a Gram-positive bacterium or recombinant nucleic acid described herein, wherein one exogenous gene encodes the light chain (VL) of an antibody or functional fragment thereof, and another exogenous gene encodes the heavy chain (VH) of an antibody or functional fragment thereof, more preferably, the functional fragment is a Fab. In certain embodiments, the exogenous gene encoding the VL or functional fragment thereof is transcriptionally coupled to the 3' end of the exogenous gene encoding the VH or functional fragment thereof.

[0301] In preferred embodiments, the recombinant bacteria of the invention express one, two, three, four, or more heterologous factors, each of which is not naturally occurring in or expressed by the bacterium used, and which are independently heterologous polypeptides or complexes thereof, preferably heterologous polypeptides or complexes thereof.

[0302] For example, if a recombinant bacterium of the invention expresses two, three, four, or more heterologous factors, the expression of each of the heterologous factors can be controlled by an individual prokaryotic chloride-inducible promoter, or more preferably, by a single prokaryotic chloride-inducible promoter that controls expression of the heterologous factors from a single operon.

[0303] For example, each nucleic acid sequence encoding each heterologous factor can be located in a separate subpopulation of recombinant bacteria, which are combined to obtain the recombinant bacterium of the invention.

[0304] Each individual subpopulation comprising each nucleic acid sequence can be from the same or different species, for example, of bacteria.

[0305] For example, different species of recombinant bacteria expressing different heterologous factors can be used to tailor the expression, and preferably release, of each heterologous factor from the recombinant bacteria.

[0306] In a preferred embodiment of the invention, each nucleic acid sequence(s) encoding at least one heterologous factor is present in various copy numbers within the recombinant bacterium, e.g., the recombinant bacterium comprises at least one copy of each nucleic acid sequence encoding at least one heterologous factor per bacterial cell.

[0307] To enhance expression of each heterologous factor, the copy number of each nucleic acid sequence present in the recombinant bacterium, preferably in each bacterial cell, can be increased independently.

[0308] For example, if two or more heterologous factors are to be expressed by a recombinant bacterium, different ratios of copies of each nucleic acid sequence encoding the heterologous factors may be present within one bacterial cell of the recombinant bacterium.

[0309] In a preferred embodiment, nucleic acid sequences encoding at least one, preferably one, two, three, four or more heterologous factor(s) are present in one population of recombinant bacteria.

[0310] In a further preferred embodiment, each nucleic acid sequence(s) encoding at least one heterologous factor is located on at least one of the chromosome and a plasmid of the recombinant bacterium.

[0311] The location of the nucleic acid sequence on the chromosome of the recombinant bacterium or on a plasmid of the recombinant bacterium determines, among other things, the amount of protein translated by the bacterium, since expression levels from a plasmid are higher compared to expression levels from the chromosome.

[0312] In a further preferred embodiment of the invention, at least one nucleic acid sequence encoding each heterologous factor is provided on the chromosome of the bacterium and at least one nucleic acid sequence encoding another heterologous factor is provided on a plasmid of the recombinant bacterium.

[0313] In alternative embodiments of the invention, each nucleic acid sequence is provided either on an individual portion of the chromosome of the recombinant bacterium or on a different plasmid in the recombinant bacterium.

[0314] In a preferred embodiment of the present invention, all nucleic acid sequences encoding at least one, preferably one, two, three, four or more heterologous factor(s) are provided on a single recombinant nucleic acid, preferably a recombinant plasmid.

[0315] More preferably, the recombinant nucleic acid, preferably the recombinant plasmid, of the invention comprises one, two, three, four or more nucleic acid sequences encoding one, two, three, four or more factors, each of which is not naturally occurring in or expressed by the bacterium used, and which is a heterologous polypeptide or complex thereof.

[0316] In another particularly preferred embodiment of the present invention, at least one nucleic acid sequence encoding at least one, preferably one, two, three, four, or more heterologous factor(s) is / are located on a single operon that is operably linked to and controlled by a single prokaryotic chloride-inducible promoter, the activity of which is controlled by at least one prokaryotic regulator gene.

[0317] More preferably, the chloride-inducible promoter and at least one regulator gene are located on a chloride-inducible gene expression cassette, wherein the chloride-inducible promoter is located downstream from the at least one regulator gene and the chloride-inducible gene expression cassette controls the transcription of at least one nucleic acid sequence encoding at least one heterologous factor, and more preferably are located on a single operon.

[0318] An operon is a functional unit of DNA containing a cluster of genes under the control of a single promoter. The genes are transcribed together from an mRNA strand and translated together in the cytoplasm.

[0319] Preferably, each nucleic acid sequence encoding each heterologous factor present on a single operon is provided with a nucleic acid sequence encoding a ribosome binding site for translation initiation.

[0320] Preferably, the nucleic acid sequence encoding the ribosome binding site is located upstream of the start codon, toward the 5' region of the same strand that is to be transcribed. The sequence is preferably complementary to the 3' end of the 16S ribosomal RNA.

[0321] Preferably, each nucleic acid sequence encoding each heterologous factor present on a single operon is provided with a nucleic acid sequence encoding a secretory signal sequence at the 5' end of the open reading frame (ORF) of the heterologous factor, thereby producing a fusion protein comprising the secretory signal sequence and the heterologous factor.

[0322] In a preferred embodiment of the recombinant bacterium of the invention or the recombinant plasmid of the invention, the chloride-inducible promoter and / or the at least one regulator gene are each independently from the same bacterial species.

[0323] In a preferred embodiment of the present invention, the chloride-inducible promoter is PgadC from a bacterial species of the taxonomic order Lactobacillales, preferably Lactococcus lactis.

[0324] In a preferred embodiment of the present invention, the regulator gene encodes GadR from a bacterial species of the taxonomic order Lactobacillales, preferably Lactococcus lactis, and preferably encodes a polypeptide comprising the amino acid sequence available under GenBank accession number AAC46186.1.

[0325] More preferably, the expression of the regulator gene, encoding GadR from a bacterial species of the taxonomic order Lactobacillales, preferably Lactococcus lactis, is controlled by a constitutive promoter.

[0326] Preferably, the nucleic acid sequences encoding the Lactococcus lactis positive regulators GadR (gadR), GadC (gadC), and glutamic acid decarboxylase (gadB) are available under GenBank accession number AAC46187.1.

[0327] In a preferred embodiment of the present invention, the chloride-inducible gene expression cassette comprises or consists of the nucleic acid sequence of SEQ ID NO: 2, also depicted in Figure 2b, and controls the transcription of at least one nucleic acid sequence encoding at least one heterologous factor, which is further preferably located on a single operon.

[0328] Preferably, the recombinant bacterium of the present invention is obtainable by transforming a bacterium, preferably selected from the taxonomic phylum Firmicutes, the taxonomic phylum Actinobacteria, or the taxonomic phylum Proteobacteria, with at least one nucleic acid sequence operably coupled to a prokaryotic chloride-inducible promoter and encoding at least one heterologous factor, which heterologous factor is independently a heterologous polypeptide or complex thereof, and the heterologous polypeptide comprises at least one eukaryotic polypeptide, at least one fragment thereof, or a combination thereof, and at least one prokaryotic regulator gene that controls the activity of the chloride-inducible promoter.

[0329] Proteobacteria are a major phylum of Gram-negative bacteria that encompass a wide variety of known bacterial genera, such as Escherichia, Salmonella, Vibrio, Helicobacter, Yersinia, and Legionellales.

[0330] Suitable bacteria for obtaining the recombinant bacteria of the present invention are preferably non-pathogenic bacteria. Preferably, the non-pathogenic bacteria are non-invasive bacteria.

[0331] In a further preferred embodiment, the recombinant bacterium is a Gram-positive bacterium, preferably a Gram-positive non-spore-forming bacterium. Even more preferably, the bacterium is a non-colonizing bacterium that lacks the ability to colonize the human gastrointestinal tract.

[0332] In a further preferred embodiment, the recombinant bacterium comprises a Gram-positive food-grade bacterial strain, preferably a facultative anaerobic bacterial strain.

[0333] According to a preferred embodiment of the present invention, the recombinant bacteria may be from the same bacterial strain or a mixture of different bacterial strains.

[0334] In another embodiment, the bacteria is classified as "Generally Recognized as Safe" (GRAS) by the US Food and Drug Administration (FDA).

[0335] In another embodiment, the bacteria have a "Qualified Presumption of Safety" (QPS status) as defined by the European Food Safety Authority (EFSA). An introduction to the Qualified Presumption of Safety (QPS) approach for the evaluation of selected microorganisms is described in EFSA Journal, Vol. 587, 2007, pages 1-16.

[0336] In a further preferred embodiment, the bacterium is a non-pathogenic bacterium belonging to the taxonomic phylum Firmicutes or Actinobacteria. Preferably, the bacterium is a non-pathogenic bacterium from at least one genera selected from the group consisting of Bifidobacterium, Corynebacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Propionibacterium, and Streptococcus.

[0337] In another preferred embodiment, the recombinant bacterium is a lactic acid bacterium (LAB). Lactic acid bacteria are a clade of Gram-positive, acid-tolerant, generally non-spore-forming, non-respiratory bacteria that share common metabolic and physiological characteristics. These bacteria produce lactic acid as the major metabolic end product of carbohydrate degradation.

[0338] Lactic acid bacteria are known and are used, for example, in food fermentation. Furthermore, proteinaceous bacteriocins are produced by some lactic acid bacterial strains.

[0339] In a further preferred embodiment, the bacteria used to obtain the recombinant bacteria of the present invention exclude pathogenic and / or opportunistic bacteria.

[0340] In another embodiment, the bacterium is from the genus Bifidobacterium sp., including Bifidobacterium actinocoroniforme, Bifidobacterium adolescentis, Bifidobacterium asclepii, Bifidobacterium anguulatum, Bifidobacterium, Bifidobacterium animalis, e.g., Bifidobacterium animalis subsp. animalis or Bifidobacterium animalis subsp. lactis, Bifidobacterium asteroides, Bifidobacterium biavati, Bifidobacterium bifidum, Bifidobacterium Bacterium bohemicum, Bifidobacterium bombyi, Bifidobacterium baum, Bifidobacterium breve, Bifidobacterium callitrichus, Bifidobacterium catenulatum, Bifidobacterium querinum, Bifidobacterium coryneforme, Bifidobacterium culdilacticis, Bifidobacterium cuniculi, Bifidobacterium denticollens, Bifidobacterium dentium, Bifidobacterium faecalis, Bifidobacterium gallicum, Bifidobacterium galli narum, Bifidobacterium globosum, Bifidobacterium indicum, Bifidobacterium infantis, Bifidobacterium inopinatum, Bifidobacterium quasiwanohens, Bifidobacterium lactis, Bifidobacterium longum, e.g. Bifidobacterium longum subsp. infantis, Bifidobacterium longum subsp. longum, or Bifidobacterium longum subsp. suis, Bifidobacterium magnum, Bifidobacterium mericicum, Bifidobacterium minima Bifidobacterium mongoliens, Bifidobacterium moucalabens, Bifidobacterium pseudocatenulatum, Bifidobacterium pseudolongum, e.g., Bifidobacterium pseudolongum subsp. globosum or Bifidobacterium pseudolongum subsp. pseudolongum, Bifidobacterium cyclerophilum, Bifidobacterium prolum, Bifidobacterium reuteri, Bifidobacterium ruminantium, Bifidobacterium seculare, Bifidobacterium saguini,Bifidobacterium skaldovii, Bifidobacterium sterlenboschens, Bifidobacterium subtilis, Bifidobacterium stercolis, Bifidobacterium suis, Bifidobacterium thermacidophilum, such as Bifidobacterium thermacidophilum subsp. porcinum or Bifidobacterium thermacidophilum subsp. thermacidophilum, Bifidobacterium thermophilum, or Bifidobacterium turumiens, but are not limited to these.

[0341] Preferably, the bacterium is not Bifidobacterium dentium.

[0342] Preferably, the bacterium is a bacterium with "Qualified Presumption of Safety" (QPS) status of the genus Bifidobacterium sp., including, but not limited to, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium longum, Bifidobacterium breve, or Bifidobacterium bifidum.

[0343] In one embodiment, the bacterium is from the genus Corynebacterium sp., such as Corynebacterium acorens, Corynebacterium afermentans, e.g., Corynebacterium afermentans subsp. afermentans or Corynebacterium afermentans subsp. lipophilum, Corynebacterium ammoniagenes, Corynebacterium amycolatum, Corynebacterium appendices, Corynebacterium aquatimens, Corynebacterium aquile, Corynebacterium argentatense, Corynebacterium thuringiensis ... Corynebacterium atypicum, Corynebacterium auris, Corynebacterium auriscanis, Corynebacterium bettae, Corynebacterium beticola, Corynebacterium bovis, Corynebacterium callunae, Corynebacterium camporealensis, Corynebacterium canis, Corynebacterium capitovis, Corynebacterium casei, Corynebacterium caspium, Corynebacterium siconiae, Corynebacterium confusum, Corynebacterium coileae , Corynebacterium cystitidis, Corynebacterium desertii, Corynebacterium diphtheriae, Corynebacterium dosanens, Corynebacterium durum, Corynebacterium efficiens, Corynebacterium epidermidicanis, Corynebacterium equi, Corynebacterium farsenii, Corynebacterium fascians, Corynebacterium farinum, Corynebacterium frachumfaciens, e.g., Corynebacterium frachumfaciens subsp. bethae, Corynebacterium frachumfaciens subsp. frachumfaciens, Corynebacterium frachumfaciens subsp. ortii, or Corynebacterium frachumfaciens subsp. poinsettiae, Corynebacterium flavescens, Corynebacterium frankenforstens, Corynebacterium freiberkens, Corynebacterium freineii, Corynebacterium glaucum, Corynebacterium glucuronolyticum, Corynebacterium glutamicum, Corynebacterium halotolerans, Corynebacterium hansenii,Corynebacterium hoagii, Corynebacterium humileduscens, Corynebacterium ilicis, Corynebacterium immitans, Corynebacterium insidiosum, Corynebacterium iranicum, Corynebacterium jeikeium, Corynebacterium kloppenstettii, Corynebacterium kutscheri, Corynebacterium lactis, Corynebacterium lilium, Corynebacterium lipophylloflavum, Corynebacterium lichaciens, Corynebacterium rubricantis, Corynebacterium Corynebacterium McGinleyi, Corynebacterium marinum, Corynebacterium maris, Corynebacterium massiliens, Corynebacterium mastitidis, Corynebacterium mattorcotii, Corynebacterium michiganens, e.g., Corynebacterium michiganens subsp. insidiosum, Corynebacterium michiganens subsp. michiganens, Corynebacterium michiganens subsp. nebrascens, Corynebacterium michiganens subsp. sepedonicum, or Corynebacterium michiganens subsp. tessellarius, Corynebacterium michiganens Bacterium minutissimum, Corynebacterium moorparkens, Corynebacterium mucifaciens, Corynebacterium mustelae, Corynebacterium mycetoides, Corynebacterium nebulusens, Corynebacterium nigricans, Corynebacterium nulli, Corynebacterium alti, Corynebacterium paulometabolum, Corynebacterium phocae, Corynebacterium pyruvalens, Corynebacterium pilosum, Corynebacterium poinsettiae, Corynebacterium propionibacterium ncum, Corynebacterium pseudodiphteriticum, Corynebacterium pseudotuberculosis, Corynebacterium pyogenes, Corynebacterium pyruviciproducens, Corynebacterium lasaii, Corynebacterium renale, Corynebacterium resistens, Corynebacterium riegerii, Corynebacterium seminale, Corynebacterium cepedonicum, Corynebacterium simulans, Corynebacterium singulare, Corynebacterium sphenisci, Corynebacterium spheniscorum,Corynebacterium spoutii, Corynebacterium stationis, Corynebacterium striatum, Corynebacterium suicordis, Corynebacterium sundosvalens, Corynebacterium turpenotabidum, Corynebacterium testudinoris, Corynebacterium somssenii, Corynebacterium timonens, Corynebacterium tritici, Corynebacterium tuberculosis Corynebacterium lucurostealicum, Corynebacterium tuscaniens, Corynebacterium ulcerans, Corynebacterium urceribovis, Corynebacterium urealyticum, Corynebacterium ureicelerivorans, Corynebacterium uterechis, Corynebacterium variabile, Corynebacterium vitaerminis, or Corynebacterium xerosis.

[0344] Preferably, the bacterium is not Corynebacterium diphtheriae, Corynebacterium amycolatum, Corynebacterium striatum, Corynebacterium jeikeium, Corynebacterium urealyticum, Corynebacterium xerosis, Corynebacterium pseudotuberculosis, Corynebacterium tenuis, Corynebacterium striatum, or Corynebacterium minutissimum.

[0345] Preferably, the bacterium is a bacterium classified as "Generally Recognized as Safe" (GRAS) of the genus Corynebacterium, including, but not limited to, Corynebacterium ammoniagenes, Corynebacterium casei, Corynebacterium flavescens, or Corynebacterium variabile.

[0346] In another embodiment, the bacterium is from the genus Enterococcus sp., including Enterococcus alcedinis, Enterococcus aquimarinus, Enterococcus assini, Enterococcus avium, Enterococcus cacae, Enterococcus camelliae, Enterococcus canintestini, Enterococcus canis, Enterococcus caselflavus, Enterococcus cecorum, Enterococcus columbae, Enterococcus debrisei, Enterococcus diestramunae, Enterococcus S. dispar, Enterococcus durans, Enterococcus eurekensis, Enterococcus faecalis, Enterococcus faecium, Enterococcus flavescens, Enterococcus gallinarum, Enterococcus gillus, Enterococcus hemoperoxidus, Enterococcus hermaniensis, Enterococcus hirae, Enterococcus italicus, Enterococcus lactis, Enterococcus lemanii, Enterococcus malodoratus, Enterococcus moravici Enterococcus ensis, Enterococcus munchii, Enterococcus olivae, Enterococcus parens, Enterococcus phoeniculicola, Enterococcus plantarum, Enterococcus porsinus, Enterococcus pseudoavium, Enterococcus quebecensis, Enterococcus raffinosus, Enterococcus ratti, Enterococcus rivorum, Enterococcus lotai, Enterococcus saccharolyticus, e.g., Enterococcus saccharolyticus subsp. saccharolyticus Enterococcus or Enterococcus saccharolyticus subsp. taiwanensis, Enterococcus saccharominimus, Enterococcus serioricida, Enterococcus silesiacus, Enterococcus solitarius, Enterococcus sulphureus, Enterococcus thermitis, Enterococcus thailandicus, Enterococcus ureilyticus, Enterococcus biekiensis, Enterococcus virorum, or Enterococcus shanfangensis.

[0347] Preferably, the bacterium is a bacterium classified as "Generally Recognized as Safe" (GRAS) of the genus Enterococcus, including, but not limited to, Enterococcus durans, Enterococcus faecalis, or Enterococcus faecium.

[0348] In another embodiment, the bacterium is from the genus Lactobacillus sp., including Lactobacillus acetotolerans, Lactobacillus acidifarinae, Lactobacillus acidipistis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus algidus, Lactobacillus alimentarius, Lactobacillus amylolyticus, Lactobacillus aminophilus, Lactobacillus amylotrophicus, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus antrii, Lactobacillus apinorum , Lactobacillus apis, Lactobacillus apodemii, Lactobacillus aquaticus, Lactobacillus arizonensis, Lactobacillus avialis, e.g. Lactobacillus avialius subsp. alafinosus or Lactobacillus avialius subsp. avialius, Lactobacillus buckii, Lactobacillus babaricus, Lactobacillus bifermentans, Lactobacillus bovarius, Lactobacillus bombi, Lactobacillus brantae, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus brueckii gallicus, Lactobacillus cacaonum, Lactobacillus camelliae, Lactobacillus capillatus, Lactobacillus carnis, Lactobacillus casei, e.g., Lactobacillus casei subsp. alactosus, Lactobacillus casei subsp. casei, Lactobacillus casei subsp. pseudoplantarum, Lactobacillus casei subsp. rhamnosus, or Lactobacillus casei subsp. tolerans, Lactobacillus catenaformis, Lactobacillus cellobiosus, Lactobacillus seti, Lactobacillus coreohominis, Lactobacillus corinoides, Lactobacillus compostii, Lactobacillus concavus, Lactobacillus confusus, Lactobacillus coliniformis, e.g., Lactobacillus coliniformis subsp. coliniformis or Lactobacillus coliniformis subsp. torkenes, Lactobacillus crispatus, Lactobacillus crustrum, Lactobacillus curiae, Lactobacillus culbatus, e.g., Lactobacillus culbatus subsp. culbatus or Lactobacillus culbatus subsp. melibosus, Lactobacillus cyplicasei,Lactobacillus delbreckii, e.g., Lactobacillus delbreckii subsp. bulgaricus, Lactobacillus delbreckii subsp. delbreckii, Lactobacillus delbreckii subsp. indicus, Lactobacillus delbreckii subsp. jacobsenii, Lactobacillus delbreckii subsp. lactis, or Lactobacillus delbreckii subsp. sunckii, Lactobacillus dextrinicus, Lactobacillus diolivorans, Lactobacillus divergens, Lactobacillus durianis, Lactobacillus excavatum Lactobacillus equicursolis, Lactobacillus equigenerosi, Lactobacillus fabifermentans, Lactobacillus faeces, Lactobacillus farciminis, Lactobacillus faraginis, Lactobacillus ferrintoschensis, Lactobacillus fermentum, Lactobacillus floricola, Lactobacillus florum, Lactobacillus fornicalis, Lactobacillus fructivorans, Lactobacillus fructosus, Lactobacillus frumentii, Lactobacillus fuchue Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus gastricus, Lactobacillus ganensis, Lactobacillus gigeriorum, Lactobacillus graminis, Lactobacillus halotolerans, Lactobacillus hamesii, Lactobacillus hamsteri, Lactobacillus harvinensis, Lactobacillus hayakitensis, Lactobacillus heilongjungensis, Lactobacillus helsingboriensis, Lactobacillus Lactobacillus helveticus, Lactobacillus heterohyotii, Lactobacillus hilgardii, Lactobacillus hokkaidonensis, Lactobacillus hominis, Lactobacillus homohyotii, Lactobacillus hordei, Lactobacillus inellus, Lactobacillus inglebiei, Lactobacillus intestinalis, Lactobacillus iwatensis, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus calizensis, Lactobacillus candleri, Lactobacillus kefiranofaciens,For example, Lactobacillus kefiranofaciens subsp. kefiranofaciens or Lactobacillus kefiranofaciens subsp. kefirgranum, Lactobacillus kefiri, Lactobacillus kefirgranum, Lactobacillus kimbradiii, Lactobacillus kimchicus, Lactobacillus kimchiensis, Lactobacillus kimchii, Lactobacillus xonensis, Lactobacillus kitasatonis, Lactobacillus coriensis, Lactobacillus claveriensis, Lactobacillus kunkeei, Lactobacillus lactis, Lactobacillus lakemannii, Lactobacillus lindneri, Lactobacillus malefermentans, Lactobacillus mali, Lactobacillus malthalomics, Lactobacillus manifotivorans, Lactobacillus mellifera, Lactobacillus melis, Lactobacillus meliventris, Lactobacillus mindensis, Lactobacillus minnow, Lactobacillus minutus, Lactobacillus mucosae, Lactobacillus murinus, Lactobacillus nagelii, Lactobacillus namurensis, Lactobacillus nantensis, La Lactobacillus nasuensis, Lactobacillus nenjangensis, Lactobacillus nodensis, Lactobacillus odoratitophi, Lactobacillus oeni, Lactobacillus oligofermentans, Lactobacillus oris, Lactobacillus oryzae, Lactobacillus otakiensis, Lactobacillus ozensis, Lactobacillus panis, Lactobacillus panseri, Lactobacillus parabrevis, Lactobacillus parabufneri, Lactobacillus paracasei, e.g. Lactobacillus paracasei subsp. paracasei or Lactobacillus paracasei subsp. tolerans, Lactobacillus paracorinoides, Lactobacillus parafaraginis, Lactobacillus parakeefili, Lactobacillus paralimentarius, Lactobacillus paraplantarum, Lactobacillus pasteurii, Lactobacillus paucivorans, Lactobacillus pentosus, Lactobacillus perorens, Lactobacillus pisicola, Lactobacillus plantarum, e.g. Lactobacillus plantarum subsp. argentatensis or Lactobacillus plantarum subsp. plantarum,Lactobacillus pobzii, Lactobacillus pontis, Lactobacillus porsinae, Lactobacillus psittaci, Lactobacillus rapi, Lactobacillus lennini, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus limae, Lactobacillus rodentium, Lactobacillus logosae, Lactobacillus rossiae, Lactobacillus ruminis, Lactobacillus saeliimnerii, Lactobacillus sakei, e.g. Lactobacillus sakei subsp. carnosus or Lactobacillus sakei subsp. sakei, Lactobacillus sakei Lactobacillus salivarius, e.g. Lactobacillus salivarius subsp. salicinius or Lactobacillus salivarius subsp. salivarius, Lactobacillus sanfrancisensis, Lactobacillus sanibili, Lactobacillus satsmensis, Lactobacillus secaliphilus, Lactobacillus selangorensis, Lactobacillus senioris, Lactobacillus senmaizukai, Lactobacillus scharpieri, Lactobacillus chenchenensis, Lactobacillus sicerae, Lactobacillus siragiei, Lactobacillus siliginis, Lactobacillus similesu, Lactobacillus sobrius, Lactobacillus songhuajiangensis, Lactobacillus spicherii, Lactobacillus ssicola, Lactobacillus suevicus, Lactobacillus sunqii, Lactobacillus santorieus, Lactobacillus taiwanensis, Lactobacillus thailandensis, Lactobacillus thermotolerans, Lactobacillus trichodes, Lactobacillus toucettii, Lactobacillus liuri, Lactobacillus altunensis, Lactobacillus uvalum, Lactobacillus Lactobacillus vacciniae, Lactobacillus vaginalis, Lactobacillus barthmordensis, Lactobacillus vini, Lactobacillus viridescens, Lactobacillus vitulinus, Lactobacillus shanfangensis, Lactobacillus xylosus, Lactobacillus yamanasiensis, such as Lactobacillus yamanasiensis subsp. mali, or Lactobacillus yamanasiensis subsp. yamanasiensis, Lactobacillus yongguinensis, Lactobacillus jiae, or Lactobacillus jimae,

[0349] Preferably, the bacterium is a bacterium classified as "Generally Recognized as Safe" (GRAS) of the genus Lactobacillus sp., such as Lactobacillus acidophilus strain NP28, Lactobacillus acidophilus strain NP51, Lactobacillus subsp. lactis strain NP7, Lactobacillus reuteri strain NCIMB30242, Lactobacillus casei strain Shirota, Lactobacillus reuteri strain DSM17938, Lactobacillus reuteri strain NCIMB30242, Lactobacillus acidophilus strain NCFM, Lactobacillus rhamnosus strain HN001, Lactobacillus Lactobacillus rhamnosus strain HN001, Lactobacillus reuteri strain DSM17938, Lactobacillus casei subsp. rhamnosus strain GG, Lactobacillus acidophilus, Lactobacillus lactis, Lactobacillus acetotolerans, Lactobacillus acidifarinae, Lactobacillus acidipistis, Lactobacillus acidophilus, Lactobacillus alimentarius, Lactobacillus amylolyticus, Lactobacillus amylovorus, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus cacaonum, Lactobacillus Casei, Lactobacillus casei, Lactobacillus corinoides, Lactobacillus compostii, Lactobacillus coriniformis subsp. coriniformis, Lactobacillus crispatus, Lactobacillus crustrum, Lactobacillus culbatus subsp. culbatus, Lactobacillus delbreckii subsp. bulgaricus, Lactobacillus delbreckii subsp. delbreckii, Lactobacillus delbreckii subsp. lactis, Lactobacillus dextrinicus, Lactobacillus diolivorans, Lactobacillus fabifermentans, Lactobacillus Bacillus farciminis, Lactobacillus fermentum, Lactobacillus fructivorans, Lactobacillus frumentii, Lactobacillus gasseri, Lactobacillus ganensis, Lactobacillus hamensii, Lactobacillus harvinensis, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus homochii, Lactobacillus hordei, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus kefiri, Lactobacillus kefiranofaciens subsp. kefiranofaciens,Lactobacillus kefiranofadens subsp. kefirgranum, Lactobacillus kimchii, Lactobacillus xonensis, Lactobacillus meiru, Lactobacillus manifotivorans, Lactobacillus mindensis, Lactobacillus mucosae, Lactobacillus naegelii, Lactobacillus namurensis, Lactobacillus nantensis, Lactobacillus nodensis, Lactobacillus oeni, Lactobacillus otakiensis, Lactobacillus panis, Lactobacillus parabrevis, Lactobacillus parabufneri, Lactobacillus paracasei subsp. paracasei, Lactobacillus parakefiri, Lactobacillus paralimentarius, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus perorens, Lactobacillus plantarum plantarum, Lactobacillus pobzii, Lactobacillus pontis, Lactobacillus rapi, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus rossiae, Lactobacillus sakei subsp. carnosus, Lactobacillus sakei subsp. sakei, Lactobacillus salivarius subsp. salivarius, Lactobacillus sanfrancisensis, Lactobacillus satsmensis, Lactobacillus secariphilus, Lactobacillus senmaizukai, Lactobacillus siliginis, Lactobacillus spicherii, Lactobacillus suevicus, Lactobacillus sunkii, Lactobacillus tucettii, Lactobacillus vaccinostercus, Lactobacillus barthmordensis, or Lactobacillus yamanashiensis.

[0350] Preferably, the bacterium is a bacterium with "Qualified Presumption of Safety" (QPS) status of the genus Lactobacillus sp., such as Lactobacillus acidophilus, Lactobacillus amylolyticus, Lactobacillus amylovorus, Lactobacillus alimentarius, Lactobacillus avialis, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus culbatus, Lactobacillus delbrueckii, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus johnsonii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus mucosae, Lactobacillus panis, Lactobacillus paracasei, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus sanfrancisensis, or Lactobacillus jiae.

[0351] In another embodiment, the bacterium is from the genus Lactococcus sp., including, but not limited to, Lactococcus chungangensis, Lactococcus formocensis, Lactococcus fuziensis, Lactococcus garvieae, Lactococcus lactis, such as Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. hordoniae, Lactococcus lactis subsp. lactis, or Lactococcus lactis subsp. torqueae, Lactococcus piscium, Lactococcus plantarum, Lactococcus raffinolactis, or Lactococcus taiwanensis.

[0352] Preferably, the bacterium is a bacterium classified as "Generally Recognized as Safe" (GRAS) of the genus Lactococcus, including, but not limited to, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, and Lactococcus raffinolactis.

[0353] Preferably, the bacterium is Lactococcus lactis, preferably Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis biovariant diacetylactis, or Lactococcus lactis subsp. cremoris, more preferably Lactococcus lactis subsp. cremoris.

[0354] In another embodiment, the bacterium is from the genus Leuconostoc sp., including Leuconostoc ameribiosum, Leuconostoc argentinum, Leuconostoc carnosum, Leuconostoc citreum, Leuconostoc cremoris, Leuconostoc dextranicum, Leuconostoc durionis, Leuconostoc fallax, Leuconostoc ficulneum, Leuconostoc fructosum, Leuconostoc gashicomitatum, Leuconostoc gerridum, such as Leuconostoc gerridum subsp. enigmaticum, Leuconostoc gerridum subsp. gashicomitatum, or Leuconostoc gerridum subsp. gerridum, ... ameribiosum, Leuconostoc ameribiosum, Leuconostoc ameribiosum, Leuconostoc ameribiosum, Leuconostoc ameribiosum, Leuconostoc ameribiosum, Leuconostoc ameribiosum, Leuconostoc Nostoc holzapfelii, Leuconostoc inhae, Leuconostoc kimchii, Leuconostoc lactis, Leuconostoc mesenteroides, such as Leuconostoc mesenteroides subsp. cremoris, Leuconostoc mesenteroides subsp. dextranicum, Leuconostoc mesenteroides subsp. mesenteroides, or Leuconostoc mesenteroides subsp. suionicum, Leuconostoc miyukimchii, Leuconostoc oeni, Leuconostoc paramesenteroides, Leuconostoc pseudoficulneum, or Leuconostoc pseudomesenteroides, but are not limited to these.

[0355] Preferably, the bacterium is a bacterium classified as "Generally Recognized as Safe" (GRAS) of the genus Leuconostoc sp., including, but not limited to, Leuconostoc carnosum, Leuconostoc citreum, Leuconostoc fallax, Leuconostoc holzapfelii, Leuconostoc inhaeae, Leuconostoc kimchii, Leuconostoc lactis, Leuconostoc mesenteroides subsp. cremoris, Leuconostoc mesenteroides subsp. dextranicum, Leuconostoc mesenteroides subsp. mesenteroides, Leuconostoc palmae, or Leuconostoc pseudomesenteroides.

[0356] Preferably, the bacterium is a bacterium with "Qualified Presumption of Safety" (QPS) status of the genus Leuconostoc sp., including, but not limited to, Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides subsp. cremoris, Leuconostoc mesenteroides subsp. dextranicum, or Leuconostoc mesenteroides subsp. mesenteroides.

[0357] In another embodiment, the bacterium is from the genus Pediococcus sp., including, but not limited to, Pediococcus acidilactici, Pediococcus argentinicus, Pediococcus coelicolor, Pediococcus claussenii, Pediococcus damnosus, Pediococcus dextrinicus, Pediococcus ethanolidurans, Pediococcus halophilus, Pediococcus inopinatus, Pediococcus rollii, Pediococcus parvulus, Pediococcus pentosaceus, Pediococcus siamensis, Pediococcus stillesii, or Pediococcus urinaequi.

[0358] Preferably, the bacterium is a bacterium with "Qualified Presumption of Safety" (QPS) status of the genus Pediococcus sp., including, but not limited to, Pediococcus acidilactici, Pediococcus dextrinicus, or Pediococcus pentosaceus.

[0359] In another embodiment, the bacterium is from the genus Propionibacterium sp., such as Propionibacterium acidifaciens, Propionibacterium acidipropionici, Propionibacterium acnes, Propionibacterium australiens, Propionibacterium avidum, Propionibacterium cyclohexanicum, Propionibacterium damnosum, Propionibacterium früdenreichii, e.g., Propionibacterium früdenreichii. früdenreichii or Propionibacterium früdenreichii subsp. schermanii, Propionibacterium granulosum, Propionibacterium innocuum, Propionibacterium jensenii, Propionibacterium lymphophilum, Propionibacterium microaerophilum, Propionibacterium oliviae, Propionibacterium propionicum, or Propionibacterium soenii.

[0360] According to one embodiment, the bacterium is not Propionibacterium acnes.

[0361] More preferably, the bacterium is a bacterium classified as "Generally Recognized as Safe" (GRAS) of the genus Propionibacterium sp., including, but not limited to, Propionibacterium acidipropionici, Propionibacterium früdenreichii subsp. früdenreichii, Propionibacterium früdenreichii subsp. schermannii, Propionibacterium jensenii, or Propionibacterium soenii.

[0362] More preferably, the bacterium is Propionibacterium früdenreichii, more preferably Propionibacterium früdenreichii subsp. früdenreichii or Propionibacterium früdenreichii subsp. schermannii.

[0363] In another embodiment, the bacterium is from the genus Streptococcus sp., including Streptococcus acidominimus, Streptococcus ajacens, Streptococcus agalactiae, Streptococcus alactolyticus, Streptococcus anginosus, Streptococcus australis, Streptococcus bovis, Streptococcus cavallii, Streptococcus canis, Streptococcus caprinus, Streptococcus castreus, Streptococcus cecorum, and Streptococcus constellatus. , for example Streptococcus constellatus subsp. constellatus, Streptococcus constellatus subsp. faringes, or Streptococcus constellatus subsp. vibogensis, Streptococcus cremoris, Streptococcus criseti, Streptococcus cristatus, Streptococcus cuniculi, Streptococcus danieriae, Streptococcus diffectivus, Streptococcus dentapli, Streptococcus dentilsetti, Streptococcus dentassini, Streptococcus dentisani, Streptococcus debrisei, Streptococcus didelphis, Streptococcus difficilis, Streptococcus downei, Streptococcus durans, Streptococcus dysgalactiae, e.g. Streptococcus dysgalactiae subsp. dysgalactiae or Streptococcus dysgalactiae subsp. equisimilis, Streptococcus entericus, Streptococcus equi, e.g. Streptococcus equi subsp. equi, Streptococcus equi subsp. luminatorum, or Streptococcus equi subsp. zooepidemicus, Streptococcus equinus, Streptococcus faecalis, Streptococcus faecium, Streptococcus fels, Streptococcus gallinaceus, Streptococcus gallinarum, Streptococcus gallolyticus, e.g. Streptococcus gallolyticus subsp. gallolyticus, Streptococcus gallolyticus subsp. macedonicus, or Streptococcus gallolyticus subsp. pasteurianus, Streptococcus garvieae,Streptococcus gordonii, Streptococcus haricoelii, Streptococcus hansenii, Streptococcus henryi, Streptococcus hongkongensis, Streptococcus hyointestinalis, Streptococcus hyovaginalis, Streptococcus ictaluri, Streptococcus infantarius, e.g. Streptococcus infantarius subsp. coli or Streptococcus infantarius subsp. infantarius, Streptococcus infantis, Streptococcus Streptococcus iniae, Streptococcus intermedius, Streptococcus intestinalis, Streptococcus lactarius, Streptococcus lactis, e.g., Streptococcus lactis subsp. cremoris, Streptococcus lactis subsp. diacetylactis, or Streptococcus lactis subsp. lactis, Streptococcus loxodontisalivarius, Streptococcus lutetiensis, Streptococcus macacae, Streptococcus macedonicus, Streptococcus marimammalium, Streptococcus lactis ... Streptococcus massiliensis, Streptococcus mellionis, Streptococcus minnows, Streptococcus mitis, Streptococcus morbilorum, Streptococcus morocensis, Streptococcus mutans, Streptococcus oligofermentans, Streptococcus oralis, Streptococcus orysassini, Streptococcus orysui, Streptococcus ovis, Streptococcus parasanguinis, Streptococcus parauberis, Streptococcus parvulus, Strep Tococcus pasteurianus, Streptococcus peroris, Streptococcus phocae, e.g., Streptococcus phocae subsp. phocae or Streptococcus phocae subsp. salmonis, Streptococcus plantarum, Streptococcus pleomorphus, Streptococcus pullanimarium, Streptococcus purrextrum, Streptococcus pneumoniae, Streptococcus porci, Streptococcus porcinus, Streptococcus porcorum, Streptococcus pseudopneumoniae,Streptococcus pseudoporsinus, Streptococcus pyogenes, Streptococcus raffinolactis, Streptococcus ratti, Streptococcus lifensis, Streptococcus rubneri, Streptococcus rupicaprae, Streptococcus saccharolyticus, Streptococcus salivarius, e.g., Streptococcus salivarius subsp. salivarius or Streptococcus salivarius subsp. thermophilus, Streptococcus salivillozodontae, Streptococcus Examples of suitable strains of Streptococcus include, but are not limited to, Streptococcus sanguinis, Streptococcus shiroi, Streptococcus sinensis, Streptococcus sobrinus, Streptococcus suis, Streptococcus thermophilus, Streptococcus thoraltensis, Streptococcus tigrinus, Streptococcus trogloditae, Streptococcus uberis, Streptococcus urinalis, Streptococcus ursolis, Streptococcus bestibularis, or Streptococcus wyus.

[0364] Preferably, the bacterium is classified as "Generally Recognized as Safe" (GRAS) of the genus Streptococcus sp., including, but not limited to, Streptococcus thermophilus strain Th4, Streptococcus gallolyticus subsp. macedonicus, Streptococcus salivarius subsp. salivarius, or Streptococcus salivarius subsp. thermophilus.

[0365] Preferably, the bacterium is a bacterium with "Qualified Presumption of Safety" (QPS) status of the genus Streptococcus sp., including, but not limited to, Streptococcus thermophilus.

[0366] In preferred embodiments, the bacteria do not produce endotoxins or other potentially toxic substances, making them safe to use and not harmful to the subject after application.

[0367] Preferably, the bacteria do not produce spores. Bacterial spores are not part of the reproductive cycle, but are resistance structures used for survival under adverse conditions. Because the recombinant bacteria preferably do not produce spores, the recombinant bacteria cannot survive, for example, without nutrients or when an auxotrophic factor is missing.

[0368] Preferably, the bacteria do not produce inclusion bodies, which often contain overexpressed proteins and whose aggregation in inclusion bodies can be irreversible.

[0369] The bacteria used to obtain the recombinant bacteria of the present invention preferably do not produce inclusion bodies, so that the amount of the at least one heterologous factor after transcription and preferably translation of the respective nucleic acid sequence is not reduced by intracellular accumulation in inclusion bodies.

[0370] More preferably, the bacterium also does not produce extracellular proteinases, which are secreted by bacteria and can break down extracellular structures such as proteins to generate nutrients such as carbon, nitrogen, or sulfur. Extracellular proteinases can also act as exotoxins and may be examples of virulence factors in bacterial pathogenesis.

[0371] Due to the absence of extracellular proteinases, the safety of the bacteria after application to a subject is preferably increased. Furthermore, the bacteria preferably does not degrade the at least one heterologous factor after release from the recombinant bacteria.

[0372] In a further preferred embodiment, the recombinant bacterium is a lactic acid bacterium, preferably a Lactobacillus or Lactococcus species. In a further preferred embodiment, the Lactococcus species is Lactococcus lactis subsp. cremoris.

[0373] Lactic acid bacteria also release lactic acid as a major metabolic end product of carbohydrate fermentation. Lactic acid is also known to stimulate endothelial growth and proliferation. Furthermore, lactic acid has antibacterial effects, which reduce the probability of bacterial infection at the site of a skin disorder.

[0374] Techniques for transforming the above-mentioned bacteria are known to those skilled in the art and are described, for example, in Green and Sambrook (2012): "Molecular cloning: a laboratory manual", fourth edition, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY, US).

[0375] After transformation, the recombinant bacterium comprises at least one nucleic acid sequence operably coupled to a prokaryotic chloride-inducible promoter and encoding at least one heterologous factor, which is independently a heterologous polypeptide or complex thereof, and at least one prokaryotic regulator gene that controls the activity of the chloride-inducible promoter, wherein the heterologous polypeptide comprises at least one eukaryotic polypeptide, at least one fragment thereof, or a combination thereof.

[0376] More preferably, the recombinant bacterium contains, after transformation, at least one copy of a recombinant plasmid of the invention.

[0377] Suitable strains for obtaining recombinant bacteria of the invention are commercially available, for example, from MoBiTec GmbH (Goettingen, Germany) or NIZO food research BV (Ede, NL). A suitable strain is, for example, Lactococcus lactis strain NZ1330, which preferably includes a plasmid selection system that is not based on antibiotic resistance.

[0378] In a further preferred embodiment, the nucleic acid sequence encoding each heterologous factor is modified to increase expression, secretion, and / or stability of the encoded at least one heterologous factor.

[0379] Suitable modifications are known to those skilled in the art and include, for example, adapting the codon usage of at least one nucleic acid sequence to the bacterium used.

[0380] For example, at least one nucleic acid sequence can be designed to fit the codon usage pattern of the bacterium being used. Furthermore, in addition to general codon optimization, a specific codon table, such as the codon table of a highly expressed ribosomal protein gene of the respective bacterium, can be used to further increase translation of the encoded at least one heterologous factor.

[0381] Suitable modifications also include, for example, the incorporation of a nucleic acid sequence encoding a secretory signal sequence at the 5' end of the open reading frame (ORF) of the heterologous factor to create a fusion protein containing the secretory signal sequence and the heterologous factor, with the secretory signal sequence preferably located N-terminally of each heterologous factor.

[0382] The secretory signal sequence preferably directs the newly synthesized protein to the plasma membrane. The secretory signal sequence, preferably a signal peptide, is preferably terminated by a stretch of amino acids that is recognized and cleaved by a signal peptidase to produce a free secretory signal sequence, preferably a signal peptide, and the mature heterologous factor that is secreted extracellularly.

[0383] Suitable secretory signal sequences include, for example, signal peptides.

[0384] Preferably, the secretory signal sequence is the native signal peptide of the respective factor, which is preferably encoded by the nucleic acid sequence that is the precursor of the respective heterologous factor.

[0385] In a further preferred embodiment, the secretory signal sequence is a homologous secretory sequence from a recombinant bacterium, preferably the secretory signal sequence is the unknown secreted protein 45 (Usp45) signal sequence of Lactococcus sp. The Usp45 secretory signal has very high secretion efficiency. Other secretory signals are known to those skilled in the art, including, for example, PrtP, SlpA, SP310, SPEXP4, and AL9 of Lactococcus sp.

[0386] The amino acid sequence of the Usp45 protein of Lactococcus lactis subsp. cremoris MG1363 is available, for example, under GenBank accession number CAL99070.1. The signal sequence spans amino acids 1 to 27 of the sequence deposited under GenBank accession number CAL99070.1.

[0387] For example, the native signal peptide of a heterologous factor can be replaced by a homologous secretory signal sequence from the recombinant bacterium used to express at least one heterologous factor.

[0388] A secretory signal sequence preferably improves the secretion efficiency of each heterologous factor. For example, in Lactococcus lactis, most proteins are secreted via the Sec pathway. Proteins are synthesized as precursors containing the mature part of the protein along with an N-terminal signal peptide. The signal peptide targets the protein to the cytoplasmic membrane and aids in protein secretion. After cleavage of the signal peptide, the mature protein is released outside the cell.

[0389] More preferably, the secretory signal comprises the amino acid sequence of SEQ ID NO: 3, which is also depicted in FIG.

[0390] In a further preferred embodiment, at least one heterologous factor, preferably one, two, three, four, or more heterologous factor(s), is expressed as a propeptide with or without a secretion-enhancing factor, which can be cleaved by a protease to release the mature form of the at least one heterologous factor, preferably one, two, three, four, or more heterologous factor(s).

[0391] Preferably, at least one heterologous factor, preferably 1, 2, 3, 4 or more heterologous factor(s) is releasable from the recombinant bacterium.

[0392] For example, in Gram-positive bacteria, such as lactic acid bacteria, proteins to be secreted from the bacteria are preferably synthesized as precursors containing an N-terminal signal peptide and the mature portion of the protein. The precursor is recognized by the bacterial secretory machinery and translocated across the cytoplasmic membrane. The signal peptide is then cleaved and degraded, and the mature protein is released from the bacteria, for example, into the culture supernatant or into areas of inflammatory skin lesions.

[0393] For example, Lactococcus lactis can secrete proteins ranging in molecular mass from low, e.g., less than 10 kDa, to high, e.g., greater than 160 kDa, via a Sec-dependent pathway.

[0394] Alternatively, at least one heterologous factor, preferably 1, 2, 3, 4 or more heterologous factor(s) is / are released from the recombinant bacterium through a leaky cytoplasmic membrane.

[0395] For example, the cytoplasmic membrane of recombinant bacteria becomes leaky due to impaired synthesis of cell wall components.

[0396] Preferably, the recombinant bacterium contains an inactivated alanine racemase (alr) gene. In the absence of D-alanine, the recombinant bacterium cannot maintain the integrity of its cytoplasmic membrane, after which at least one heterologous factor, preferably one, two, three, four, or more heterologous factor(s), is / are released from the recombinant bacterium.

[0397] In a further preferred embodiment, the recombinant bacterium of the invention comprises at least one inactivated gene encoding an essential protein required for viability of the bacterium.

[0398] In a preferred embodiment, the essential protein required for viability of the recombinant bacterium is a protein, more preferably an enzyme, required for the synthesis of an organic compound required for viability of the recombinant bacterium.

[0399] After inactivation of the essential proteins, preferably enzymes, each organic compound is an auxotrophic factor that is required for viability of the recombinant bacterium and must be replenished to sustain viability of the recombinant bacterium.

[0400] Preferably, the auxotrophic factor is a vitamin, an amino acid, a nucleic acid, and / or a fatty acid.

[0401] For example, amino acids and nucleotides are biologically important organic compounds that are precursors of proteins and nucleic acids, respectively.

[0402] Preferably, genes encoding essential proteins required for bacterial viability are provided on recombinant nucleic acids and / or recombinant plasmids according to the invention.

[0403] In a further preferred embodiment, the at least one gene required for viability of the recombinant bacterium is selected from the group consisting of alanine racemase (alr), thymidylate synthase (thyA), asparagine synthase (asnH), CTP synthase (pyrG), tryptophan synthase (trbBA), and combinations thereof.

[0404] For example, alanine racemase is an enzyme that catalyzes the racemization of L-alanine to L- and D-alanine. The D-alanine produced by alanine racemase is used in the synthesis of peptidoglycan, which is found in the cell walls of all bacteria.

[0405] It is known to those skilled in the art that inactivating the bacterial alanine racemase (alr) gene results in the bacteria needing to use an exogenous source of D-alanine to maintain cell wall integrity.

[0406] For example, it is known to those skilled in the art that Lactococcus lactis and Lactobacillus plantarum contain a single alr gene, and inactivation of the gene affects the incorporation of D-alanine into the cell wall.

[0407] For example, Lactococcus lactis bacteria with an inactivated alanine racemase (alr) gene are dependent on an exogenous supply of D-alanine to synthesize peptidoglycan and to incorporate D-alanine into lipoteichoic acid (LTA). These bacteria rapidly lyse when D-alanine is deprived, e.g., during mid-exponential growth.

[0408] Alanine racemase-deficient strains of lactic acid bacteria can be generated, for example, by methods known in the art.

[0409] Thymidylate synthase is an enzyme that catalyzes the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidylate monophosphate (dTMP). dTPM is one of the three nucleotides that form thymine (dTMP, dTDP, and dTTP). Thymidine is a nucleotide in DNA.

[0410] It is known to those skilled in the art that thymidylate synthase plays a crucial role in DNA biosynthesis. Furthermore, bacterial growth requires the synthesis of DNA.

[0411] Inactivation of the bacterial thymidylate synthase (thyA) gene results in the bacteria needing to use an exogenous source of thymidine to maintain DNA integrity.

[0412] Asparagine synthetase is an enzyme that produces the amino acid asparagine from aspartic acid. Inactivation of the asparagine synthetase (asnH) gene results in the inability to synthesize the respective amino acid, making it an auxotrophic factor.

[0413] CTP synthase is an enzyme involved in pyrimidine synthesis. CTP synthase interconverts uridine-5'-triphosphate (UTP) and cytidine-5'-triphosphate (CTP). Inactivation of the CTP synthase (pyrG) gene prevents bacteria from synthesizing cytosine nucleotides from both de novo synthesis and the uridine cell wall pathway.

[0414] CTP becomes an auxotrophic factor that must be replenished for RNA and DNA synthesis.

[0415] Tryptophan synthase is the enzyme that catalyzes the last two steps in the biosynthesis of the amino acid tryptophan.

[0416] Inactivation of the tryptophan synthase (trpBA) gene renders the bacterium unable to synthesize the respective amino acid, which becomes an auxotroph.

[0417] Methods for inactivating genes necessary for bacterial viability are known to those of skill in the art and include gene deletion, gene mutation, RNA interference (RNAi)-mediated gene silencing, translational inhibition of the gene, or a combination thereof.

[0418] In a further preferred embodiment, each auxotrophic factor is provided together with the recombinant bacterium.

[0419] In a further preferred embodiment of the present invention, the inactivated genes necessary for viability of the recombinant bacterium are used for environmental containment.

[0420] After application of the recombinant bacterium containing at least one inactivated gene necessary for bacterial viability, each auxotrophic factor must be provided exogenously, e.g., with a reconstituted medium, an applied medium, or a growth medium.

[0421] In the case where the recombinant bacteria are released into the environment, they will lack the auxotrophic factor and preferably die.

[0422] Furthermore, the application of exogenously supplied auxotrophic factors allows for the control of the biosynthesis and release of the respective heterologous factor(s).

[0423] More preferably, genes encoding essential proteins required for bacterial viability, preferably selected from the group consisting of alanine racemase (alr), thymidylate synthase (thyA), asparagine synthase (asnH), CTP synthase (pyrG), tryptophan synthase (trbBA), and combinations thereof, are provided on recombinant nucleic acids and / or recombinant plasmids according to the invention.

[0424] Thereby, it is possible to provide a highly stable system for maintaining the recombinant nucleic acid and / or recombinant plasmid according to the invention in the recombinant bacterium even during in vivo growth, since selective pressure for maintaining the recombinant nucleic acid and / or recombinant plasmid according to the invention can be achieved, preferably in the absence of exogenously supplied auxotrophic factors.

[0425] The expression of genes encoding essential proteins required for bacterial viability, preferably selected from the group consisting of alanine racemase (alr), thymidylate synthase (thyA), asparagine synthase (asnH), CTP synthase (pyrG), tryptophan synthase (trbBA), and combinations thereof, on a recombinant nucleic acid and / or recombinant plasmid according to the invention not only allows complementation of auxotrophy, but can also be used as a selection marker for the presence of a recombinant nucleic acid and / or recombinant plasmid according to the invention in the respective bacterium.

[0426] According to a preferred embodiment, the recombinant bacteria is to be used in medicine, more preferably for use in the treatment of chronic inflammatory wounds or degenerative conditions, or for use in the treatment of tumors, preferably malignant tumors.

[0427] During administration of the recombinant bacteria of the present invention to a subject, at least one heterologous factor, preferably one, two, three, four, or more heterologous factor(s), that is released may undergo degradation, e.g., by proteinase cleavage, which may lead to loss of biological activity of the heterologous factor(s).

[0428] The degradation or depletion of the heterologous factor(s) can be compensated for by the sustained release of at least one heterologous factor, preferably one, two, three, four, or more heterologous factor(s), from the recombinant bacteria of the invention in the presence of chloride ions.

[0429] Preferably, the recombinant bacterium expresses at least one heterologous factor, preferably 1, 2, 3, 4 or more heterologous factor(s) in a constant manner. Preferably, the recombinant bacterium releases the heterologous factor(s) in a constant manner.

[0430] Pharmaceutical compositions of the present invention comprise a recombinant bacterium according to the present invention and at least one pharmaceutically acceptable excipient.

[0431] In a preferred embodiment, the recombinant bacteria of the present invention are provided in a pharmaceutical composition for medical use, more preferably for use in the treatment of chronic inflammatory wounds or degenerative conditions, or for use in the treatment of tumors, preferably malignant tumors.

[0432] Excipients are substances formulated with the active ingredient of a drug and are included for purposes of long-term stabilization, bulking up a small amount of a potent active ingredient in a solid formulation (hence often referred to as a "bulking agent," "filler," or "diluent"), or imparting a therapeutic enhancement to the active ingredient in the final dosage form, such as aiding drug absorption, reducing viscosity, or enhancing solubility.

[0433] The term "excipient" preferably also includes at least one diluent.

[0434] Excipients may also be useful in the manufacturing process, for example, to aid in in vitro stability, e.g., to prevent denaturation or aggregation during the expected shelf life, as well as to aid handling of the active agent by aiding powder flow or non-adherence properties, and / or to contribute to bacterial cell viability of recombinant bacteria during freezing and thawing.

[0435] Preferably, the pharmaceutical composition comprises at least one pharmaceutically acceptable excipient, more preferably suitable for the intended route of administration.

[0436] Preferably, the pharmaceutically acceptable excipient is known to those skilled in the art and comprises at least one polymer carrier, preferably selected from the group consisting of polysaccharides, polyesters, polymethacrylamides, and mixtures thereof. For example, the pharmaceutically acceptable excipient is a hydrogel, which, for example, additionally provides an optimal moist environment for promoting wound healing. Furthermore, after application, the pharmaceutically acceptable excipient preferably supports the adhesion of the recombinant bacteria to the site of a chronic inflammatory wound or degenerative condition, or a tumor, preferably a malignant tumor.

[0437] Preferably, the pharmaceutical composition further comprises at least one nutrient for the recombinant bacterium, preferably selected from the group consisting of carbohydrates, vitamins, minerals, amino acids, trace elements, and mixtures thereof.

[0438] More preferably, the pharmaceutical composition further comprises at least one, preferably one, two, three, four or more heterologous factor(s) and / or at least one component for stabilizing the recombinant bacterium of the present invention. The stabilizing compound preferably comprises a metal cation, preferably a divalent metal cation, such as Mg. 2+ , Ca 2+ and mixtures thereof, antimicrobial agents, cryoprotectants, protease inhibitors, reducing agents, metal chelators, and mixtures thereof.

[0439] In a preferred embodiment, the recombinant bacterium of the invention is provided in the form of a kit according to the invention for medical use, more preferably for use in the treatment of, preferably, chronic inflammatory wounds or degenerative conditions, or for the treatment of tumors, preferably malignant tumors, wherein the recombinant bacterium is capable of expressing at least one heterologous factor under the control of a prokaryotic chloride-inducible promoter, and the kit further comprises at least one inducer comprising chloride ions.

[0440] Preferably the kit is provided as a combined preparation for separate, sequential or simultaneous use in medical treatment, more preferably for use in the treatment of chronic inflammatory wounds or degenerative conditions, or for the treatment of tumors, preferably malignant tumors.

[0441] Preferably, the inducer comprising chloride ions is provided in the form of a liquid, preferably a culture medium, comprising chloride ions, more preferably together with at least one of the pharmaceutically acceptable excipients mentioned above.

[0442] In a preferred embodiment, the recombinant bacterium of the invention is provided in the form of a medical device of the invention for medical use, more preferably for use in the treatment of, preferably, chronic inflammatory wounds or degenerative conditions, or for the treatment of tumors, preferably malignant tumors, wherein the recombinant bacterium is capable of expressing at least one heterologous factor under the control of a chloride-inducible promoter in a prokaryotic system.

[0443] Suitable medical devices preferably further comprise at least one support and / or container for the presentation of the recombinant bacteria, such as a cell encapsulation system and / or microbeads.

[0444] The suitable medical device is preferably a stent, an implant, an inhaler, an encapsulation system, or a medical dressing.

[0445] Preferably, the pharmaceutical composition and / or kit and / or medical device according to the present invention comprises the recombinant bacterium in solution, frozen, or dried, preferably freeze-dried or spray-dried. More preferably, the recombinant bacterium is reconstituted with a liquid, preferably a culture medium, containing chloride ions prior to administration of the recombinant bacterium and / or pharmaceutical composition and / or kit to a subject.

[0446] The recombinant bacterium of the present invention may preferably be contacted with at least one inducer to induce expression of at least one, preferably 1, 2, 3, 4 or more heterologous factor(s).

[0447] Furthermore, preferably, after induction of expression, at least one, preferably one, two, three, four or more heterologous factor(s) are released from the bacterium.

[0448] Preferably, at least one, preferably one, two, three, four, or more heterologous factor(s) are expressed with a secretory signal sequence, preferably an N-terminal signal peptide. After expression of each factor, the secretory signal sequence, preferably an N-terminal signal peptide, can be removed. Alternatively, the first, second, and / or third heterologous factor can be expressed in a mature form, preferably without a secretory signal sequence, preferably without an N-terminal signal peptide.

[0449] The chloride ion-containing reconstituted medium used to reconstitute the recombinant bacteria according to the invention can be in the form of a solution or dispersion, such as an emulsion, suspension, gel, preferably a hydrogel, or colloidal solution.

[0450] Preferably, the reconstituted medium contains chloride ions in an amount sufficient to initiate expression of at least one heterologous factor under the control of a chloride-inducible promoter in a prokaryotic system.

[0451] Suitable polymers for obtaining gels, preferably hydrogels, are known to those skilled in the art and include natural and / or synthetic polymers.

[0452] Preferably, the recombinant bacteria of the present invention are provided in an effective amount to treat and / or alleviate the course of a disease in a subject, such as, preferably, a chronic inflammatory wound, or a degenerative condition, or a tumor, preferably a malignant tumor.

[0453] Preferably, the pharmaceutical composition and / or kit and / or medical device according to the present invention comprises an effective amount of the recombinant bacterium of the present invention.

[0454] In a preferred embodiment of the present invention, the recombinant bacteria of the present invention and / or the pharmaceutical composition and / or kit and / or medical device according to the present invention are to be used for the treatment of inflammatory wounds, preferably chronic inflammatory wounds.

[0455] In a preferred embodiment, the inflammatory, preferably chronic inflammatory wounds preferably include frostbite, dermatitis, ulcers, and combinations thereof, more preferably ulcers.

[0456] Inflammatory wounds can also include inflammatory skin lesions, which can progress to a chronic inflammatory condition.

[0457] Frostbite is a medical condition in which freezing causes localized damage to the skin and other tissues, and tissue destruction may be involved. Frostbite may also be chilblains (pernio), which are superficial ulcers on the skin resulting from exposure to cold and humidity. Damage to the capillary beds of the skin causes redness, itching, inflammation, and sometimes blisters. More preferably, the frostbite is chilblains.

[0458] Chronic inflammatory wounds are known to those skilled in the art and include, for example, chronic venous ulcers, chronic arterial ulcers, chronic diabetic ulcers, and chronic pressure ulcers. Preferably, the chronic wound is at least one of chronic venous ulcers, chronic arterial ulcers, chronic pressure ulcers, and chronic pre-ulceration stages thereof, preferably chronic venous ulcers, chronic arterial ulcers, and chronic pressure ulcers.

[0459] Chronic wounds occur in a variety of forms and prevalences. Compared to acute wounds, chronic wounds do not heal in an orderly set of stages and over a predictable amount of time. Wounds that do not heal within 1 to 3 months or that do not respond to initial treatment are typically classified as chronic. In acute wounds, there is a precise balance between the production and degradation of molecules in the microenvironment and the activation levels of immune cells, resulting in a well-controlled, stepwise healing process. In chronic wounds, this balance is lost, leading to degradation and aberrant activation of the local immune system. Typically, chronic wounds remain stuck in one or more phases of the wound healing process, such as the inflammatory stage. This type of wound is referred to as a chronic inflammatory wound.

[0460] Chronic wounds are usually the result of a variety of underlying diseases and medical conditions, such as tumors, diabetes (leading to DFUs), poor blood circulation (leading to VLUs), surgery, and burns. Ischemia (i.e., lack of oxygen in the limb), bacterial infection and colonization, increased proteolytic enzymes, and inflammation are common underlying pathophysiological causes of non-healing wounds.

[0461] The ulcer may be a pressure ulcer or a leg ulcer. The ulcer may also be a venous ulcer, an arterial ulcer, a diabetic ulcer, or a pressure ulcer. The ulcer may also be the pre-ulceration stage of the ulcers mentioned above, without any visible signs of an open skin wound. Without medical intervention, the pre-ulceration stage may progress to ulceration.

[0462] Chronic venous ulcers usually occur on the legs, account for approximately 70% to 90% of chronic wounds, and mostly affect older patients.

[0463] Another major cause of chronic inflammatory wounds is diabetes. Patients with diabetes have a 15% higher risk of amputation due to chronic ulcers than the general population. Diabetes causes neuropathy, which inhibits nociception and pain perception. Therefore, patients may not notice small wounds on their legs and feet, and therefore may fail to prevent infection or repetitive injury.

[0464] An additional problem is that diabetes causes immune suppression and damage to small blood vessels, resulting in reduced tissue oxygenation. Preventing adequate tissue oxygenation significantly increases the prevalence of chronic inflammatory wounds.

[0465] Pressure sores, also known as decubitus ulcers or bed sores, can occur with or without diabetic conditions. Pressure sores are localized injuries to the skin and / or underlying tissue that can occur over bony prominences as a result of pressure, or pressure in combination with shear or friction.

[0466] Currently, the standard of care management of chronic inflammatory wounds, including diabetic wounds such as leg ulcerations, focuses primarily on controlling infection and promoting revascularization. Despite these strategies, amputation rates remain unacceptably high in patients with leg ulcerations.

[0467] Furthermore, when the underlying disease state or cause of ulceration, such as diabetes or chronic venous insufficiency, is improved and / or treated, for example, by controlling blood sugar levels or administering blood pressure medication, respectively, existing ulcers can still take a very long time to heal.

[0468] Therefore, to overcome the lack of a definitive non-invasive treatment for chronic inflammatory wounds, including diabetic wounds such as leg ulcerations, new strategies are urgently needed to revitalize and promote wound healing in patients with chronic wounds.

[0469] Preferably, in the case of inflammatory wounds, preferably chronic inflammatory wounds, the unique combination of factors preferably released from the bacteria allows reprogramming of the chronic inflammatory wound into an acute wound, which subsequently undergoes wound closure.

[0470] In a preferred embodiment, in the case of inflammatory wounds, preferably chronic inflammatory wounds, the recombinant bacteria should be administered systemically, topically and / or by subcutaneous injection, more preferably topically.

[0471] The recombinant bacteria should preferably be administered locally to the inflamed, preferably chronically inflammatory, wound to be treated.

[0472] The recombinant bacteria may be administered topically to the inflamed, preferably chronically inflammatory wound and / or by subcutaneous injection in the vicinity of the wound, preferably at the edge or cavity of the inflamed, preferably chronically inflammatory wound.

[0473] Furthermore, topical application or subcutaneous injection of the recombinant bacteria of the present invention to and / or within the site of pre-ulceration may prevent the progression of the pre-ulceration to an open, inflamed wound.

[0474] In a preferred embodiment, in the case of tumors, preferably malignant tumors, the recombinant bacteria should be administered systemically, for example by intravenous injection, or locally, preferably by intratumoral and / or intraperitoneal injection.

[0475] The recombinant bacteria are preferably administered to a tumor, preferably a malignant tumor, by intratumoral injection of the recombinant bacteria of the invention into the tumor and / or by injection in the vicinity of the tumor and / or by intraperitoneal injection.

[0476] In a preferred embodiment, the recombinant bacterium according to claim 1 comprises at least one nucleic acid sequence encoding a first heterologous factor, at least one nucleic acid sequence encoding a second heterologous factor, and at least one nucleic acid sequence encoding a third heterologous factor, wherein the first factor, the second factor, and the third factor are functionally distinct from one another, and wherein the first factor is a growth factor, the second factor is selected from the group consisting of M2 polarization factors, and the third factor is selected from the group consisting of M2 polarization factors and growth factors.

[0477] In a preferred embodiment, the second heterologous factor and the third heterologous factor are selected from the group consisting of M2 polarizing factors, and the second factor and the third factor are functionally distinct M2 polarizing factors.

[0478] That is, the second heterologous factor is a first M2-polarizing factor, and the third heterologous factor is a second M2-polarizing factor that is functionally distinct from the first M2-polarizing factor.

[0479] Preferably, the first M2 polarizing factor is an M2 polarizing factor selected from the group consisting of colony stimulating factor-1 (CSF-1), interleukin 34 (IL-34), interleukin 4 (IL-4), and interleukin 13 (IL-13), and the second M2 polarizing factor is an M2 polarizing factor selected from the group consisting of colony stimulating factor-1 (CSF-1), interleukin 34 (IL-34), interleukin 4 (IL-4), interleukin 10 (IL-10), and interleukin 13 (IL-13), provided that the second M2 polarizing factor is functionally distinct from the first M2 polarizing factor.

[0480] More preferably, the first M2 polarizing factor is a colony-stimulating factor-1 receptor (CSF1R) ligand and the second M2 polarizing factor is an M2 polarizing factor selected from the group consisting of interleukin 4 (IL-4), interleukin 10 (IL-10), interleukin 13 (IL-13), functional analogs thereof, biosimilars thereof, and mixtures thereof.

[0481] Further preferred combinations of M2 polarizing factors are: Colony-stimulating factor-1 and interleukin-4, Colony-stimulating factor-1 and interleukin-13, Colony-stimulating factor-1 and interleukin-10, Interleukin-34 and interleukin-4, Interleukin-34 and interleukin-13, Interleukin-34 and interleukin-10, Interleukin-4 and interleukin-10, or Interleukin-13 and interleukin-10, is.

[0482] Further preferred combinations of the above-mentioned M2-polarizing factors are those in which at least one of the above-mentioned growth factors is combined with a growth factor, preferably selected from the group consisting of fibroblast growth factor 1, fibroblast growth factor 2, fibroblast growth factor 7, fibroblast growth factor 10, hepatocyte growth factor, transforming growth factor beta (TGF-beta), epidermal growth factor (EGF), heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor-alpha (TGF-alpha), and platelet-derived growth factor BB.

[0483] Preferably, the first, second, and third heterologous factors are: Fibroblast growth factor 2, colony-stimulating factor-1, and interleukin-4, Fibroblast growth factor 2, interleukin-34, and interleukin-4, Fibroblast growth factor 2, colony-stimulating factor-1, and interleukin-13, Fibroblast growth factor 2, interleukin-34, and interleukin-13, Fibroblast growth factor 2, colony-stimulating factor-1, and interleukin-10, Fibroblast growth factor 2, interleukin-34, and interleukin-10, Fibroblast growth factor 7, colony-stimulating factor-1, and interleukin-4, Fibroblast growth factor 7, interleukin-34, and interleukin-4, Fibroblast growth factor 7, colony-stimulating factor-1, and interleukin-13, Fibroblast growth factor 7, interleukin-34, and interleukin-13, Fibroblast growth factor 7, colony-stimulating factor-1, and interleukin-10, Fibroblast growth factor 7, interleukin-34, and interleukin-10, Transforming growth factor beta, colony-stimulating factor-1, and interleukin-4, Transforming growth factor beta, interleukin-34, and interleukin-4, Transforming growth factor beta, colony-stimulating factor-1, and interleukin-13, Transforming growth factor beta, interleukin-34, and interleukin-13, Transforming growth factor beta, colony-stimulating factor-1, and interleukin-10, Transforming growth factor beta, interleukin-34, and interleukin-10, Transforming growth factor alpha, colony-stimulating factor-1, and interleukin-4, Transforming growth factor alpha, interleukin-34, and interleukin-4, Transforming growth factor alpha, colony-stimulating factor-1, and interleukin-13, Transforming growth factor alpha, interleukin-34, and interleukin-13, Transforming growth factor alpha, colony-stimulating factor-1, and interleukin-10, Transforming growth factor alpha, interleukin-34, and interleukin-10, Platelet-derived growth factor BB, colony-stimulating factor-1, and interleukin-4, Platelet-derived growth factor BB, interleukin-34, and interleukin-4, Platelet-derived growth factor BB, colony-stimulating factor-1, and interleukin-13, Platelet-derived growth factor BB, interleukin-34, and interleukin-13, Platelet-derived growth factor BB, colony-stimulating factor-1, and interleukin-10, or Platelet-derived growth factor BB, interleukin-34, and interleukin-10, It is a combination of:

[0484] More preferably, the first, second, and third heterologous factors are a combination of fibroblast growth factor 2, colony stimulating factor-1, and interleukin-4, functional analogs thereof, and biosimilars thereof.

[0485] Preferably, release of two or more M2 polarizing factors from the bacteria of the present invention further promotes M2 polarization of unpolarized macrophages, M1 polarized macrophages, and undifferentiated monocytes, as well as other macrophage precursor cells.

[0486] In an alternative embodiment, the first factor is a first growth factor selected from the group consisting of the growth factors mentioned above, and the third factor is a second growth factor selected from the group consisting of the growth factors mentioned above and functionally different from the first growth factor. Preferably, the second growth factor is transforming growth factor beta (TGF-beta).

[0487] A more preferred combination of growth factors is: fibroblast growth factor 1 and transforming growth factor beta, Fibroblast growth factor 2 and transforming growth factor beta, Fibroblast growth factor 7 and transforming growth factor beta, Fibroblast growth factor 10 and transforming growth factor beta, Platelet-derived growth factor BB and transforming growth factor beta, Transforming growth factor alpha and transforming growth factor beta, epidermal growth factor and transforming growth factor beta, Heparin-binding EGF-like growth factor and transforming growth factor beta, Hepatocyte growth factor and transforming growth factor beta, or Vascular endothelial growth factor A and transforming growth factor beta, is.

[0488] The above mentioned further preferred combinations of growth factors are preferably combined with an M2 polarizing factor selected from the group consisting of colony stimulating factor-1 (CSF-1), interleukin-34 (IL-34), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-13 (IL-13), and mixtures thereof, preferably colony stimulating factor-1 (CSF-1), interleukin-34 (IL-34), interleukin-4 (IL-4), interleukin-13 (IL-13), and mixtures thereof.

[0489] In another preferred embodiment of the present invention, the recombinant bacterium of the present invention and / or the pharmaceutical composition and / or kit and / or medical device according to the present invention is to be used for the treatment of tumors, preferably malignant tumors, preferably peritoneal cancer, more preferably peritonitis carcinomatosa, more preferably metastatic ovarian cancer, colorectal cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma, gallbladder cancer, renal cell carcinoma, transitional cell carcinoma, endometrial cervical cancer, and / or extra-abdominal conditions such as breast cancer, lung cancer, and malignant melanoma.

[0490] A tumor is an abnormal growth of cells / tissues that has no physiological function and is usually due to uncontrolled and rapid cell proliferation. Tumors can be benign or malignant. Different types of tumors are named after the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas.

[0491] Peritoneal cancer can be divided into two categories: primary peritoneal cancer, where the primary tumor originates within the peritoneum, and peritoneal carcinomatosis (PC), which is defined as the intraperitoneal dissemination of any tumor originating elsewhere.

[0492] Peritoneal carcinomatosis is one of the most common diffuse peritoneal diseases, with ovarian cancer being the most common cause (46%), followed by colorectal cancer (31%), pancreatic cancer, gastric cancer, and other malignancies, including hepatocellular carcinoma, gallbladder cancer, renal cell carcinoma, transitional cell carcinoma, endometrial carcinoma, cervical carcinoma, and unknown primary carcinoma. Extra-abdominal conditions, such as breast cancer, lung cancer, and malignant melanoma, can involve the peritoneal cavity via hematogenous spread (Singh, S. et al. "Peritoneal Carcinomatosis: Pictorial Review of Computed Tomography Findings," International Journal of Advanced Research (2016), Volume 4, Issue 7, pages 735 to 748; doi:10.21474 / IJAR01 / 936).

[0493] In 10% to 35% of patients with recurrent colorectal cancer (CRC) and up to 50% of patients with recurrent gastric cancer (GC), tumor recurrence is limited to the peritoneal cavity (Coccolini, F. et al., World J Gastroenterol. 2013;19(41):pages 6979 to 6994, doi:10.3748 / wjg.v19.I41.6979).

[0494] Peritoneal metastases can also form due to distant metastases from extraperitoneal cancers, such as pleural mesothelioma, breast, and lung. Tumor cell colonization of the diaphragm or abdominal lymphatic vessels causes obstruction of lymphatic drainage and reduced outflow of peritoneal fluid, leading to carcinomatosis and / or the formation of ascites.

[0495] The immune system preferably plays a major role in the pathophysiology of cancer. Many tumors are heavily infiltrated by different types of immune cells. However, the composition of these cells within tumors can vary. Both cells from the adaptive immune system, such as cytotoxic T cells and regulatory T cells, and cells from the innate immune system, such as macrophages, dendritic cells, and natural killer (NK) cells, are central to the progression of disease. These immune cells appear to be linked to tumor growth, invasion, and metastasis.

[0496] In particular, tumor growth is paralleled by the recruitment and accumulation of macrophages, as these cells acquire the function of supporting tumor growth and cancer cell dissemination from the primary tumor. Furthermore, clinical evidence demonstrates a strict correlation between increased numbers of macrophages with disease progression and poor prognosis. Because macrophages can produce a wide spectrum of different cytokines (Th1- and Th2-associated cytokines), they are central players in orchestrating the immune microenvironment.

[0497] Macrophages can be divided into M1 (classically activated) or M2 (alternatively activated) phenotypes. M1 macrophages exhibit a pro-inflammatory phenotype and are characterized by the expression of pro-inflammatory cytokines such as IL-1, IL-6, TNF-α, and IFN-γ. Monocytes can be differentiated into M1 macrophages by bacterial components, such as lipopolysaccharide (LPS) and interferon-γ (IFN-γ).

[0498] In contrast, M2 macrophages have an immunosuppressive phenotype and release factors that promote Th2 responses, such as IL-10, TGF-β, and VEGF.

[0499] Macrophages within tumors, also called tumor-associated macrophages (TAMs), often express many genes typical of the M2 phenotype. Chemoattractants secreted by tumor and stromal cells recruit monocytes into tumor tissue and drive their differentiation into M2 tumor-associated macrophages, thereby supporting tumor growth and metastasis.

[0500] After administration of the recombinant bacteria of the present invention to a tumor, the recombinant bacteria produce and release the therapeutic protein(s) into the tumor environment.

[0501] Preferably, the released therapeutic protein(s) induces these immune cells to become T H1 enhances and drives the immune response, which in turn leads to a reduction in the production of vascular endothelial growth factor (VEGF).

[0502] Studies have shown that the expression of VEGF and its receptors on macrophages plays a role in regulating the immune system and its surrounding microenvironment. In particular, M2 tumor-associated macrophages express large amounts of VEGF, supporting angiogenesis and immune regulation.

[0503] VEGF has also been described to inhibit dendritic cell differentiation. In contrast, VEGF has also been shown to inhibit T cell development, thereby contributing to tumor-induced immunosuppression.

[0504] The reduction in the production of VEGF preferably further supports the anti-tumor effect by preventing immunosuppression and angiogenesis.

[0505] In a preferred embodiment, the recombinant bacterium according to claim 1 is preferably a cell receptor activator, such as glucocorticoid-inducible TNFR-related protein (GITR), tumor necrosis factor receptor superfamily member 9 (TNFRSF9), also known as 4-1BB, tumor necrosis factor receptor superfamily member 4 (TNFRSF4), also known as cluster of differentiation 40 (CD40), CD134 or OX40 receptor, and combinations thereof, costimulatory receptor activators, immune checkpoint inhibitors, such as cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-1L), programmed death ligand 2 (PD-1L2), and combinations thereof, chimeric antigen receptors, such as CAR-T, and combinations thereof, prodrug-activating enzymes, such as HSV-tk, cytidine deaminase, and combinations thereof, cytokines, chemokines, growth factors, decoys, Receptor ligands, antibodies, soluble receptors, decoy receptors, and combinations thereof, preferably granulocyte-macrophage-colony stimulating factor (GM-CSF), interferon alpha, preferably interferon alpha 2 (IFNA2), interferon beta, interferon gamma, granulocyte-colony stimulating factor (G-CSF), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-23 (IL-23), interleukin-24 (IL-24), interleukin-32 (IL-32), and combinations thereof.

[0506] array SEQ ID NO: 1 corresponds to the nucleic acid sequence of the expression plasmid pAUC1010, which is also shown in Figure 1. SEQ ID NO: 2 corresponds to the nucleic acid sequence of the PgadC promoter used in the examples, which is also shown in Figure 2b. SEQ ID NO: 3 corresponds to the amino acid sequence of the L. lactis Usp45 signal sequence, which is also shown in Figure 3. SEQ ID NO: 4 corresponds to the 3' end of the L. lactis 16S RNA, which is also shown in Figure 4.

[0507] SEQ ID NO: 5 corresponds to the amino acid sequence of the mature form of hFGF-2-155, which is also shown in Figure 5a. SEQ ID NO: 6 corresponds to the amino acid sequence of the hFGF-2-153 variant used in the examples, which is also shown in Figure 5b. SEQ ID NO: 7 corresponds to the amino acid sequence of the recombinant hFGF-2-153 precursor protein used in the examples, which is also shown in Figure 5c.

[0508] SEQ ID NO: 8 corresponds to the amino acid sequence of the mature form of hIL-4, which is also shown in Figure 6a. SEQ ID NO: 9 corresponds to the amino acid sequence of the hIL-4 variant used in the examples, which is also shown in Figure 6b. SEQ ID NO: 10 corresponds to the amino acid sequence of the recombinant hIL-4 precursor protein used in the examples, which is also shown in Figure 6c.

[0509] SEQ ID NO: 11 corresponds to the amino acid sequence of the mature form of hCSF1, which is also shown in Figure 7a. SEQ ID NO: 12 corresponds to the amino acid sequence of the hCSF1 variant used in the examples, which is also shown in Figure 7b. SEQ ID NO: 13 corresponds to the amino acid sequence of the recombinant hCSF1 precursor protein used in the examples, which is also shown in Figure 7c.

[0510] SEQ ID NO: 14 corresponds to the nucleic acid sequence of the synthetic CFI construct used in the examples, which is also shown in Figure 8b. SEQ ID NO: 15 corresponds to the nucleic acid sequence of the expression plasmid pC-CFI used in the examples, which is also shown in Figure 9b.

[0511] SEQ ID NO: 16 corresponds to the amino acid sequence of the mature form of mIL-18, which is also shown in Figure 10a. SEQ ID NO: 17 corresponds to the amino acid sequence of the mIL-18 variant used in the examples, which is also shown in Figure 10b. SEQ ID NO: 18 corresponds to the amino acid sequence of the recombinant mIL-18 precursor protein used in the examples, which is also shown in Figure 10c.

[0512] SEQ ID NO: 19 corresponds to the amino acid sequence of the mature form of mGM-CSF, which is also shown in Figure 10a. SEQ ID NO: 20 corresponds to the amino acid sequence of the mGM-CSF variant used in the examples, which is also shown in Figure 10b. SEQ ID NO: 21 corresponds to the amino acid sequence of the recombinant mGM-CSF precursor protein used in the examples, which is also shown in Figure 10c.

[0513] SEQ ID NO: 22 corresponds to the nucleic acid sequence of the synthetic mEG construct used in the examples, which is also shown in Figure 12b. SEQ ID NO: 23 corresponds to the nucleic acid sequence of the expression plasmid pC-mEG used in the examples, which is also shown in Figure 12c.

[0514] SEQ ID NO:24 corresponds to the amino acid sequence of the mature form of mIL-12 beta, which is also shown in Figure 13a. SEQ ID NO:25 corresponds to the amino acid sequence of the mature form of mIL-12 alpha isoform 2, which is also shown in Figure 13b. SEQ ID NO:26 corresponds to the amino acid sequence of the mature form of a recombinant murine interleukin-12 fusion protein used in the examples, which is also shown in Figure 13c. SEQ ID NO:27 corresponds to the amino acid sequence of a recombinant murine interleukin-12 precursor protein used in the examples, which is also shown in Figure 13d.

[0515] SEQ ID NO: 28 corresponds to the amino acid sequence of the mature form of murine interferon alpha-2, which is also shown in Figure 14a. SEQ ID NO: 29 corresponds to the amino acid sequence of the murine interferon alpha-2 variant used in the examples, which is also shown in Figure 14b. SEQ ID NO: 30 corresponds to the amino acid sequence of the recombinant murine interferon alpha-2 precursor protein used in the examples, which is also shown in Figure 14c.

[0516] SEQ ID NO: 31 corresponds to the nucleic acid sequence of the synthetic mTEA construct used in the examples, which is also shown in Figure 15a. SEQ ID NO: 32 corresponds to the nucleic acid sequence of the expression plasmid pC-mTEA used in the examples, which is also shown in Figure 15c.

[0517] SEQ ID NO: 33 corresponds to the nucleic acid sequence of the synthetic mGTE construct used in the examples, which is also shown in Figure 16a. SEQ ID NO: 34 corresponds to the nucleic acid sequence of the expression plasmid pC-mGTE used in the examples, which is also shown in Figure 16c.

[0518] SEQ ID NO: 35 corresponds to the nucleic acid sequence of the synthetic mCherry construct used in the examples, which is also shown in Figure 17b. SEQ ID NO: 36 corresponds to the nucleic acid sequence of the expression plasmid pC-mCherry used in the examples, which is also shown in Figure 18b.

[0519] The following figures and examples are given for illustrative purposes only and the present invention should not be construed as being limited to the following examples.

[0520] figure: In the figure, the following abbreviations are used: "alr" refers to the alanine racemase gene used for growth of the host strain in the absence of d-alanine; "T" refers to the terminator sequence; "-35" and "-10" refer to the -35 and -10 elements of the promoter, respectively; "IR" refers to the inverted repeat sequence; "repC" and "repA" are prokaryotic genes required for replication of the plasmid by bacterial cells, "gadR" refers to the gadR gene, "RBS" refers to the ribosome binding site of the indicated gene, e.g., the ATP synthase subunit gamma (atpG) gene and / or the galactoside O-acetyltransferase (lacA) gene; and "ssUsp45" refers to the Usp45 secretion signal. [Brief explanation of the drawings]

[0521] Figure 1a shows the plasmid map of the expression plasmid pAUC1010. The corresponding nucleic acid sequence is shown in Figure 1b. Figure 2a shows a schematic of the control elements encompassing the chloride-inducible promoter PgadC. The corresponding nucleic acid sequence is shown in Figure 2b, which includes the control elements encompassing the PgadC promoter region, the gadR gene, and the ribosome binding site (RBS) and start codon of the gadC gene. FIG. 3 shows the amino acid sequence of the secretion signal of the lactococcal protein Usp45. FIG. 4 shows the nucleic acid sequence of the 3′ end of the 16S rRNA of L. lactis. Figure 5a shows the amino acid sequence of the mature form of human FGF-2-155. The amino acid sequence of the hFGF2-153 variant used in the examples is shown in Figure 5b. The amino acid sequence of the recombinant hFGF-2-153 precursor protein used in the examples is shown in Figure 5c. Figure 6a shows the amino acid sequence of the mature form of human IL-4. The amino acid sequence of the hIL-4 variant used in the examples is shown in Figure 6b. The amino acid sequence of the recombinant hIL-4 precursor protein used in the examples is shown in Figure 6c. Figure 7a shows the amino acid sequence of the mature form of human CSF-1. The amino acid sequence of the hCSF-1 variant used in the examples is shown in Figure 7b. The amino acid sequence of the recombinant hCSF-1 precursor protein used in the examples is shown in Figure 7c. Figure 8a shows a schematic diagram of the de novo synthesized CFI construct used in the examples. The corresponding nucleic acid sequence of the de novo synthesized CFI construct is shown in Figure 8b. The nucleic acid sequence of the expression plasmid designated pC-CFI is shown in Figure 9b. A schematic diagram of the expression plasmid pC-CFI is shown in Figure 9a. Figure 10a shows the amino acid sequence of the mature form of murine IL-18. The amino acid sequence of the mIL-18 variant used in the examples is shown in Figure 10b. The amino acid sequence of the recombinant mIL-18 precursor protein used in the examples is shown in Figure 10c. Figure 11a shows the amino acid sequence of the mature form of murine GM-CSF. The amino acid sequence of the mGM-CSF variant used in the examples is shown in Figure 11b. The amino acid sequence of the recombinant mGM-CSF precursor protein used in the examples is shown in Figure 11c. Figure 12a shows the nucleic acid sequence of the synthetic mEG construct used in the examples. The nucleic acid sequence of the expression plasmid designated pC-mEG is shown in Figure 12c, and a schematic diagram of the corresponding expression plasmid pC-mEG used in the examples is shown in Figure 12b. Figure 13a shows the amino acid sequence of the mature form of murine IL-12 subunit beta. The amino acid sequence of mature interleukin-12 subunit alpha isoform 2 is shown in Figure 13b. The amino acid sequence of the mature form of the recombinant interleukin-12 fusion protein used in the examples is shown in Figure 13c. The amino acid sequence of the recombinant mIL-12 precursor protein used in the examples is shown in Figure 13d. Figure 14a shows the amino acid sequence of mature murine mIFNa2. The amino acid sequence of the mIFNa2 variant used in the examples is shown in Figure 14b. The amino acid sequence of the synthetic mIFNa2 precursor protein used in the examples is shown in Figure 14c. Figure 15a shows the nucleic acid sequence of the synthetic mTEA construct used in the examples. The nucleic acid sequence of the expression plasmid designated pC-mTEA is shown in Figure 15c. A schematic diagram of the expression plasmid pC-mTEA is shown in Figure 15b. Figure 16a shows the nucleic acid sequence of the synthetic mGTE construct used in the examples. The nucleic acid sequence of the expression plasmid designated pC-mGTE is shown in Figure 16c. A schematic diagram of the corresponding expression plasmid pC-mEG used in the examples is shown in Figure 16b. Figure 17a shows a schematic of the synthetic mCherry construct used in the examples, and the corresponding nucleic acid sequence of the synthetic mCherry construct is shown in Figure 17b. The nucleic acid sequence of the expression plasmid used in the examples, designated pC-mCherry, is shown in Figure 18b. A schematic diagram of the expression plasmid pC-mCherry is shown in Figure 18a. Figure 19a shows a comparison of the growth curves of L. lactis NZ1330 (pC-mCherry) after induction of expression of the mCherry gene with 100 mM NaCl ("L. lactis NZ1330::pC-mCherry+NaCl") and without induction ("L. lactis NZ1330::pC-mCherry+mq"), each determined by measuring the optical density at a wavelength of 600 nm (OD600) at the indicated time points (T) in Example 3. Figure 19b shows a comparison of mCherry fluorescence obtained from L. lactis NZ1330(pC-mCherry) after induction of expression of the mCherry gene with 100 mM NaCl ("NZ1330::pC-mCherry+NaCl") and without induction ("NZ1330::pC-mCherry+MQ"), in artificial units, measured at the indicated time points in minutes under the conditions described in Example 3. Figure 20a shows a Western blot analysis of recombinant bacteria designated AUP1602-C expressing human CSF-1, human FGF-2, and human IL-4 from the expression plasmid pC-CFI obtained in Example 1.4. "Anti-CSF-1," "anti-FGF-2," and "anti-IL-4" refer to the primary antibodies used in Example 3 to detect the expression of human CSF-1, human FGF-2, and human IL-4, respectively. Figures 20b and 20c show Western blot analysis of recombinant bacteria designated AUP5563-C expressing murine IL-12, murine IL-18, and murine GM-CSF from the expression plasmid pC-mGTE obtained in Example 1.7. "Anti-GM-CSF" and "anti-IL-18" refer to the primary antibodies used in Example 3 to detect the expression of murine GM-CSF and murine IL-18, respectively. Figure 20d shows Western blot analysis of recombinant bacteria designated AUP555m-C, which expresses mouse IL-12, mouse IL-18, and mouse IFNa from the expression plasmid pC-mTEA obtained in Example 1.6, and AUP5563-C, which expresses mouse IL-12, mouse IL-18, and mouse GM-CSF from the expression plasmid pC-mGTE obtained in Example 1.7. "Anti-IL-12" refers to the primary antibody used in Example 3 to detect the expression of mouse IL-12. Figure 21 shows fluorescence imaging of tumor-induced BALB / c mice 48 hours after it injection of recombinant L. lactis containing the AUC1000(pC-mCherry) construct ("PGAD-mCherry") and control bacteria without the expression plasmid ("L. lactis (control)") under the conditions described in Example 4. The blue circle indicates the location of the tumor. FIG. 22 shows the mean percentage wound area for all treatment groups in the wound closure experiment described in Example 5. FIG. 23 shows the mean percentage wound contraction for all treatment groups in the wound closure experiment described in Example 5. FIG. 24 shows the percentage of responding wounds for each treatment group on day 1 of the wound closure experiment described in Example 5. Figures 25a to 27b show the results described in Example 6 of the detection of human FGF-2, human IL-4, and human CSF-1 in wound fluid from mice treated in the wound closure experiment described in Example 5. Figures 28a to 28c show the results described in Example 7 for the specific tumor volumes of vehicle-treated control mice, AUP5563-C4-treated mice, and anti-m-CTLA-4-treated control mice at the indicated time points. Figure 29 shows the survival curves of C57BL76 mice treated in Example 8 with the respective combinations of drug products AUP2059 (mIL18 / mGM-CSF) and AUP5551-C (mIL12 / mIL18 / mIFNa2b) obtained in Example 3, and vehicle-treated control mice. [Example]

[0522] example: I) General Experimental Procedure: Unless otherwise stated, the examples were performed according to the analytical system manufacturer's protocols. Unless otherwise stated, the indicated chemicals were commercially obtained from Sigma-Aldrich Chemie GmbH (Munich, DE), Merck KGaA (Darmstadt, DE), Thermo Fisher Scientific Inc. (Waltham, MA, US), or Becton, Dickinson and Company (Franklin Lakes, NJ, US).

[0523] I.1 Growth medium For different purposes, cells were grown in different media.

[0524] For general cloning procedures, M17 medium (Oxoid Deutschland GmbH, Wesel, DE) containing 1 wt % glucose or lactose, respectively, was used.

[0525] E. coli strains were grown in TY medium. The recipe for TY medium used was as follows: 1wt% tryptone 0.5wt% yeast extract 0.5wt% NaCl

[0526] For fermentation and other functional growth experiments, IM1 medium was used, which is free of animal-derived components, the only remaining animal-derived component being lactose, which can be obtained in pharmaceutical quality.

[0527] The recipe for IM1 medium used is as follows: 1wt% lactose 2wt% Sodium β-glycerophosphate 1.5wt% soy peptone 1wt% yeast extract 1 mM MgSO4 x 7H2O 0.1 mM MnSO4 x 4H2O pH 6.7 Sterilization: 110℃, 15min

[0528] During fermentation, no buffer (e.g., Na-β-glycerophosphate) was added because the pH was automatically controlled with 2.5 M NaOH. This buffer neutralized the lactic acid produced and prevented the culture from reaching OD. 600 Allow to reach a cell density of 10–15.

[0529] For growth of strains L. lactis NZ1330 and NZ9130, D-alanine was added to the medium at a final concentration of 200 μg / mL.

[0530] I.2 Bacterial strains The following commercial bacterial strains were used: Lactococcus lactis strains NZ3900 and NZ1330 were obtained from MoBiTec GmbH (Goettingen, DE).

[0531] L. lactis strain NZ1330 contains a deleted gene encoding alanine racemase (alr) (Δalr). Deletion of the alr gene results in auxotrophy for the essential component D-alanine. The respective strain cannot grow on medium without D-alanine unless the alr gene is provided on a plasmid.

[0532] I.3 Molecular cloning techniques Standard techniques were used for molecular cloning, as described, for example, in Green and Sambrook (2012): “Molecular cloning: a laboratory manual”, fourth edition, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY, US).

[0533] The different synthetic DNA constructs were produced by BaseClear (Leiden, NL). The constructs were obtained as purified plasmids and clones in E. coli.

[0534] Plasmid DNA was isolated using standard DNA isolation according to Birnboim, H.C. and Doly, J. (1979). Synthetic gene constructs were excised from their plasmids using selected restriction enzymes, purified, and ligated into their respective target plasmids. Ligations were performed using Anza T4 ligase master mix (ThermoFisher Scientific Inc.) according to the supplied protocol.

[0535] The different ligation mixtures were transformed into selected L. lactis strains ( L. lactis NZ1330 or L. lactis NZ9130) by electroporation and plated on appropriate media.

[0536] I.4 Preparation of electrocompetent cells of strains and electroporation Electrocompetent cells were prepared according to the following protocol: L. lactis NZ1330 and NZ9130 were grown overnight at 30°C in 10 mL of SMGG medium, which was used to inoculate 100 mL of SMGG medium.

[0537] OD of ~0.5 600 The cells were grown until they reached a viable density of 1000kJ / ml. The cells were washed three times with ice-cold wash buffer and resuspended in 1 mL of wash buffer. Aliquots of 40 μL were stored at -80°C. The electrocompetent properties of the cells were tested using standard electroporation procedures.

[0538] To this end, 40 μL of cells were thawed on ice and mixed with 0.5 μL of plasmid DNA in an ice-cold electroporation cuvette, and 2500 volts, 25 μF, 200 ohms were applied using an Eporator® electroporator (Eppendorf AG, Hamburg, DE).

[0539] The cells were resuspended in 4 mL of SMG17MC medium and incubated for 2 hours at 30°C. Different amounts of the culture were plated on GSM17 agar and incubated at 30°C.

[0540] Buffers used: SMGG medium: commercially available M17 medium +0.5M sucrose +0.5wt% glucose +1wt% glycine SMG17MC medium: commercially available M17 medium +0.5M sucrose +0.5wt% glucose + 20 mM MgCl2 + 2mM CaCl2 GSM17 agar: M17 agar obtained from Oxoid Deutschland GmbH (Wesel, DE) +0.5M sucrose +0.5wt% glucose Wash buffer: 0.5M sucrose +10wt% glycerol

[0541] I.5 Selection of transformants Transformants were transferred from plates to new plates and into tubes with 3 ml of medium M17 (supplemented with 1% glucose or lactose), incubated at 30°C, and DNA isolated as outlined above in section I.3. Clones were screened for the presence and orientation of inserts using selected restriction endonucleases and agarose gel electrophoresis.

[0542] Positive clones were selected, cultured, and stored at -80°C.

[0543] I.6 Cultivation and induction of gene expression Selected clones were inoculated into IM1 medium supplemented with 0.5% glucose or lactose and grown overnight at 30°C. At t = 0, the culture was inoculated 1:100 into 45 mL of medium and incubated. OD 600 At = 0.5, the culture was split into three separate 15 ml cultures, which were induced by the addition of NaCl (0 mM, 100 mM, or 500 mM).

[0544] After 3 hours of gene induction, cells and supernatant were separated by centrifugation (6,000 rpm for 10 minutes).

[0545] Prior to further processing, the cells were frozen at -20°C. The supernatant was mixed with one-third volume of TCA (see below) and stored at -20°C.

[0546] I.7 Preparation of cell-free extracts and trichloroacetic acid (TCA) precipitation Cell-free extracts were prepared by bead-beating according to standard working protocols. The procedure is based on the use of 50-100 μm glass beads. Samples were kept on ice whenever possible to prevent proteolytic degradation of proteins. After a final centrifugation step, cell-free extracts were collected and stored at -20°C.

[0547] TCA precipitation was performed by adding 1 volume of TCA to 4 volumes of culture supernatant. The mixture was incubated at -20°C until further use. To obtain TCA-precipitated proteins, a 1 mL sample was taken and centrifuged at 14,000 rpm for 10 min. The supernatant was decanted, and the pellet was dried in a stove at 65-70°C. After that, SDS-PAGE sample buffer containing 2% β-mercaptoethanol was added, and the sample was denatured by incubation at 100°C for 10 min.

[0548] I.8 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting SDS-PAGE (15 wt% running gels) was performed essentially according to Laemmli, UK (1970). After electrophoresis, gels were stained using Coomassie R-250 (Bio-Rad Laboratories, Inc., Hercules, CA, US), or proteins were transferred to polyvinylidene fluoride (PVDF) or nitrocellulose membranes by means of a semi-dry blotting system (Owl®, Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.

[0549] PVDF or nitrocellulose membranes were treated by blocking with Tris-buffered saline containing 2 wt% bovine serum albumin (BSA) in 0.05 wt% Tween 20 (TBST), followed by incubation with specific primary antibodies raised against each protein diluted in TBST containing 1 wt% BSA for 30 min.

[0550] The blot was then washed three times for 10 minutes with TBST. After washing, an alkaline phosphatase-conjugated anti-rabbit secondary antibody (e.g., goat-derived, from Santa Cruz Biotechnology, Inc., Dallas, TX, US, catalogue no. sc-2004) was applied (1:7500 in TBST + 1 wt% BSA). Unbound antibody was washed off with TBST (3 x 10 min.), followed by enzymatic staining using NBT and BCIP according to standard procedures.

[0551] After sufficient color development, the reaction was terminated by rinsing with water, and the membrane was dried and photographed or scanned.

[0552] Alternatively, donkey anti-goat antibody IRDye® 680RD (LI-COR, Inc., Lincoln, NE, US), conjugated with a near-infrared (NIR) fluorescent dye, was used according to the manufacturer's protocol. Detection of bound secondary antibodies was performed using the Odyssey® imaging system (LI-COR, Inc.) according to the manufacturer's protocol.

[0553] Tris-buffered saline with 0.05 wt% Tween 20 was prepared according to the manufacturer's instructions using Roti® fair TBST7.6 tablets obtained from Carl Roth GmbH+Co. KG (Karlsruhe, DE).

[0554] The following primary antibodies from Abcam PLC (Cambridge, UK), Bio-Rad Laboratories Inc. (Hercules, CA, US), and LifeSpan BioSciences, Inc. (Seattle, WA, US) were used to detect each recombinant protein. Anti-FGF-2 antibody - rabbit polyclonal antibody against FGF-2 (Abcam PLC, ab126861), Anti-IL-4 antibody - rabbit polyclonal antibody against IL-4 (Abcam PLC, ab9622), Anti-M-CSF antibody - rabbit polyclonal antibody against M-CSF (Abcam PLC, ab9693), Anti-mouse GM-CSF antibody - rabbit polyclonal antibody against mouse GM-CSF (Bio-Rad Laboratories Inc., AAM16G), Anti-mouse IL-18 antibody - rabbit polyclonal antibody against mouse IL-18 (LifeSpan BioSciences, Inc., LS-C147100), Anti-mouse IL-12 antibody - goat anti-mouse antibody against IL-12 (Biorad Laboratories Inc., AAM33).

[0555] I.9 Fermentation Fermentation was carried out in a 0.5 L Multifors 6 fermentor array (Infors Benelux, Velp, NL). Inoculation was performed with a 1% preculture in IM-1 medium; the stirrer speed was 200 rpm, and the incubation temperature was 30°C. During the run, pH was controlled with 2.5 M NaOH. pH, temperature, and NaOH addition were continuously monitored.

[0556] I.10 Fluorescent protein measurement Strains containing constructs with the mCherry reporter gene under the control of different promoters were grown and treated as described above, and mCherry activity was measured using a Synergy HT (BioTek Instruments, Inc., Winooski, VT, US) fluorescence spectrophotometer.

[0557] Excitation was at 530 nm, emission was measured at 590 nm, and a gain of 120 was applied as described by Tauer et al. (2014).

[0558] Example 1: Creation of an expression plasmid 1.1 Construction of plasmid pAUC1010 Plasmid pAUC1010 was used in the following experiments for the expression of heterologous genes in L. lactis. Plasmid pAUC1010 is based on the native rolling-circle plasmid of Lactococcus lactis pSH71, as described in de Vos, W. M. (1987) and de Vos, W. M. and Simons, G. (1994).

[0559] Plasmid pAUC1010 contains the alanine racemase (alr) gene, including its terminator, of L. lactis as a selection marker, which can be used to generate L. lactis alr knockout mutant strains, such as NZ1330 (Bron, PA et al. (2002); Hols, P. et al. (1999)). The plasmid also contains the artificial promoter Pcp14 (Ruhdal Jensen, P. and Hammer, K. (1998)).

[0560] Furthermore, the plasmid pAUC1010 contains the terminator sequence of the L. lactis aminopeptidase N (pepN) gene (Tan, PS et al. (1992)) downstream of the promoter and multiple cloning site (MCS). The terminator sequence allows for the termination of transcription.

[0561] The nucleic acid sequence of the Lactococcus lactis subsp. cremoris alr gene for alanine racemase (EC 5.1.1.1) is available, for example, under NCBI accession number Y18148.2.

[0562] The respective amino acid sequence of the alanine racemase of Lactococcus lactis subsp. cremoris can be found, for example, at UniProt accession number Q9RLU5 version 99 or NCBI reference sequence number WP_011835506.1.

[0563] The nucleic acid sequence of pAUC1010 is provided in SEQ ID NO: 1 and Figure 1b.

[0564] Figure 1a shows a schematic overview of the plasmid pAUC1010. "alr" refers to the alanine racemase gene used for growth of the host strain in the absence of d-alanine; "T" refers to the terminator sequence; and "repC" and "repA" are prokaryotic genes required for replication of the plasmid by bacterial cells.

[0565] 1.2 Construction of a chloride-inducible expression cassette In the next experiment, a chloride-inducible gene expression cassette was created in which the PgadC promoter from L. lactis subsp. cremoris was used to control the expression of different heterologous genes by L. lactis.

[0566] The PgadC promoter was used in combination with the activator gene gadR and the ribosome binding site and start codon of the gadC gene, both of which were placed downstream of the PgadC promoter, as described by Sanders et al. (1997) and Sanders et al. (1998).

[0567] The PgadC promoter is controlled by the gadR protein, a positive regulator of Pgad, which activates the PgadC promoter in the presence of chloride ions.

[0568] Expression of the gadR gene itself is not activated by chloride ions, but instead is controlled by a constitutive promoter, which is also provided on the chloride-inducible gene expression cassette used in the present invention, along with a ribosome binding site (RBS) located upstream of the ATG start codon of the nucleic acid sequence encoding gadR.

[0569] The nucleic acid sequence of the PgadC promoter, including the gadR gene and the ribosome binding site and start codon of the gadC gene, was extracted from the genome sequence of the strain L. lactis subsp. cremoris MG1363. The respective genome sequences are available, for example, under NCBI accession number NC_009004.1.

[0570] The nucleic acid sequence of the chloride-inducible promoter, including the PgadC promoter region, the gadR gene, and the ribosome binding site and start codon of the gadC gene, is provided in SEQ ID NO: 2 and Figure 2b. The ATG start codon of gadC is shown underlined and in bold capital letters in Figure 2b.

[0571] A schematic diagram of the control elements encompassing the chloride-inducible promoter is shown in Figure 2a.

[0572] Each target gene(s) encoding at least one protein to be expressed by the recombinant bacterium of the present invention was attached to the ATG start codon of gadC. Furthermore, each gene encoding a protein to be expressed by the recombinant bacterium of the present invention was provided with a secretion signal for secretion of the protein from the expression host.

[0573] The secretion signal was preferably derived from the lactococcal protein Usp45, which is described, for example, in van Asseldonk et al. (1993) and van Asseldonk et al. (1990). The amino acid sequence of the secretion signal of lactococcal protein Usp45 is shown in Figure 3 and SEQ ID NO:3.

[0574] Cells have mechanisms to ensure that this signal sequence is cleaved from the target protein after secretion: this reaction is catalyzed by signal peptidases that have specific preferences for certain amino acids at positions surrounding the cleavage site.

[0575] At least one protein to be expressed by the recombinant bacterium of the present invention is expressed as a recombinant precursor protein comprising the respective secretion signal of the lactococcal protein Usp45 at the N-terminus of the respective precursor protein.

[0576] The codon usage of each gene(s) encoding at least one protein to be expressed by the recombinant bacterium of the invention was adapted to the general codon usage of L. lactis.

[0577] Furthermore, when more than one protein was to be expressed by a recombinant bacterium of the present invention, the same Usp45 signal peptide sequence was used for each protein.

[0578] To avoid large regions of identical nucleotides on closely adjacent plasmids that could lead to recombination / deletion within the plasmid, the same signal peptide was encoded by different codons based on the codon degeneracy and codon usage of L. lactis.

[0579] Furthermore, the web-based program SignalP4.1 (http: / / www.cbs.dtu.dk / services / SignalP / ) was used to optimize the cleavage site at which the signal peptide is cleaved from the synthetic precursor protein to generate the mature protein.

[0580] Also, when more than one protein is to be expressed by the recombinant bacterium of the present invention, preferably from at least one operon, each of the respective genes is provided with a ribosome binding site (RBS) to improve protein expression.

[0581] Preferably, the ribosome binding site of the gadC gene was used for the first gene of an operon containing nucleic acid sequences encoding two or more proteins, and the ribosome binding sites of endogenous L. lactis genes, such as the ATP synthase subunit gamma (atpG) gene and / or the galactoside O-acetyltransferase (lacA) gene, were used for the other genes of the operon.

[0582] Suitable ribosome binding sites can be selected from known L. lactis nucleic acid sequences, for example from the nucleotide sequence of the L. lactis subsp. lactis IL1403 genome available from NCBI under accession no. AE005176.1 or the L. lactis subsp. cremoris MG1363 genome available under NCBI accession no. NC_009004.1, as described by Bolotin et al. (2001), based on, for example, a good fit with the 3' end of the 16S rRNA of L. lactis.

[0583] The 3' end of the L. lactis 16S rRNA (5'GGAUCACCUCCUUUCU3') is shown in SEQ ID NO: 4 and in FIG.

[0584] A synthetic nucleic acid construct comprising a chloride-inducible promoter and at least one nucleic acid sequence encoding at least one protein to be expressed by the recombinant bacterium of the present invention was synthesized de novo and ligated into respective expression plasmids as outlined above, which were then transformed into a suitable bacterial strain, preferably L. lactis.

[0585] The following expression plasmids were created:

[0586] 1.3 Construction of an expression plasmid for mCherry expression A different construct containing the mCherry gene under the control of PgadC was prepared. mCherry is a fluorescent protein first described by Beilharz K, et al. (2015).

[0587] The nucleic acid sequence encoding the mCherry protein is available under NCBI accession number KJ908190.1. The corresponding amino acid sequence is available under NCBI accession number AIL28759.1.

[0588] The mCherry coding sequence was fused to the PgadC promoter. A schematic of each insert is shown in Figure 17a.

[0589] The nucleic acid sequence of the synthetic mCherry construct is shown in Figure 17b and SEQ ID NO: 33. A schematic of the synthetic mCherry construct is shown in Figure 17a.

[0590] The completed gene synthesis product was obtained from BaseClear as a fragment cloned onto the E. coli vector pUC57.

[0591] The pUC57 plasmid carrying each gene synthesis product was digested with the restriction enzymes SphI and BglII. The SphI and BglII fragments containing each gene synthesis product were isolated by phenol extraction and ethanol precipitation and ligated into the SphI- and BglII-cut plasmid pAUC1010, resulting in the generation of an expression plasmid designated pC-mCherry.

[0592] The nucleic acid sequence of the expression plasmid, designated pC-mCherry, is shown in Figure 18b and SEQ ID NO: 34. A schematic diagram of the expression plasmid pC-mCherry is shown in Figure 18a.

[0593] 1.4 Creation of expression plasmids for expression of hFGF-2, hIL-4, and hCSF-1 from a single operon The following nucleic acid sequences were generated for expression of human fibroblast growth factor 2 (hFGF-2), human interleukin 4 (hIL-4), and human colony-stimulating factor 1 (hCSF-1) by L. lactis NZ1330.

[0594] 1.4.1 Human Fibroblast Growth Factor 2 (hFGF-2) The amino acid sequence used for expression of hFGF-2 is derived from the 288 amino acid sequence of the human FGF-2 precursor available under NCBI accession number NP_001997.5.

[0595] The mature form of hFGF-2 contains 155 amino acids and is hereafter referred to as hFGF-2-155. It has a molecular weight of 17.3 kDa and a pI of 9.85. The molecule contains four cysteine ​​residues.

[0596] The hFGF-2 variant to be expressed by L. lactis lacks the first two amino acids, methionine and alanine, and contains 153 amino acids. Therefore, this variant was subsequently designated hFGF-2-153. hFGF-2-153 has a molecular weight of 17.1 kDa and a pI of 9.85. This variant contains four cysteine ​​residues.

[0597] The corresponding amino acid sequence of human FGF-2-155 is shown in Figure 5a and SEQ ID NO: 5. The underlined amino acids shown in Figure 5a refer to the first two amino acids, methionine and alanine, which are absent in variant hFGF2-153. The amino acid sequence of the hFGF2-153 variant is shown in Figure 5b and SEQ ID NO: 6.

[0598] The sequence of FGF-2-155 is the mature human FGF-2 sequence after secretion. In vivo, this sequence is further processed. However, various existing recombinant products use this 155 amino acid sequence with or without an N-terminal methionine residue.

[0599] To express and secrete hFGF2, for example, into the supernatant of the growth medium, a signal peptide was added to the N-terminus as described above, creating recombinant hFGF-2-153 precursor protein.

[0600] The amino acid sequence of each of the recombinant hFGF-2-153 precursor proteins is shown in Figure 5c and SEQ ID NO: 7. The underlined amino acids shown in Figure 5c refer to the secretion signal of the lactococcal protein Usp45.

[0601] 1.4.2 Human interleukin-4 (hIL-4) The mature protein of hIL-4 contains 129 amino acids, which correspond to amino acids 25 to 153 of the amino acid sequence available under NCBI accession number NP_000580.1. The mature protein has a molecular weight of 14.96 kDa and a pI of 9.36. The molecule contains six cysteine ​​residues.

[0602] The amino acid sequence of mature hIL-4 is shown in FIG. 6a and SEQ ID NO:8.

[0603] The hIL-4 variant to be expressed by L. lactis contains an additional alanine residue at the N-terminus of the mature protein, thus containing 130 amino acids. It has a molecular weight of 15.03 kDa and a pI of 9.36. This variant also contains six cysteine ​​residues.

[0604] The corresponding amino acid sequence of the human hIL-4 variant to be expressed is shown in Figure 6b and SEQ ID NO: 9. The underlined amino acids shown in Figure 6b refer to an additional alanine residue at the N-terminus of the amino acid sequence.

[0605] To express and secrete hIL-4, for example, into the supernatant of the growth medium, a signal peptide was added to the N-terminus as described above to create recombinant hIL-4 precursor proteins, the amino acid sequences of which are shown in Figure 6c and SEQ ID NO: 10. The underlined amino acids shown in Figure 6c refer to the secretion signal of the lactococcal protein Usp45.

[0606] 1.4.3 Human colony-stimulating factor 1 (hCSF1) The amino acid sequence of hCSF1 used for expression was derived from the 554 amino acid sequence of human CSF1 precursor isoform a, which is available under NCBI accession number NP_000748.3.

[0607] The mature form of hCSF1 spans amino acids 33 to 450 of the precursor. The amino acid sequence is shown in Figure 7a and SEQ ID NO:11.

[0608] The amino acid sequence to be expressed begins with amino acid 33 (E), the first amino acid of mature hCSF1, and ends with amino acid 181 (Q).

[0609] Furthermore, nine additional amino acids, occupying positions 480-488 of the native protein, were added to the C-terminus (GHERQSEGS). An additional alanine residue was added to the N-terminus to improve removal of the signal peptide by signal peptidase. The resulting amino acid sequence is shown in Figure 7b and SEQ ID NO:12.

[0610] The additional alanine residue at the N-terminus in Figure 7b is underlined. The additional 9 amino acids added to the C-terminus are shown in bold in Figure 7b. The protein encoded by this sequence contains 159 amino acids and has a molecular weight of 18.5 kDa and a pI of 4.72. The protein contains seven cysteine ​​residues.

[0611] To express and secrete hCSF1, for example, into the supernatant of the growth medium, a signal peptide was added to the N-terminus as described above to create recombinant hCSF1 precursor proteins. The amino acid sequences of each of the synthetic hCSF1 precursor proteins are shown in Figure 7c and SEQ ID NO: 13. The underlined amino acids shown in Figure 7c refer to the secretion signal of the lactococcal protein Usp45.

[0612] 1.4.4 Creation of expression constructs The amino acid sequences of the synthetic hFGF-2-153 precursor protein shown in Figure 5c and SEQ ID NO:7, the synthetic hIL-4 precursor protein shown in Figure 6c and SEQ ID NO:10, and the synthetic hCSF1 precursor protein shown in Figure 7c and SEQ ID NO:13 were translated into the corresponding nucleic acid sequences, whereby the codon usage was adapted to the general codon usage of L. lactis.

[0613] Two constructs were made with two of six permutations of three genes (FGF2, IL4, CSF1): a) hCSF1, hFGF2, hIL4 (CFI) b) hIL4, hCSF1, hFGF2 (ICF)

[0614] For the design of the operon, each gene was provided with its own ribosome binding site for translation initiation and its own signal peptide for protein secretion, but with the same signal sequence (ssUsp45) encoded by different codons based on the codon degeneracy and codon usage of L. lactis.

[0615] The ribosome binding site of the gadC gene was used for the first gene in each of the two constructs, and the ribosome binding sites of the ATP synthase subunit gamma (atpG) gene and the galactoside O-acetyltransferase (lacA) gene were used for the other two genes in each of the two constructs.

[0616] The nucleic acid sequence of the de novo synthesized CFI construct is shown in Figure 8b and SEQ ID NO: 14. A schematic diagram of the de novo synthesized CFI construct is depicted in Figure 8a.

[0617] The completed gene synthesis products were each obtained from BaseClear as fragments cloned into the E. coli vector pUC57.

[0618] The pUC57 plasmid carrying each gene synthesis product was digested with the restriction enzymes SphI and BglII, respectively, obtained from New England Biolabs (Ipswich, MA, USA). The SphI and BglII fragments containing each gene synthesis product were isolated by phenol extraction and ethanol precipitation and ligated into the SphI- and BglII-cut plasmid pAUC1010, resulting in the generation of an expression plasmid designated pC-CFI.

[0619] The nucleic acid sequence of the expression plasmid, designated pC-CFI, is shown in Figure 9b and SEQ ID NO: 15. A schematic diagram of the expression plasmid pC-CFI is shown in Figure 9a.

[0620] 1.5 Creation of expression plasmids for expression of mIL-18 and mGM-CSF from a single operon The following nucleic acid sequences were engineered for expression of murine interleukin 18 (mIL18) and murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) by L. lactis NZ1330.

[0621] 1.5.1 Murine interleukin-18 (mIL-18) The mature protein of mIL-18 contains 157 amino acids, which correspond to amino acids 36 to 192 of the amino acid sequence of the mouse interleukin-18 isoform a precursor available under NCBI accession number NP_032386.1.

[0622] The amino acid sequence of mature murine IL-18 is shown in Figure 10a and SEQ ID NO:16.

[0623] The mIL-18 variant to be expressed by L. lactis contains an additional alanine residue at the N-terminus of the mature protein. The corresponding amino acid sequence of the murine IL-18 variant to be expressed is shown in Figure 10b and SEQ ID NO: 17. The underlined amino acid shown in Figure 10b refers to the additional alanine residue at the N-terminus of the amino acid sequence.

[0624] To express and secrete mIL-18, for example, into the supernatant of the growth medium, a signal peptide was added to the N-terminus as described above to create synthetic mIL-18 precursor proteins. The amino acid sequences of each of the synthetic mIL-18 precursor proteins are shown in Figure 10c and SEQ ID NO: 18. The underlined amino acids shown in Figure 10c refer to the secretion signal of the lactococcal protein Usp45.

[0625] 1.5.2 Murine Granulocyte-Macrophage Colony-Stimulating Factor (mGM-CSF) Mature mGM-CSF contains 129 amino acids, which correspond to amino acids 18 to 141 of the amino acid sequence of the murine granulocyte-macrophage colony-stimulating factor precursor available in NCBI reference sequence: NP_034099.2.

[0626] The amino acid sequence of mature mouse GM-CSF is shown in FIG. 11a and SEQ ID NO:19.

[0627] The mGM-CSF variant to be expressed by L. lactis contains an additional alanine residue at the N-terminus of the mature protein, replacing the N-terminal serine to optimize cleavage of the signal peptide. The corresponding amino acid sequence of the murine GM-CSF variant to be expressed is shown in Figure 11b and SEQ ID NO: 20. The underlined amino acid shown in Figure 11b indicates the additional alanine residue at the N-terminus of the amino acid sequence.

[0628] To express and secrete mGM-CSF, e.g., into the growth medium supernatant, a signal peptide was added to the N-terminus as described above to create recombinant mGM-CSF precursor proteins. The amino acid sequences of each of the synthetic mGM-CSF precursor proteins are shown in Figure 11c and SEQ ID NO: 21. The underlined amino acids shown in Figure 11c refer to the secretion signal of the lactococcal protein Usp45.

[0629] 1.5.3 Creation of expression constructs The amino acid sequences of the recombinant mIL-18 precursor protein shown in Figure 10c and SEQ ID NO: 18 and the recombinant mGM-CSF precursor protein shown in Figure 11c and SEQ ID NO: 21 were translated into the corresponding nucleic acid sequences, whereby the codon usage was adapted to the general codon usage of L. lactis.

[0630] For the design of the operon, each of the two genes encoding the respective precursor proteins was provided with its own ribosome binding site for translation initiation and its own signal peptide for protein secretion, but with the same signal sequence (ssUsp45) encoded by different codons based on the codon degeneracy and codon usage of L. lactis as outlined above.

[0631] The ribosome binding site of the gadC gene was used for the first gene of the operon (mIL-18), and the ribosome binding site of the ATP synthase subunit gamma (atpG) gene was used for the second gene (mGM-CSF).

[0632] The nucleic acid sequence of the synthetic mEG construct is shown in Figure 12a and SEQ ID NO:22.

[0633] The completed gene synthesis product was obtained from BaseClear as a fragment cloned onto the E. coli vector pUC57.

[0634] The pUC57 plasmid carrying each gene synthesis product was digested with the restriction enzymes SphI and BglII, respectively, obtained from New England Biolabs (Ipswich, MA, USA). The SphI and BglII fragments containing each gene synthesis product were isolated by phenol extraction and ethanol precipitation and ligated into the SphI- and BglII-cut plasmid pAUC1010, resulting in the generation of an expression plasmid designated pC-mEG.

[0635] The nucleic acid sequence of the expression plasmid, designated pC-mEG, is shown in Figure 12c and SEQ ID NO: 23. A schematic diagram of the expression plasmid pC-mEG is shown in Figure 12b.

[0636] 1.6 Creation of expression plasmids for expression of mIL-12, mIL-18, and mIFNa from a single operon The following nucleic acid sequences were generated for expression of murine interleukin 12 (mIL-12), murine interleukin 18 (mIL-18), and murine interferon alpha-2 (mIFNa) by L. lactis NZ1330.

[0637] 1.6.1 Murine interleukin-12 (mIL-12) Interleukin-12 (IL-12) is a heterodimeric cytokine encoded by two separate genes, IL-12A (p35) and IL-12B (p40).

[0638] The mIL-12 variant to be expressed by L. lactis was designed as a fusion protein with mature interleukin-12 subunit beta forming the N-terminal part of the fusion protein and mature interleukin-12 subunit alpha forming the C-terminal part of the fusion protein, both separated by a peptide linker.

[0639] The mature protein of mouse interleukin-12 subunit beta contains 313 amino acids, which correspond to amino acids 23 to 335 of the amino acid sequence of mouse interleukin-12 subunit beta precursor available under NCBI accession number NP_001290173.1.

[0640] The amino acid sequence of mature interleukin-12 subunit beta is shown in Figure 13a and SEQ ID NO:24.

[0641] Murine interleukin-12 subunit alpha exists as at least two isoforms, 1 and 2. The encoded isoform 2 has a shorter N-terminus compared to isoform 1.

[0642] The amino acid sequence of mouse interleukin-12 subunit alpha isoform 1 precursor is available under NCBI accession number NP_001152896.1.

[0643] The mature protein of mouse interleukin-12 subunit alpha isoform 2, whose amino acid sequence was used for expression in the following examples, contains 199 amino acids, which correspond to amino acids 23 to 215 of the amino acid sequence of the mouse interleukin-12 subunit alpha isoform 2 precursor available under NCBI accession number NP_032377.1.

[0644] The amino acid sequence of mature interleukin-12 subunit alpha isoform 2 is shown in Figure 13b and SEQ ID NO:25.

[0645] The amino acid sequence of the mature form of the recombinant interleukin-12 fusion protein is shown in Figure 13c and SEQ ID NO: 26. Furthermore, two additional amino acids (alanine and aspartic acid) were added to the N-terminus of the recombinant fusion protein, which are indicated by underlining in Figure 13c. The linker sequence separating the two IL.12 subunits is marked by bold letters in 13c.

[0646] To express and secrete the mIL-12 fusion protein, for example, into the supernatant of the growth medium, a signal peptide was added to the N-terminus as described above to create a recombinant mIL-12 precursor protein.

[0647] The amino acid sequences of each of the recombinant mIL-12 fusion protein precursors are shown in Figure 13d and SEQ ID NO: 27. The underlined amino acids shown in Figure 13d mark the secretion signal of the lactococcal protein Usp45.

[0648] 1.6.2 Murine interleukin-18 (mIL-18) The amino acid sequences of the mIL-18 variants to be expressed by L. lactis are shown in Figure 10b and SEQ ID NO: 17. The amino acid sequences of the respective recombinant mIL-18 precursor proteins are shown in Figure 10c and SEQ ID NO: 18. The underlined amino acids shown in Figure 10c refer to the secretion signal of the lactococcal protein Usp45.

[0649] 1.6.3 Murine interferon alpha-2 (mIFNa2) The mature form of mIFNa2 contains 167 amino acids, which correspond to amino acids 24 to 190 of the amino acid sequence of the murine interferon alpha-2 precursor available in NCBI reference sequence: NP_034633.2.

[0650] The amino acid sequence of mature mouse IFNa2 is shown in Figure 14a and SEQ ID NO:28.

[0651] The mIFNa2 variant to be expressed by L. lactis contains an additional alanine residue at the N-terminus of the mature protein to optimize cleavage of the signal peptide. The corresponding amino acid sequence of the recombinant murine IFNa2 variant to be expressed is shown in Figure 14b and SEQ ID NO: 29. The underlined amino acid shown in Figure 14b refers to the additional alanine residue at the N-terminus of the amino acid sequence.

[0652] To express and secrete mIFNa2, e.g., into the supernatant of the growth medium, a signal peptide was added to the N-terminus as described above to create recombinant mIFNa2 precursor proteins. The amino acid sequences of each of the synthetic mIFNa2 precursor proteins are shown in Figure 14c and SEQ ID NO: 30. The underlined amino acids shown in Figure 14c refer to the secretion signal of the lactococcal protein Usp45.

[0653] 1.6.4 Creation of expression constructs The amino acid sequences of the recombinant mIL-12 precursor protein shown in Figure 13d and SEQ ID NO: 27, the amino acid sequence of the recombinant mIL-18 precursor protein shown in Figure 10c and SEQ ID NO: 18, and the amino acid sequence of the recombinant mIFNa2 precursor protein shown in Figure 14c and SEQ ID NO: 30 were translated into the corresponding nucleic acid sequences, whereby the codon usage was adapted to the general codon usage of L. lactis.

[0654] For the design of the operon, each of the three genes encoding the respective precursor proteins was provided with its own ribosome binding site for translation initiation and its own signal peptide for protein secretion, but with the same signal sequence (ssUsp45) encoded by different codons based on the codon degeneracy and codon usage of L. lactis as outlined above.

[0655] The first gene of the operon (mIL-12) used the ribosome binding site of the gadC gene, the second gene (mIL-18) used the ribosome binding site of the ATP synthase subunit gamma (atpG) gene, and the third gene (mIFNa2) used the ribosome binding site of the galactoside O-acetyltransferase (lacA) gene.

[0656] The nucleic acid sequence of the synthetic mTEA construct is shown in Figure 15 and SEQ ID NO:31.

[0657] The completed gene synthesis product was obtained from BaseClear as a fragment cloned onto the E. coli vector pUC57.

[0658] The pUC57 plasmid carrying the gene synthesis products was digested with the restriction enzymes SphI and BglII. The SphI and BglII fragments containing each gene synthesis product were isolated by phenol extraction and ethanol precipitation and ligated into the SphI- and BglII-cut plasmid pAUC1010, resulting in the generation of an expression plasmid designated pC-mTEA.

[0659] The nucleic acid sequence of the expression plasmid, designated pC-mTEA, is shown in Figure 16b and in SEQ ID NO: 32. A schematic diagram of the expression plasmid pC-mTEA is shown in Figure 16a.

[0660] 1.7. Creation of expression plasmids for expression of mGM-CSF, mIL-12, and mIL-18 from a single operon The following nucleic acid sequences were engineered for expression of murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), murine interleukin-12 (mIL-12), and murine interleukin-18 (mIL-18) by L. lactis NZ1330.

[0661] 1.7.1 Murine Granulocyte-Macrophage Colony-Stimulating Factor (mGM-CSF) The mGM-CSF variant to be expressed by L. lactis is shown in Figure 11b and SEQ ID NO: 20. The underlined amino acids shown in Figure 11b refer to the additional alanine residue at the N-terminus of the amino acid sequence, as described above in Section 1.5.2. The amino acid sequence of each of the synthetic mGM-CSF precursor proteins is shown in Figure 11c and SEQ ID NO: 21. The underlined amino acids shown in Figure 11c refer to the secretion signal of the lactococcal protein Usp45.

[0662] 1.7.2 Murine interleukin-12 (mIL-12) The mIL-12 variants to be expressed by L. lactis were designed as fusion proteins with the mature interleukin-12 subunit beta forming the N-terminal part of the fusion protein and the mature interleukin-12 subunit alpha forming the C-terminal part of the fusion protein, both separated by a peptide linker as described above in Section 1.6.1. The amino acid sequences of each of the recombinant mIL-12 fusion protein precursors are shown in Figure 13d and SEQ ID NO: 27. The underlined amino acids shown in Figure 13d mark the secretion signal of the lactococcal protein Usp45.

[0663] 1.7.3 Murine Interleukin-18 (mIL-18) The amino acid sequences of the mIL-18 variants to be expressed by L. lactis are shown in Figure 10b and SEQ ID NO: 17. The amino acid sequences of the respective recombinant mIL-18 precursor proteins are shown in Figure 10c and SEQ ID NO: 18. The underlined amino acids shown in Figure 10c refer to the secretion signal of the lactococcal protein Usp45.

[0664] 1.7.4 Creation of expression constructs The amino acid sequences of the recombinant mGM-CSF precursor protein shown in Figure 11c and SEQ ID NO: 21 and the recombinant mIL-12 fusion protein precursor shown in Figure 13d and SEQ ID NO: 27 were translated into the corresponding nucleic acid sequences, whereby the codon usage was adapted to the general codon usage of L. lactis.

[0665] For the design of the operon, each of the two genes encoding the respective precursor proteins was provided with its own ribosome binding site for translation initiation and its own signal peptide for protein secretion, but with the same signal sequence (ssUsp45) encoded by different codons based on the codon degeneracy and codon usage of L. lactis as outlined above.

[0666] The ribosome binding site of the gadC gene was used for the first gene (mGM-CSF), the ribosome binding site of the ATP synthase subunit gamma (atpG) gene for the second gene (mIL-12), and the ribosome binding site of the galactoside O-acetyltransferase (lacA) gene for the third gene (mIL-18).

[0667] The nucleic acid sequence of the synthetic mGTE construct is shown in Figure 16a and SEQ ID NO:33.

[0668] The completed gene synthesis product was obtained from BaseClear as a fragment cloned onto the E. coli vector pUC57.

[0669] The pUC57 plasmid carrying each gene synthesis product was digested with the restriction enzymes SphI and BglII, respectively, obtained from New England Biolabs (Ipswich, MA, USA). The SphI and BglII fragments containing each gene synthesis product were isolated by phenol extraction and ethanol precipitation and ligated into the SphI- and BglII-cut plasmid pAUC1010, resulting in the generation of an expression plasmid designated pC-mGTE.

[0670] The nucleic acid sequence of the expression plasmid, designated pC-mGTE, is shown in Figure 16c and SEQ ID NO: 34. A schematic diagram of the expression plasmid pC-mGTE is shown in Figure 16b.

[0671] Example 2: Creating recombinant bacteria The expression plasmids obtained in Example 2 were transformed into L. lactis strains by means of electroporation as outlined above. The respective L. lactis strains used in the following examples are summarized in Table 1 below.

[0672] [Table 1]

[0673] For further testing, approximately 1 × 10 11 Stocks of each recombinant bacterium with a cell density of colony-forming units (CFU) / ml were prepared by growth in IM1 or CDM3 medium containing lactose and sodium beta-glycerophosphate followed by the addition of glycerol to a final concentration of 20 wt%. The stocks were stored at -80°C.

[0674] The composition of CDM3 medium is based on ZMB3 medium described by Zhang, G. and Block, DE ("Using highly efficient nonlinear experimental design methods for optimization of Lactococcus lactis fermentation in chemically defined media", Biotechnol. Prog. 2009, 25(6): pages 1587 to 1597, doi:10.1002 / btpr.277).

[0675] Example 3 Determination of Pgad promoter activity in vitro For induction studies, IM1 medium was used for growth of each L. lactis NZ1330 strain indicated in Table 1. IM1 medium was supplemented as described below for the following experiments: 2 wt% β-glycerophosphate (Sigma, Catalog no. 50020-500G) 1.5 wt% soy peptone (BD, catalog no. 211906) 1wt% yeast extract (BD, catalog no. 212750) 1 mM MgSO4.7H2O (Merck, catalog no. 1058860500) 0.1 mM MgSO4.7H2O (Sigma, catalog no. M-7634) 3 wt% glucose (Natural Spices BV, catalog no. ES212)

[0676] In pH-controlled fermentations, 3.5 M NH3OH was used for base addition.

[0677] Induction Protocol Frozen stocks from the cell bank were used to inoculate 50 ml of IM1 medium supplemented with 3% glucose, followed by a 16-hour incubation at 30°C (preculture). These overnight cultures were used to inoculate 100 ml fermentors (1 vol% inoculum) set at 30°C in IM1 medium with 3% glucose, pH 6.5. The cultures were mixed using a magnetic stirrer at a continuous speed of 200 rpm.

[0678] The expression of each gene is 600 = 0.5, and were induced by 100 mM NaCl. Control cells were grown in medium without added NaCl, and therefore expression of the respective genes was not induced.

[0679] The effect of chloride-induced expression of heterologous genes on the viability of L. lactis NZ1330 was assessed by comparing the growth curves of NaCl-induced L. lactis NZ1330 expressing mCherry under the control of Pgad with uninduced controls.

[0680] Cultures were sampled at 0.5, 1, 2, 3, 4, 5, and 6 hours. These samples were taken at OD 600 The induced bacterial cells were used for measurement and mCherry activity determination. Bacterial growth was monitored by measuring the optical density at 600 nm (OD600) of either the induced bacterial cells or the uninduced control. A comparison of the respective growth curves is shown in Figure 19a.

[0681] mCherry fluorescence activity was measured using a Synergy HT fluorescence spectrophotometer (Biotek, Winooski, Vermont, United States). Excitation was at 530 nm, emission was measured at 590 nm, and a gain of 120 was applied.

[0682] The results are shown in Figure 19b. As can be seen in Figure 19b, induction of mCherry gene expression by adding 100 mM NaCl resulted in a significant increase in fluorescence, which corresponded to the increased amount of mCherry protein produced by chloride-induced expression of the heterologous gene in L. lactis NZ1330. The increased amount of mCherry protein expression did not significantly impact the viability of each bacterium after induction of mCherry gene expression, because, as can be seen in Figure 19a, the growth curves of the induced bacteria did not show a significantly reduced growth rate compared to the uninduced bacteria.

[0683] Chloride-induced expression of FGF-2, IL-4, and CSF-1 from the recombinant strain designated AUP-1602-C was determined by Western blotting from TCA precipitates of 1 ml supernatant obtained by centrifugation of each culture at 14000 g for 10 min at +4°C using the protocol indicated above.

[0684] Commercially available purified preparations of recombinant human CSF-1 protein (Abcam-PLC, 5 ng), recombinant human FGF-2 protein (R&D Systems, 7.5 ng), and recombinant human IL-4 protein (Sigma Aldrich, 5 ng) were used as positive controls. The primary antibodies used are indicated above. The secondary antibody used was an anti-rabbit IgG HRP-linked antibody (catalog number #7074) from New England Biolabs.

[0685] The respective Western blots are shown in Figure 20a. As can be seen in Figure 20a, all three recombinant proteins were expressed by AUP-1602-C and released into the supernatant.

[0686] Chloride-induced expression of GM-CSF from the recombinant strain designated AUP5563-C was determined by Western blotting from TCA precipitates of 1 ml supernatants obtained by centrifugation of each culture at 14,000 g for 10 min at +4° C., using the protocol indicated above. A commercially available purified preparation of recombinant mouse GM-CSF protein (Sigma Aldrich) was used as a positive control.

[0687] The respective Western blots are shown in Figure 20b. As can be seen in Figure 20b, GM-CSF is expressed by AUP5563-C and released into the supernatant.

[0688] Chloride-induced expression of IL-18 from the recombinant strain designated AUP5563-C was determined by Western blotting from TCA precipitates of 1 ml supernatants obtained by centrifugation of each culture at 14,000 g for 10 min at +4° C., using the protocol indicated above. A commercially available purified preparation of recombinant mouse IL-18 protein (R&D Systems) was used as a positive control.

[0689] The respective Western blots are shown in Figure 20c. As can be seen in Figure 20c, IL-18 is expressed by AUP5563-C and released into the supernatant.

[0690] Chloride-induced expression of IL-12 from the recombinant strains designated AUP5551m-C and AUP5563-C was determined by Western blotting from TCA precipitates of 1 ml supernatants obtained by centrifugation of the respective cultures at 14,000 g for 10 min at +4° C., using the protocol indicated above. A commercially available purified preparation of recombinant mouse IL-12 protein (R&D Systems) was used as a positive control.

[0691] The respective Western blots are shown in Figure 20d. As can be seen in Figure 20d, IL-12 is expressed by AUP5551m-C and AUP5563-C and released into the supernatant.

[0692] Example 4 Determination of Pgad promoter activity in vivo The activity of the Pgad promoter system in vivo was assessed by utilizing a chloride-inducible mCherry construct and measuring the in situ fluorescent signal after intratumoral (it) injection.

[0693] Murine CT26 is an N-nitroso-N-methylurethane (NNMU)-induced undifferentiated colon carcinoma cell line from Mus musculus that was cloned to create a cell line designated CT26.WT (ATCC® CRL-2638™), which is commercially available from LGC Standards GmbH.

[0694] When inoculated into BALB / c mice, CT26.WT cells can induce lethal tumors.

[0695] According to Wang, M. et al. (1995), BALB / c mice subcutaneously inoculated with CT26 cells were incubated for 10 3 In cells, it occurs at 80% frequency, and 4 The cells developed lethal tumors in 100% of mice. 4 When cells were inoculated intravenously, lung metastases developed. BALB / cJ mice (genotype: A / A Tyrp1b / Tyrp1b Tyrc / Tyrc, Stock Code 000651) commercially obtained from The Jackson Laboratory (Bar Harbor, ME, USA) were used in this study.

[0696] BALB / c mice were inoculated with CT26.WT cells by the following procedure.

[0697] 1 x 10 in 0.1 ml PBS -5Tumor cells were injected subcutaneously into the left flank of mice. Tumors grew to approximately 100–200 mm. 3 Treatment was initiated when tumors reached a mean tumor volume of 1000 mg / kg, after which mice were assigned to their treatment groups with uniform mean tumor volumes between groups.

[0698] After a single intratumoral injection of L. lactis NZ1330 containing the pC-mCherry construct, tumor-bearing mice were imaged using an IVIS Spectrum CT (excitation filter: 535-570 nm / emission filter: 580-680 nm). Animals were imaged 48 h after intratumoral injection of L. lactis NZ1330 (pC-mCherry) bacteria or control bacteria, i.e., L. lactis NZ1330 without the mCherry plasmid. The time length and binning (sensitivity) of image acquisition depended on the intensity of the lesions present. Images were captured and processed using Living Image 4.3.1 software (PerkinElmer, Inc., Waltham, MS, US).

[0699] The results are shown in Figure 21, which shows fluorescence imaging 48 hours after it injection of L. lactis NZ1330 (pC-mCherry) and control bacteria. The black circle indicates the location of the tumor. Higher mCherry activity is indicated by the black area.

[0700] As can be seen in Figure 21, 2 x 10 cells obtained in Example 3 and expressing mCherry protein under the control of the PgadC promoter system 10 A single 50 μl intratumoral injection of CFU / ml recombinant bacteria provided sufficient localization and survival of each bacterium in the tumor environment.

[0701] The results clearly demonstrate that the recombinant bacteria of the present invention can survive long enough in the tumor environment to express and secrete at least one therapeutic protein, thereby influencing cells of the innate and / or adaptive immune system in the tumor environment to achieve an anti-tumor response.

[0702] Furthermore, the chloride concentration within the tumor environment is sufficiently high to provide for expression of each of the at least one therapeutic protein to be expressed by the recombinant bacteria of the present invention.

[0703] Example 5: Wound closure experiment Diabetic patients are prone to impaired wound healing, with foot ulcerations being particularly prevalent. This delayed wound healing also applies to diabetic animals, including spontaneously diabetic (db / db) mice obtained commercially from The Jackson Laboratory (Bar Harbor, ME, US).

[0704] Sixty diabetic mice (strain name BKS.Cg-Dock7m+ / +Leprdb / J-Stock Code00642), all male and approximately 10 weeks old, were used in the first study.

[0705] The consequences of application of bacteria expressing a combination of human FGF-2, CSF1, and IL-4, each under the control of the PgadC promoter, to full-thickness excision wounds in db / db diabetic mice were investigated by a pharmacokinetic (PK) / pharmacodynamic (PD) combination efficacy study.

[0706] In this study, a recombinant bacterium designated AUP16-C obtained in Example 3, which expresses FGF-2, IL-4, and CSF-1 in a single bacterial cell, was evaluated and compared with vehicle treatment. The composition of each vehicle used in the application is summarized below: Vehicle 1: 5% dextrose in 0.9% saline Vehicle 2: 5% dextrose + 0.9% saline + 200 mM Na acetate (pH 6.5) Vehicle 3: 5% dextrose + 2.5% saline Vehicle 4: 5% dextrose + 2.5% saline + 200 mM Na acetate (pH 6.5) Vehicle 5: 10% dextrose + 0.9% saline Vehicle 6: 10% dextrose + 0.9% saline + 200 mM Na acetate (pH 6.5)

[0707] Animals were randomized to one of seven treatment regimens according to Table 2.

[0708] [Table 2]

[0709] Recombinant bacteria expressing FGF-2, IL-4, and CSF-1 by a single bacterial cell were applied to the wounds on the day of wounding (day 0), and their survival was examined 6 hours after application.

[0710] To determine the production of FGF-2, CSF-1, and / or IL-4 by bacteria applied to the wound, wound fluid samples were obtained from the wound at 6 hours and 1, 2, and 7 days. To aid in PK / PD analysis, systemic blood and major organs were harvested at 6 and 24 hours and 7 days. Wounds receiving Vehicle 1 alone served as controls for the PK / PD component of this study.

[0711] To examine the impact of these recombinant bacteria on the wound healing process, bacteria expressing human FGF-2, CSF-1, and IL-4 were applied to wounds on the day of wounding (day 0) and daily thereafter until post-wounding day 6. Healing of wounds receiving these recombinant bacteria was compared to that of similar wounds exposed to fresh vehicle 1 alone.

[0712] Wound healing was studied at both the macroscopic and histological levels. Wound healing was studied at the macroscopic level in terms of the initiation of a neodermal repair response, wound contraction, and wound closure.

[0713] After wounding, wound closure and its components, wound contraction and wound re-epithelialization, were determined from digital photographs taken on days 0, 4, and 7 after wounding. Histological assessments of granulation tissue formation (depth) and wound width (cranial-caudal contraction) were performed on routine (H&E) stained sections. These histological assessments were performed on tissue harvested on day 7 after wounding.

[0714] The occurrence of adverse effects was monitored and fully recorded.

[0715] Creation of full-thickness experimental wounds and application of treatments All mice were anesthetized with isoflurane and air; their dorsal flank skin was depilated and cleaned. A single, standardized, full-thickness wound (10 mm × 10 mm) was created on the left flank approximately 10 mm from the spine. Each wound was then photographed with an identification plate and a calibrated ruler.

[0716] All wounds were then covered with a transparent film dressing, Tegaderm® Film (3M Deutschland GmbH, Neuss, DE). The animals were then allowed to recover in a warm environment (34°C). The animals were then restrained and administered one of the treatments listed in Table 2, each of which was applied by injection through the Tegaderm® film using a 27-gauge needle.

[0717] Treatment was similarly reapplied to the wounds daily until post-wounding day 6, therefore each wound received seven applications. All wounds were closely monitored for excessive buildup of applied agent and excessive wound site hydration; excess product / fluid was removed by aspiration on post-wounding days 4, 6, and 8, as well as prior to reapplication on days 4 and 6, and prior to dressing removal on day 8.

[0718] On days 4, 8, 12, and 16 after wounding, all animals were re-anesthetized, the film dressings and any loose debris were gently removed, and the wounds were cleaned with sterile saline-soaked gauze. The wounds were then photographed and re-covered with Bioclusive® film dressings (as above), and the animals were allowed to recover in a warm environment (34°C).

[0719] Immediately after wounding and then at 4, 8, and 12 days, all wounds were digitally photographed with a calibrated / identifying plate after film dressing removal and wound cleansing.

[0720] All animals were sacrificed on day 20 after wound photography. Sacrificing was achieved by methods that complied with UK Home Office Schedule 1.

[0721] Image analysis of wound closure Each wound, along with an identification / calibration plate, was digitally photographed immediately after injury and at 4, 8, 12, 16, and 20 days thereafter, and the open wound area was measured and expressed as % wound area relative to day 0.

[0722] Image Pro Plus image analysis software (version 4.1.0.0, Media Cybernetics, Inc., Rockville, MD, US) was used to calculate wound closure from scaled wound images taken at each evaluation time point.

[0723] The results for each treatment group are summarized in Table 3 and FIG.

[0724] For a given wound at a given time point, wound closure was expressed as the percentage wound area remaining relative to the initial wound area immediately after injury (i.e., day 0). Table 3 shows the mean percentage wound area remaining for all treatment groups.

[0725] [Table 3]

[0726] As can be seen from the data presented in Table 3, all groups receiving AUP1602-C bacteria were found to close significantly more rapidly than the control group (5% dextrose in 0.9% saline) at all time points, regardless of delivery vehicle.

[0727] Wound contraction Contraction is the central movement of the wound periphery due to compaction of granulation tissue within the "body" of the wound.

[0728] The "compaction" forces that drive this process are thought to reside in cells of the fibroblast lineage. In this study, % contraction was: % contraction = area defined by the border of normal dermis and "repair neodermis" x 100 / original wound area (day 0) was calculated as:

[0729] The mean percentage wound contraction data for all treatment groups is set forth in Table 4 and in FIG.

[0730] [Table 4]

[0731] As can be seen from Table 4, all groups receiving AUP1602-C bacteria were found to contract significantly more rapidly than the control group (5% dextrose in 0.9% saline) at all time points, regardless of delivery vehicle (p≦0.007).

[0732] Wounds in non-diabetic mice close primarily by contraction, whereas those in diabetic mice have a significantly reduced ability to contract, likely due to impaired granulation tissue formation. As a result, wounds in diabetic animals tend to close by re-epithelialization to a greater extent than those in non-diabetic animals. The force driving the contraction process is thought to derive from the activity of fibroblasts resident in the neodermal compartment of skin wounds.

[0733] The observation of enhanced contraction after application of AUP1602-C clearly suggests improved granulation tissue function; this, in turn, may be explained by an increased amount of granulation tissue formed, an increased speed at which it forms, and / or an increased contractile capacity of the tissue.

[0734] Initiation of wound healing (neodermal tissue formation) To establish a "healed" status, wounds were visually evaluated daily until day 8, and every other day thereafter. Each wound was scored as to whether it displayed "neodermal tissue formation activity," i.e., whether the wound had initiated the dermal healing process. Each wound was evaluated by two independent observers, and the percentage of wounds displaying "neodermal tissue formation activity" was compared between treatment groups at each evaluation time point.

[0735] The number of responding wounds for each treatment group on each day is displayed in Table 5.

[0736] The percentage of wounds responding for each treatment group on day 1 is displayed in Figure 24. In the majority of wounds receiving AUP1602-C, a healing response was evident after the first day of dosing on day 1 post-wounding, regardless of the delivery vehicle used.

[0737] On the first day after wounding, 100% of the wounds in treatment groups AUP1602-C(Veh2), AUP1602-C(Veh3), and AUP1602-C(Veh6), 88% in AUP1602-C(Veh4), 80% in AUP1602-C(Veh1), and 75% in AUP1602-C(Veh5) were found to have initiated a healing response.

[0738] No significant differences were detected between these treatment groups (Fisher's exact test).

[0739] It was found that 100% of wounds receiving AUP1602-C demonstrated a healing response by day 2 post-wounding.

[0740] None of the wounds that received Vehicle 1 alone (5% dextrose in 0.9% saline) demonstrated a healing response during the 20 day study period.

[0741] Compared to the vehicle control group, a significantly greater proportion of wounds were found to respond in all AUP1602-C treatment groups at all time points evaluated (p≦0.001, Fisher's exact test).

[0742] [Table 5]

[0743] Compared to that observed in the Vehicle 1 control group (5% dextrose alone in 0.9% saline), a significantly greater proportion of wounds receiving AUP1602-C bacteria initiated "neodermal tissue formation," regardless of formulation; i.e., 100% of wounds receiving AUP bacteria responded; wounds receiving Vehicle 1 alone did not appear to respond.

[0744] For all formulation vehicles, AUP1602-C treatment initiated a response in the wound bed very quickly (within 1 to 2 days) after application.

[0745] Angiogenic response To quantify the level of angiogenesis, wound images were visually assessed on days 4 and 8. The mean scores of the angiogenic response on days 4 and 8 for all groups are shown in Table 6, where the scores from two independent observers were averaged.

[0746] [Table 6]

[0747] All treatments involving AUP1602-C scored significantly higher for angiogenesis than vehicle 1 alone when observed on days 4 and 8 (p≦0.002).

[0748] Wounds receiving AUP1602-C bacteria, regardless of vehicle, demonstrated significantly increased vascularization compared to the vehicle 1 control group (5% dextrose in 0.9% saline alone) at both the 4th and 8th day assessment time points.

[0749] All bacterial treatment regimens tested in this study, regardless of vehicle, were found to promote wound repair in diabetic db / db mice, one of the widely accepted and best-validated animal models of delayed wound healing in humans.

[0750] Excisional wounds in db / db mice show a statistically significant delay in wound closure, reduced granulation tissue formation, decreased wound bed vascularity, and markedly diminished proliferation, as described, for example, by Michaels et al. (2007) (“db / db mice exhibit severe wound-healing impairments compared with other murine diabetic strains in a silicone-splinted excisional wound model”, Wound Rep. Reg. 15, pages 665 to 670).

[0751] The experimental results clearly demonstrate that application of the recombinant bacteria of the present invention provides a significant improvement in the treatment of human wounds, especially chronic wounds.

[0752] Application of the recombinant bacteria of the present invention induces granulation tissue formation, increased wound bed vascularity, and proliferation, thus leading to improved and accelerated wound closure even in models displaying severely impaired wound healing.

[0753] Therefore, these results indicate that inflammatory, preferably chronic inflammatory, skin disorders such as frostbite, eczema, psoriasis, dermatitis, ulcers, wounds, systemic lupus erythematosus, neurodermatitis, and combinations thereof, preferably dermatitis, ulcers, wounds, and combinations thereof, more preferably ulcers, will benefit from the application of the recombinant bacteria of the present invention.

[0754] For example, chronic wounds such as chronic venous ulcers, chronic arterial ulcers, chronic diabetic ulcers, and chronic pressure ulcers can be treated by application of the recombinant bacteria of the present invention, since the impaired wound healing observed in each chronic wound is overcome after application of the recombinant bacteria of the present invention, which preferably express human FGF-2, human IL-4, and human CSF-1.

[0755] Example 6: Detection of human FGF-2, human IL-4, and human CSF-1 in wound fluid. As in Example 5, all male, approximately 10-week-old diabetic mice (strain BKS.Cg-Dock7m+ / +Leprdb / J-Stock Code00642) were used in the subsequent studies.

[0756] In this study, the detection of each recombinant protein was investigated after application of bacteria expressing a combination of human FGF2, CSF1, and IL4, each under the control of the PgadC promoter, to full-thickness excision wounds of db / db diabetic mice. In this study, the recombinant bacteria obtained in Example 3, designated AUP16-C, which expresses FGF-2, IL-4, and CSF-1 in a single bacterial cell, was evaluated for the composition of the vehicle. The composition of each vehicle used for application is summarized below: Vehicle 1: 5% dextrose in 0.9% saline Vehicle 4: 5% dextrose in 2.5% saline +200mM Na acetate (pH 6.5) Vehicle 7: 5% dextrose in 2.5% saline + 200mM Sodium Acetate + 50mM L-glutamic acid (pH 6.0)

[0757] Wound fluid samples were prepared using a commercially available 5 kDa cut-off concentrator from Sigma-Aldrich (Vivaspin® sample concentrator) with 0.5% SDS according to the manufacturer's protocol.

[0758] Samples were separated by SDS PAGE gel and blotted onto nitrocellulose as described above in section I.8.

[0759] The recombinant proteins were detected using the following antibodies:

[0760] For detection of human FGF-2, purified mouse anti-human FGF-2 monoclonal antibody (BD Transduction Laboratories, Heidelberg, DE) was used at a dilution of 1:250. The secondary antibody was horseradish peroxidase (HRP)-linked anti-mouse IgG antibody obtained from Bioke BV (Leiden, NL) at a dilution of 1:10,000.

[0761] For detection of human IL-4, monoclonal mouse anti-human IL-4 IgG1 (clone #3007) from R&D systems was used at a dilution of 1:500. The secondary antibody was an HRP-linked anti-mouse IgG antibody from Bioke BV (Leiden, NL) at a dilution of 1:10,000.

[0762] For detection of human CSF-1, a rabbit polyclonal antibody against human MCSF (clone ab9693) from Abcam PLC was used at a dilution of 1:2,000. The secondary antibody was an HRP-linked anti-rabbit IgG antibody from Bioke BV (Leiden, NL) at a dilution of 1:2,000.

[0763] The amount of each recombinant protein in the wound fluid was determined by comparison with the respective reference protein.

[0764] Human FGF-2 reference protein was obtained from Sigma-Aldrich and dissolved in 5 mM Tris pH 7.5 to give a final concentration of 100 μg / μl. As a reference, total amounts of 10 mg, 3.3 ng, 1.1 ng, 0.37 ng, and 0.12 ng were applied to different lanes of a Western blot. The results are shown in Figures 25a and 25b.

[0765] Human IL-4 reference protein was obtained from R&D systems (Minneapolis, MN, US). Human IL-4 was dissolved in PBS (5.8 mM NaHPO, 4.2 mM NaHPO, 145 mM NaCl) to give a final concentration of 100 μg / mL. As a reference, total amounts of 10 mg, 3.3 ng, 1.1 ng, 0.37 ng, and 0.12 ng were applied to different lanes of a Western blot. The results are shown in Figures 26a and 26b.

[0766] Human CSF-1 reference protein was obtained from Abcam PLC (Cambridge, GB). Human CSF-1 was dissolved in sterile water to give a final concentration of 100 μg / mL. As a reference, total amounts of 10 mg, 3.3 ng, 1.1 ng, 0.37 ng, and 0.12 ng were applied to different lanes of a Western blot. The results are shown in Figures 27a and 27b.

[0767] Recombinant bacteria using the PgadC system properly expressed human FGF-2, human IL-4, and human CSF-1 in vivo, and all three target proteins could be detected in wound fluid.

[0768] Under in vivo conditions, AUP1602-C harboring the PgadC promoter system produced sufficient amounts of each target protein after topical application of each recombinant bacterium without the requirement for additional administration of exogenous inducers and / or excipients, such as β-galactosidase or nisin.

[0769] Example 7 Inhibition of cancer cell growth in vivo A prerequisite for successful immunotherapy is the existence of a fully functional immune system, in which tumor immunogenicity is considered a strong predictor of response to immunotherapy.

[0770] Among the different mouse models developed to evaluate the properties of anti-cancer drugs and / or treatments, syngenic models are tumor-bearing mice of genetically identical murine strains. These models provide an intact immune and microenvironmental system for testing immunotherapeutic and / or anti-angiogenic drugs and treatment regimens.

[0771] The background mouse strain used to generate each murine model has been shown to influence tumor immunogenicity, with tumors established in BALB / c mice generally being more immunogenic than tumors established in, for example, C57BL / 6 mice.

[0772] Furthermore, a study of murine solid tumor models conducted by Lechner, M., Get et al. (2013) demonstrated that among different cell lines, CT26, RENCA, and 4T1 tumors exhibited the most significant positive responses to immunotherapy regimens, including significantly reduced tumor growth and increased survival.

[0773] Therefore, CT26 cells were selected as the cell line to be used in BALB / C mice to initially evaluate the efficacy of AUP5563-C4 administration in vivo.

[0774] An efficacy study with AUP5563-C4 expressing IL-12, IL-18, and GM-CSF was conducted to evaluate the effect of intratumoral administration of the bacterial preparation AUP5563-C4 in treating a subcutaneous murine CT-26 cancer model.

[0775] As described above in Example 4, CT26.WT cells were used to induce lethal tumors in BALB / c mice, with 1 × 10 cells in 0.1 ml of PBS. 5 CT26.WT cells were injected subcutaneously into the left flank of mice using a 27-gauge needle.

[0776] All comments regarding weight, medication, and clinical status were captured in real time using the study management software StudyDirector (StudyLog Systems, Inc., San Francisco, CA, US).

[0777] Tumors were measured three times weekly during treatment, and apparent tumor volume was calculated using the formula V = 0.5 × (length × width 2 ) where V is tumor volume, L is tumor length, and W is tumor width, by measuring the tumor in two dimensions with electronic calipers, as described, for example, in Tomayko, M. and Reynolds, C. (1989).

[0778] Mice were randomly assigned to treatment groups. Treatments were administered to mice with tumors between 50 and 100 mm 3 Treatment was initiated when the tumor reached a mean tumor volume of 100 mg / kg / day. Mice were assigned to their treatment groups 1 through 3 with uniform mean tumor volumes across groups. Treatment duration was up to 2 weeks. Treatment groups are summarized in Table 7.

[0779] [Table 7]

[0780] Three weekly doses of AUP5563-C4 (1 × 10 8 Efficacy was assessed after intratumoral (it) injection of 1000 CFU.

[0781] Vehicle-treated mice served as a negative control, and anti-CTLA-4-treated mice served as a positive control. The monoclonal anti-mouse CTLA-4 antibody clone 9D9 was obtained from Bio X Cell (West Lebanon, NH, US) and is directed against the murine cytotoxic T lymphocyte-associated protein 4 (CTLA-4), a protein receptor that downregulates the immune system.

[0782] The results are summarized in Figures 28a to 28c, which show assessment of tumor growth following intratumoral (it) administration of (a) saline (negative control), (b) AUP5563-C4, and (c) murine anti-CTLA4 (positive control), respectively.

[0783] As can be seen in Figures 28a and 28b, three weekly doses of AUP5563-C4 (1 x 10 8 CFU) significantly reduced tumor growth compared to vehicle-treated control mice.

[0784] Three weekly doses of AUP5563-C4 (1 × 10 8 Administration of m-CTLA-4 antibodies resulted in a reduction in tumor growth comparable to treatment with anti-m-CTLA-4 antibody, which was used as a positive control.

[0785] The results demonstrate that it administration of AUP5563-C4 had similar efficacy to the immune checkpoint inhibitor anti-CTLA4.

[0786] Tumors that received intratumoral injections of saline (negative control) grew normally.

[0787] Example 8 Treatment of intraperitoneal tumors in mice To evaluate the feasibility and efficacy of the recombinant bacteria of the present invention expressing at least one therapeutic protein under the control of the Pgad promoter after intraperitoneal administration, B16-F1 cells were inoculated intraperitoneally into C57BL / 6 mice, and survival was measured as the primary endpoint.

[0788] B16 melanoma cells were selected for tumor inoculation because, as described by Fu Q. et al. (2016), C57BL / 6 mice are syngenic hosts for B16-F1 cells, which have a high tumor uptake rate.

[0789] Furthermore, this model has been shown to respond to immunotherapeutic treatment regimens as described by Lesinski, GB et al (2003).

[0790] Inoculation of C57BL / 6 mice with murine B16-F1 cells leads to the development of malignant tumors, which, without any treatment, can kill the mice before day 20 after implantation.

[0791] Mouse B16-F1 (KCLB no. 80007) cells were obtained from the Korean Cell Line Bank. Cells were seeded in 96-well microplates and incubated for 24 hours under the following conditions: ·Culture conditions: 37℃. Culture medium: Dulbecco's modified Eagle's medium (DMEM) supplemented with penicillin 50 U / ml, streptomycin 50 μg / ml, and fetal bovine serum at a final concentration of 10% by volume (Life Technologies Corporation, Carlsbad, CA, US).

[0792] B16-F1 cells are a well-known model for the study of metastasis and solid tumor formation.

[0793] A total of 66 C57BL / 6N mice commercially obtained from Orient Bio Inc. (Gyeonggi-do, KR) were used in this study.

[0794] 1×10 5 of B16F1 cells were inoculated into each individual C57BL / 6 mouse by intraperitoneal (ip) injection (day 0). Treatment with either vehicle (5% dextrose in 0.9% saline) or AUP2059 / AUP5551-C (1:1) was administered intraperitoneally (ip) three times weekly (300 μl per injection) starting on day 0 for up to 5 weeks (up to 15 doses between days 0 and 35).

[0795] Animals were randomized to one of six treatment regimens according to Table 8.

[0796] [Table 8]

[0797] Treatment was carried out by administering the combination of drug products AUP2059 (mIL18 / mGM-CSF) and AUP5551-C (mIL12 / mIL18 / mIFNa2b), respectively, obtained in Example 3.

[0798] Recombinant bacteria were cultured at 2 x 10 per 300 μl dose. 5 , 2 × 10 6 , 2 × 10 7 , or 2 × 10 8 The total amount of CFU was mixed in a 1:1 ratio based on CFU / mL.

[0799] Treatment was administered by ip injection of a 1:1 combination of AUP2059 (mIL18 / mGM-CSF) and AUP5551-C2 (mIL12 / mIL18 / mIFNa2b) in commercially available 5% dextrose in 0.9% saline solution obtained from Dai Han Pharm. CO. (Dai Han, KR).

[0800] All dosing was initiated on day 0 after inoculation with B16F1 cells.

[0801] The mean survival times compared to vehicle treatment are summarized in FIG.

[0802] As can be seen in Figure 29, treatment with the combination of AUP2059 (mIL18 / mGM-CSF) and AUP5551-C2 (mIL12 / mIL18 / mIFNa2b) leads to a significant increase in survival time compared to vehicle-treated mice.

[0803] Syngenic cancer cell implantation induced rapid mortality in the vehicle-treated control group, with all animals dying by day 19.

[0804] In comparison, treatment with a combination of AUP5551-C2 and AUP2059 increased mouse survival in a dose-dependent manner. Survival improved by more than 70%, with mean and median survival times of 28.3 and 28 days, respectively. These results clearly demonstrate that administration of the recombinant bacteria of the present invention, e.g., a combination of AUP5551-C2 and AUP2059, is highly effective in delaying tumor growth in syngenic mice bearing intraperitoneal (IP) tumors.

[0805] literature Beilharz K, et al. (2015): “Red fluorescent proteins for gene expression and protein localization studies in Streptococcus pneumoniae and efficient transformation with DNA assembled via the gibson assembly method”, Appl.Environ.Microbiol.81(20), pages 7244 to 7252. Birnboim,H.C.and Doly,J.(1979):“A rapid alkaline extraction procedure for screening recombinant plasmid DNA”,Nucleic Acids Res.7(6),pages 1513 to 1523. Bron,P.A.et al.(2002):“Use of the alr gene as a food-grade selection marker in lactic acid bacteria”,Appl.Environ.Microbiol.68(11),pages 5663 to 5670 Bolotin et al.(2001):“The complete genome sequence of the lactic acid bacterium lactococcus lactis ssp.lactis IL1403”,Genome Res.Volume 11,pages 731 to 753. de Vos,W.M.(1987):“Gene cloning and expression in lactic streptococci”,FEMS Microbiol.Lett.46(3),page 281 to 295. de Vos,W.M.and Simons,G.(1994):Gene cloning and expression systems in Lactococci.In Genetics and Biotechnology of Lactic Acid Bacteria.Edited by Gasson MJ,de Vos WM.Oxford:Chapman and Hall;pages 52 to 105 - ISBN:978-0-7514-0098-4. Hols,P.et al.(1999):“Conversion of Lactococcus lactis from homolactic to homoalanine fermentation through metabolic engineering”,Nat.Biotechnol.17(6),pages 588 to 592. Ruhdal Jensen,P.and Hammer,K.(1998):“The Sequence of Spacers between the Consensus Sequences”,Appl.Environ.Microbiol.64(1),pages 82 to 87. Tan,P.S.T.et al.(1992):“Characterization of the Lactococcus lactis pepN gene encoding an aminopeptidase homologous to mammalian aminopeptidase N”,FEBS Lett.306(1),pages 9 to 16. Sanders,J.W.et al.(1998):“A chloride-inducible acid resistance mechanism in Lactococcus lactis and its regulation”,Mol Microbiol.27(2)pages 299 to 310. Sanders,J.W.et al.(1997):“A chloride-inducible gene expression cassette and its use in induced lysis of Lactococcus lactis”,Appl.Environ.Microbiol.63(12),pages 4877 to 4882. van Asseldonk et al.(1993):“Functional analysis of the Lactococcus lactis usp45 secretion signal in the secretion of a homologous proteinase and a heterologous alpha-amylase”,Mol.Gen.Genet.240(3),1993,pages 428-434. van Asseldonk et al.(1990):“Cloning of usp45,a gene encoding a secreted protein from Lactococcus lactis subsp.lactis MG1363”,Gene 95(1),1990,pages 155 to 160. Laemmli,U.K.(1970):“Cleavage of structural proteins during the assembly of the head of bacteriophage T4”,Nature 227 (5259),pages 680 to 685. Tauer,C.et al.(2014),“Tuning constitutive recombinant gene expression in Lactobacillus plantarum”,Microbial.Cell Factories 13,150,doi:10.1186 / s12934-014-0150-z: Wang,M.et al.(1995):“Active immunotherapy of cancer with a nonreplicating recombinant fowlpox virus encoding a model tumor-associated antigen”,J.Immunol.154(9),pages 4685 to 4692. Tomayko,M.and Reynolds,C.(1989):“Determination of subcutaneous tumor size in athymic(nude)mice”,Cancer Chemother.Pharmacol.24(3),pages 148 to 154. Lechner M.G.(2013);“Immunogenicity of murine solid tumor models as a defining feature of in vivo behavior and response to immunotherapy”,J.Immunother.36(9),pages 477 to 489. Fu,Q.et.al(2016):“Novel murine tumour models depend on strain and route of inoculation”,Int.J.Exp.Path.(2016),97,pages 351 to 356. Lesinski,G.B.et al(2003:“The antitumor effects of IFN-α are abrogated in a STAT1-deficient mouse”,J.Clin.Invest.112(2),pages 170 to 180.

Claims

1. A recombinant bacterium comprising: a) at least one nucleic acid sequence operably coupled to a chloride-inducible promoter in a prokaryotic system and encoding at least one heterologous factor having a therapeutic and / or prophylactic effect against a tumor in a subject after administration of the recombinant bacterium to the subject, the heterologous factor independently being a heterologous polypeptide or complex thereof; and b) at least one prokaryotic regulator gene that controls the activity of the chloride-inducible promoter; a chloride-inducible promoter and at least one regulator gene are disposed on a chloride-inducible gene expression cassette, wherein the chloride-inducible promoter is disposed downstream from the at least one regulator gene, and the chloride-inducible gene expression cassette controls the transcription of at least one nucleic acid sequence encoding at least one heterologous factor; the heterologous polypeptide is selected from the group consisting of granulocyte-macrophage-colony stimulating factor (GM-CSF), interferon alpha, interferon beta, interferon gamma, granulocyte-colony stimulating factor (G-CSF), interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-23 (IL-23), interleukin-24 (IL-24), interleukin-32 (IL-32), and combinations thereof, from a eukaryotic species, but is not an antimicrobial peptide; The term "heterologous" as used above means heterologous to the recombinant bacterium. Recombinant bacteria.

2. A recombinant nucleic acid used to produce a recombinant bacterium, comprising: a) at least one nucleic acid sequence operably coupled to a prokaryotic chloride-inducible promoter and encoding at least one heterologous factor having a therapeutic and / or prophylactic effect against a tumor in a subject, the heterologous factor independently being a heterologous polypeptide or complex thereof; and b) at least one prokaryotic regulator gene that controls the activity of the chloride-inducible promoter; a chloride-inducible promoter and at least one regulator gene are disposed on a chloride-inducible gene expression cassette, wherein the chloride-inducible promoter is disposed downstream from the at least one regulator gene, and the chloride-inducible gene expression cassette controls the transcription of at least one nucleic acid sequence encoding at least one heterologous factor; the heterologous polypeptide is selected from the group consisting of granulocyte-macrophage-colony stimulating factor (GM-CSF), interferon alpha, interferon beta, interferon gamma, granulocyte-colony stimulating factor (G-CSF), interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-23 (IL-23), interleukin-24 (IL-24), interleukin-32 (IL-32), and combinations thereof, from a eukaryotic species, but is not an antimicrobial peptide; The term "heterologous" as used above means heterologous to the recombinant bacterium. Recombinant nucleic acids.

3. 2. The recombinant bacterium of claim 1, wherein at least one nucleic acid sequence operably coupled to the prokaryotic chloride-inducible promoter and encoding at least one heterologous factor, and / or at least one prokaryotic regulatory factor gene controlling the activity of the chloride-inducible promoter, are each independently located on the chromosome and / or at least one plasmid of the recombinant bacterium.

4. 4. The recombinant bacterium of claim 1, wherein the chloride-inducible promoter and / or at least one regulator gene are each independently from a Gram-positive or Gram-negative bacterial species.

5. 5. The recombinant bacterium of claim 1, wherein the chloride-inducible promoter is PgadC.

6. 5. The recombinant bacterium of any one of claims 1 or 3-4, wherein the chloride-inducible promoter is PgadC from a bacterial species of the taxonomic order Lactobacillales.

7. 5. The recombinant bacterium of any one of claims 1 or 3-4, wherein the chloride-inducible promoter is PgadC from Lactococcus lactis.

8. 5. The recombinant bacterium of claim 1, wherein the regulator gene encodes gadR.

9. 5. The recombinant bacterium of claim 1, wherein the regulator gene encodes gadR from a bacterial species of the taxonomic order Lactobacillales.

10. 5. The recombinant bacterium of claim 1, wherein the regulator gene encodes gadR from Lactococcus lactis.

11. 2. The recombinant bacterium of claim 1, wherein the chloride-inducible gene expression cassette comprises or consists of the nucleic acid sequence of SEQ ID NO: 2 and controls the transcription of at least one nucleic acid sequence encoding at least one heterologous factor.

12. 3. The recombinant nucleic acid of claim 2, wherein the chloride-inducible gene expression cassette comprises or consists of the nucleic acid sequence of SEQ ID NO: 2 and controls the transcription of at least one nucleic acid sequence encoding at least one heterologous factor.

13. 12. The recombinant bacterium of claim 1, 3, or 11, wherein at least one heterologous factor is from a mammalian species.

14. 12. The recombinant bacterium of claim 1, 3, or 11, wherein at least one heterologous factor is from a human.

15. 15. The recombinant bacterium of any one of claims 1, 3-10, 11, or 13-14, further comprising at least one inactivated gene encoding an essential protein required for viability of the recombinant bacterium.

16. 16. The recombinant bacterium of claim 15, wherein the gene encoding the essential protein is inactivated by gene deletion, gene mutation, RNA interference (RNAi)-mediated gene silencing, translational inhibition of the gene, or a combination thereof.

17. 13. The recombinant nucleic acid of claim 2 or 12, further comprising at least one gene encoding an essential protein required for viability of the recombinant bacterium.

18. 17. The recombinant bacterium of any one of claims 15-16, wherein at least one gene encoding an essential protein required for viability is selected from the group consisting of alanine racemase (alr), thymidylate synthase (thyA), asparagine synthase (asnH), CTP synthase (pyrG), tryptophan synthase (trpBA), and combinations thereof.

19. 18. The recombinant nucleic acid of claim 17, wherein the at least one gene encoding an essential protein required for viability is selected from the group consisting of alanine racemase (alr), thymidylate synthase (thyA), asparagine synthase (asnH), CTP synthase (pyrG), tryptophan synthase (trpBA), and combinations thereof.

20. 20. The nucleic acid of any one of claims 2, 12, 17, or 19, wherein the recombinant nucleic acid is in the form of a plasmid.

21. 21. A recombinant bacterium according to any one of claims 1, 3 to 10, 11, 13 to 16, or 18, comprising at least one recombinant nucleic acid according to any one of claims 2, 12, or 19 to 20.

22. 22. The recombinant bacterium of any one of claims 1, 3 to 10, 11, 13 to 16, 18, or 21, wherein the recombinant bacterium is a non-pathogenic bacterium.

23. 23. The recombinant bacterium of any one of claims 1, 3 to 10, 11, 13 to 16, 18, or 21 to 22, wherein the recombinant bacterium is a lactic acid bacterium.

24. 23. The recombinant bacterium of any one of claims 1, 3 to 10, 11, 13 to 16, 18, or 21 to 22, wherein the recombinant bacterium is a species belonging to the genus Lactobacillus or Lactococcus.

25. 25. The recombinant bacterium of claim 24, wherein the species belonging to the genus Lactococcus is Lactococcus lactis.

26. 25. The recombinant bacterium of claim 24, wherein the species belonging to the genus Lactococcus is Lactococcus lactis subsp. cremoris.

27. 27. The recombinant bacterium of any one of claims 1, 3 to 10, 11, 13 to 16, 18, or 21 to 26 for medical use.

28. 30. A pharmaceutical composition comprising the recombinant bacterium of any one of claims 1, 3 to 10, 11, 13 to 16, 18, or 21 to 27, and at least one pharmaceutically acceptable excipient.

29. a) the recombinant bacterium of any one of claims 1, 3 to 10, 11, 13 to 16, 18, or 21 to 27, which is capable of expressing at least one heterologous factor under the control of a chloride-inducible promoter in a prokaryotic system; b) at least one inducer comprising chloride ions; 10. A kit for medical use comprising:

30. a) the recombinant bacterium of any one of claims 1, 3 to 10, 11, 13 to 16, 18, or 21 to 27, which is capable of expressing at least one heterologous factor under the control of a chloride-inducible promoter in a prokaryotic system; 2. A medical device, comprising:

31. 29. The pharmaceutical composition of claim 28, wherein the recombinant bacteria is in solution, frozen, or dried.

32. 29. The pharmaceutical composition of claim 28, wherein the recombinant bacterium is freeze-dried or spray-dried.

33. 30. The kit of claim 29, wherein the recombinant bacteria is in solution, frozen, or dried.

34. 31. The medical device of claim 30, wherein the recombinant bacteria is in solution, frozen, or dried.

35. 27. Use of a reconstituted medium containing chloride ions to reconstitute the recombinant bacterium of any one of claims 1, 3 to 10, 11, 13 to 16, 18, or 21 to 26.

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