WO2023118327A1 - Live bacteria as excipients for proteins - Google Patents

Abstract

The present invention provides a pharmaceutical composition or a pharmaceutical product or kit comprising (i) a recombinant protein and (ii) live wild-type or non-wild type bacteria, particularly wherein the bacteria do not express the recombinant protein. Also provided are wound dressings and devices comprising the recombinant protein and bacteria, and the use of such compositions, products and kits in therapy, especially in the treatment of wounds.

Description

LIVE BACTERIA AS EXCIPIENTS FOR PROTEINS

Field

The invention relates generally to using live lactic acid bacteria as excipients in the formulation of protein-based biological pharmaceutical products and medical devices. The invention further relates to live Lactobacillus stabilizing recombinant chemokines and allowing sufficient tissue bioavailability of the chemokines when given topically to skin wounds or to mucosal surfaces in humans and animals.

Background

The process of wound healing has overlapping phases (coagulation phase, inflammatory phase and proliferative/remodelling phase) where constituents of the local microenvironment change over time and distinct cell types play different roles. Key cell players in the healing process are platelets, keratinocytes/epithelial cells, fibroblasts/myofibroblasts, different immune cells and endothelial cells. All tissues in the body can be injured and the healing process is somewhat specific to the organ, however the initial signals elicited by the damaged cells are similar. The most studied form of wound healing is in skin.

Tissue injury disrupts homeostasis, which initiates the coagulation process and activates the sympathetic nervous system. The platelets forming the blood clots release signals, mainly PDGF (platelet derived growth factor) and TGF (transforming growth factor) changing the local environment (Ref. 1). Injured and stressed cells release alarm signals that initiate the recruitment of immune cells such as neutrophils and monocytes. Within the wounded tissue, the immune cells secrete various chemokines, growth factors like VEGF-A, FGF, and EGF (vascular endothelial growth factor A, fibroblast growth factor, epidermal growth factor), ROS (reactive oxygen species) and matrix digestive enzymes, which change the microenvironment and allow the healing process to enter the proliferative phase where failing and dead tissue is removed by macrophages. Cells from the wound edges, such as fibroblasts and keratinocytes, migrate inwards to the wound centre and cover the wound surface with a layer of collagen and extracellular matrix. The fibroblasts within the wound are then transformed into myofibroblasts expressing contractile a-SMA (a-smooth muscle actin) allowing the wound to contract and finally close. The transition from fibroblasts into myofibroblasts is dependent on signals from the microenvironment, some of which originate from immune cells, mainly macrophages. During this process, blood vessels are growing into the newly formed tissue, the granulation tissue. Blood flow to the adjacent area is normally increased during this phase to increase the availability of oxygen and nutrients, in addition to immune cell recruitment and migration to the afflicted site.

Following wound closure, the afflicted site becomes re-epithelialized by keratinocytes/epithelial cells whereby the integrity of the organ barrier is regained. Even after wound closure, some tissue remodelling occurs to normalize the matrix structure and the majority of involved immune cells either die or leave the site. Also, at this stage dead or dying cells are ingested and cleared (phagocytosed) by the remaining tissue macrophages (Ref. 1). Faster wound healing reduces complications and discomfort to the patient.

Impaired or delayed cutaneous or mucosal wound healing is a worldwide clinical problem causing pain, direct exposure to pathogens of the environment, loss of tissue function and loss of temperature and fluid balance regulation. There are several conditions where the tightly regulated wound healing process is impaired and the cutaneous or mucosal wounds remain unhealed for longer time periods than normal, which in worst case become chronic.

Reduced blood flow or impaired blood flow regulation in the skin, especially in extremities, significantly reduces the efficiency of the healing process. There are several clinical conditions where the skin perfusion is either reduced or the function of the vasculature is impaired such as PAD (peripheral artery disease), intermittent claudication, vein insufficiency or vessel obstruction by arteriosclerotic plaques. Impaired blood flow to the wound area results in shortage of oxygen and nutrients and the cells aiding in the tissue remodeling either die from necrosis or are unable to perform their tasks on site. Also, the surrounding tissue will if not sufficiently supplied lose functionality and ultimately start to die. Tissues are during the remodeling phase very metabolically active and have high oxygen consumption.

Another factor impairing cutaneous wound healing is hyperglycemia and diabetes mellitus. During hyperglycemic conditions cell signaling and immune system functions are impaired. Complications resulting from diabetes include microvascular changes and damage to peripheral neurons. As a result, diabetic patients often develop chronic wounds on their feet, commonly called diabetic foot. The available treatment for these patients today is removal of dead tissue using surgical debridement or collagenase together with systemic antibiotic treatment and closed wound dressing. There are experimental studies where growth factors and biomaterials have been applied to chronic wounds (Ref. 2).

The stromal cell-derived factor 1 (SDF-1) also known as C-X-C motif chemokine 12 (CXCL12) is a chemokine protein that in humans is encoded by the CXCL12 gene. WO 2009/079451 discloses a method for promoting wound healing in a subject, comprising administering directly to the wound or an area proximate the wound an amount of SDF-1 effective to promote healing of the wound of the subject.

Certain probiotics (Lactobacillus re ute ri ATCC PTA 6475) have been shown to facilitate wound healing if supplemented in the drinking water during the healing process (Ref. 3), i.e., the bacteria were ingested. Further, supernatants from culture of Lactobacillus plantarum have been demonstrated to inhibit biofilm production by Pseudomonas aeruginosa, commonly infecting chronic wounds (Ref. 4). It has been found that lactic acid bacteria which are modified to express specific proteins, such as cytokines, are useful for promoting wound healing. W02016102660 A1 , belonging to the applicant, discloses the use of Lactobacillus reuteri modified to express CXCL12, CXCL17 and YM1 for treatment of cutaneous and mucosal wounds (Ref 5).

It is known within the field that strains of lactic acid bacteria are sparsely present on the human skin (Ref. 6) and is thus not the expected choice of bacteria to use for cutaneous wound treatment. Lactobacilli are also more difficult to work with since they grow relatively slowly and require special medium and conditions in comparison with more commonly used bacteria like E. coli and S. aureus. Additionally, for Lactobacilli, fewer molecular tools have been developed for genetic engineering and industrial use and control.

The different phases of wound healing comprise distinct key events that could be altered to change the healing process. Vascular remodeling during the healing process is highly dependent on induction of hypoxia inducible factor 1a (HIF-1 a) that regulates the expression of VEGF-A (vascular endothelial growth factor A) and a range of chemokines, such as CXCL12 (also known as SDF-1 ; SEQ ID NO: 3 and 6), as well as most proteins of the secretome having proximal and local effects. CXCL12 is constitutively expressed in tissues and acts through the receptor CXCR4 found on leukocytes and endothelial cells inducing multiple cellular actions (Ref. 7). CXCL12 is found in high levels in macrophages specialized in tissue remodeling (Ref. 8). Dermal overexpression of CXCL12 using lentiviral vectors improves wound healing in diabetic mice (Ref. 9).

The beneficial role of CXCL12 in wound healing is known and there is prior art describing CXCL12 given topically to wounds in a pharmaceutical formulation (WO2014145236 A2). The pharmaceutical formulations mentioned here are not tested and the majority of them are not feasible for CXCL12 or viral vectors expressing CXCL12. There is evidence that CXCL12 directly administrated to wounds does not affect the rate of healing and CXCL12 is estimated to a have half-life of 6 seconds in tissue in vivo.

Summary

The invention inter alia relates to specific pharmaceutical formulations to allow administered proteins e.g., chemokines, being the active pharmaceutical ingredient, longer half lives in vivo in wounds. Said formulations also comprise live wildtype or non-wild-type organisms, particularly bacteria, and more particularly lactic acid bacteria. The lactic acid bacteria are acting as the excipient in the pharmaceutical formulation. It is novel to use live organisms as excipients in pharmaceutical formulations.

Consequently, the invention relates to a therapeutic recombinant protein and a live wild-type or non-wild-type organism as excipient, wherein the said protein is useful for improving wound healing, such as cutaneous or mucosal wound healing, in a human or animal subject. The organism is particularly bacteria.

The invention can thus be seen to relate to the use of live bacteria (which may be wild-type or non-wild-type), as excipients for proteins, particularly for therapeutic proteins, more particularly for therapeutic proteins which are to be administered to a subject. Put another way, the invention relates to the use of live bacteria as aids, or additives, in the delivery, or administration of a therapeutic protein to a subject.

Accordingly, in a first aspect, the invention provides a pharmaceutical composition comprising (i) a recombinant protein and (ii) live wild-type or non-wild-type bacteria.

The bacteria are included, or used, in the composition as an excipient.

In an embodiment, the bacteria do not express the recombinant protein of (i). In particular, in such an embodiment, the non-wild type bacteria are not engineered to express the recombinant protein.

Particularly, the recombinant protein is a therapeutic protein.

Preferably, the said protein is useful for wound healing due to its capability of targeting immune cells such as macrophages and its precursors. Thus, in an embodiment, the recombinant protein is a wound-healing protein or antiinflammatory protein. In other words, the protein is a protein capable of promoting wound healing and/or resolution of inflammation. Preferably, the said recombinant protein is an interleukin, a cytokine or a chemokine. More preferably, the said recombinant protein is a protein which occurs in the secretome (i.e. it is a protein of the secretome, or a “secretome protein”).

This first aspect of the invention more particularly provides a pharmaceutical composition comprising a recombinant protein, preferably selected from an interleukin, a cytokine or a chemokine, more preferably from a CXC protein, in particular selected from CXCL12 and CXCL17. Most preferably, the said recombinant protein is chosen from the group consisting of murine CXCL12, in particular murine CXCL12-1O (SEQ ID NO: 3); human CXCL12, in particular human CXCL12-1a (SEQ ID NO: 6); murine CXCL17 (SEQ ID NO: 9); human CXCL17 (SEQ ID NO: 12).The pharmaceutical composition particularly comprises, a live wild-type or non-wild-type bacteria strain, and more particularly a live wild-type or non-wild-type lactic acid bacteria strain. In embodiments of the invention, the lactic acid bacteria strain is Lactobacillus, particularly Lactobacillus reuteri, and more particularly Lactobacillus reuteri R2LC.

Further aspects of the invention provide said pharmaceutical composition for use in therapy, and more particularly for use in wound healing, including ulcer healing, wherein the pharmaceutical composition is administrated to a wound in a subject to be treated. The pharmaceutical composition may also comprise at least another pharmaceutically acceptable carrier or excipient.

Such a pharmaceutical composition, or product, may conveniently take the form of a stand-alone product produced in vials, prefilled syringes or a wound dressing or wet formulated wound dressing comprising the therapeutic protein and excipient bacteria of the invention. Thus, in a further aspect the invention provides a wound dressing comprising recombinant protein and bacteria of the invention as hereinbefore defined, together with at least one dressing material.

Thus, according to a further aspect of the invention provides use of the pharmaceutical composition of the invention as defined herein in a wound dressing.

This aspect also provides a wound dressing comprising the pharmaceutical composition as defined herein.

Yet another aspect of the invention provides use of the pharmaceutical composition as defined herein in a medical device.

This aspect also provides a medical device comprising the pharmaceutical composition as defined herein.

A further aspect of the invention provides use of the pharmaceutical composition as defined herein for the manufacture of a medicament for use in wound healing. This aspect can also be seen to provide use of live wild-type or non-wild- type bacteria for the manufacture of a medicament for use in wound healing, wherein said medicament comprises a recombinant protein.

The medicament may be a pharmaceutical composition as defined herein, or it may be product comprising (i) the recombinant protein and (ii) the bacteria as separate entities, or in separate preparations/compositions. Again, the bacteria are included, or used, in the medicament as an excipient.

Also provided is a method of treating a subject to heal a wound, said method comprising administering to said subject, or to the wound in said subject, an amount of a recombinant protein, more preferably selected from CXC proteins, and most preferably from the group consisting of CXCL12 and CXCL17, effective to promote healing of the wound, and a live wild-type or non-wild type bacteria strain, preferably live wild-type or non-wild type lactic acid bacteria.

Another aspect of the invention provides a kit for healing wounds, said kit comprising a recombinant protein, preferably a therapeutic protein, more preferably selected from CXC proteins, and most preferably selected from the group consisting of CXCL12 and CXCL17, and live wild-type or non-wild type bacteria, preferably live wildtype or non-wild type lactic acid bacteria.

Such a kit may be provided for use in wound healing.

Accordingly, a still further aspect of the invention provides a pharmaceutical product (e.g., a kit or combination product) comprising a recombinant protein and live wild-type or non-wild type bacteria as a combined preparation for separate, sequential or simultaneous use in wound healing (or for treating a wound, which includes an ulcer, in a subject).

In embodiments of the various aspects above, the bacteria do not express the recombinant protein of (i). In particular, in such embodiments, the non-wild type bacteria are not engineered to express the recombinant protein.

Detailed description

The products (i.e. the pharmaceutical compositions, products, kits etc.) and medical uses and methods provided herein are directed to, or based on, the use of live bacteria as excipients for recombinant proteins, to enhance the delivery and/or effect, or therapeutic function, of the recombinant protein to or in a subject. The recombinant protein may be any therapeutic protein. By this, it is generally meant that the protein may be any protein having a desired or beneficial therapeutic effect in a subject to whom it is administered. In an embodiment, as noted above the protein is effective or beneficial in promoting the healing of wounds, and/or in resolving inflammation. In particular, it may achieve this anti-inflammatory or wound healing effect by an effect on immune cells, and in particular on macrophages or their precursors. It may thus be referred to as an immune-effective protein.

As noted above, the bacteria may be wild-type or non-wild-type. The term “wild-type” as used herein means that the bacteria are native bacteria, or bacteria which occur in nature, and which have not been engineered or modified. In particular, they have not been modified by human intervention. The term “non-wild-type” on the other hand means that the bacteria are non-native, or do not occur in nature. Non-wild- type bacteria are bacteria which have been subject to modification or engineering. In particular, non-wild-type-bacteria have been altered or modified by human intervention. For example, they have been subjected to mutation, or genetic engineering, which includes all forms of genetic modification or gene editing etc. Thus, non-wild type bacteria may be modified to introduce a heterologous nucleic acid sequence or molecule into their genome, for example to express a heterologous protein, or they may be subject to gene knock-out, or gene inactivation, or gene duplication etc. They may be genetically modified in any way. Furthermore, non-wild type bacteria include those that have been subjected to adaptation or evolution by culture methods. Such modifications may be made for example to introduce or improve a function or property of the bacteria, for example to improve growth, or viability, or nutrient usage or assimilation etc., or to introduce or modify a biological, e.g. biosynthetic or degradative, pathway etc.

The terms “bacteria” and “bacterial strain” are used interchangeably herein to refer generally to bacteria, without limitation to any particular genus, species or strain. Thus, the terms refer generally to bacterial organisms. In an embodiment, the bacteria will be of a single population or type, i.e. of a particular strain, rather than a mixed culture or mixed population.

As noted above, in an embodiment the bacteria do not express the recombinant protein with which they are used. In particular, they are not modified or engineered to express the protein.

In another embodiment, the bacteria have not been modified or engineered to express or produce or accumulate or secrete a therapeutic product (or in other words, any therapeutic product). The therapeutic product may be a therapeutic protein, and in such an embodiment, the bacteria have not been modified or engineered to express or produce a therapeutic protein. More particularly, they have not been modified to introduce a nucleic acid molecule encoding a therapeutic protein. Alternatively, the therapeutic product may be the product of a biological (e.g. biosynthetic) pathway, or the substrate of a transporter molecule. The key feature of this embodiment is that the bacteria have not been modified or engineered to provide to the subject a therapeutic product for use in conjunction with the recombinant protein. In such an embodiment, the bacteria may be non-wild-type bacteria, and may have subjected to genetic modification, but the modification does not include the introduction of the capacity or ability to express, produce, secrete or accumulate a therapeutic product.

In another embodiment, the non-wild-type bacteria have not been modified or engineered to express a eukaryotic protein, in particular a mammalian or human protein.

In another embodiment, the non-wild-type bacteria have not been modified or engineered to express a transporter protein. In particular in such an embodiment, the bacteria have not been modified or engineered to express a heterodimeric MsbA1-MsbA2-like transporter protein, and especially such a transporter protein comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO. 1 and a second polypeptide having an amino acid sequence of SEQ ID NO. 2 as set out in WO2021/1148661.

The pharmaceutical composition, wound dressing, medical device or kit according to the invention may in various embodiments be used for treatment of cutaneous or mucosal wounds, particularly cutaneous treatment of skin wounds and treatment of gastrointestinal wounds.

Thus, in certain embodiments, the pharmaceutical compositions, kits, products, wound dressings and devices are provided in a form suitable for topical administration to the skin.

The term “wound healing” is used broadly herein to include any aspect of promoting or improving the healing of a wound. Thus, the various aspects of the invention set out above may alternatively be defined with respect to a utility of the recombinant protein and bacteria in promoting or enhancing or improving wound healing.

Wound healing may accordingly include or encompass any effect which results in faster wound healing, or more complete healing of a wound or indeed any amelioration or improvement in the healing of a wound, e.g., reduced healing time, reduced time to achieve partial or complete closure of a wound, improved wound appearance (e.g., the appearance of a healed or healing wound), reduced or improved scar formation, the promotion of healing of a chronic or recalcitrant wound, etc., i.e. the application of the recombinant protein and bacteria of the invention to a wound may induce, or cause, or start, the healing of a wound which has up to now not healed or shown any signs of healing). Wounds are discussed in more detail below.

The subject having a wound to be treated may be any human or animal subject, including for example domestic animals, livestock animals, laboratory animals, sports animals or zoo animals. The animal is preferably a mammalian animal, but other animals, e.g., birds are included. Thus, the animal may be a primate, a rodent (e.g., a mouse or rat), or a horse, dog or cat. Most preferably the subject is a human.

Lactic acid bacteria (LAB) is a group of Gram-positive, low-GC, acid-tolerant, generally nonsporulating, non-respiring, either rod-shaped (bacilli), or spherical (cocci) bacteria which share common metabolic and physiological characteristics. These bacteria produce lactic acid as the major metabolic end-product of carbohydrate fermentation and are characterized by an increased tolerance to acidity (low pH range). These characteristics of LAB allow them to outcompete other bacteria in a natural fermentation because LAB can withstand the increased acidity from organic acid production (e.g., lactic acid). Thus, LAB play an important role in food fermentations, as acidification inhibits the growth of spoilage microorganisms. Several LAB strains also produce proteinaceous bacteriocins which further inhibit spoilage and growth of pathogenic microorganisms. LAB often have a generally recognized safe (GRAS) status and are amongst the most important groups of microorganisms used in the food industry.

The core genera that comprise the lactic acid bacteria group are Lactobacillus*, Leuconostoc, Pediococcus, Lactococcus, Enterococcus, Weissella, and Streptococcus, as well as the more peripheral Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Sporolactobacillus, Tetragenococcus, and Vagococcus. Any lactic acid bacterium from these genera is included within the scope of the present invention, but particularly bacteria from the genera Lactobacillus or Lactococcus, and more particularly from Lactobacillus. (*Lactobacillus, Pediococcus, Weissella and Leuconostoc have recently been unified but together also been divided into 25 genera. For the sake of simplicity, herein the old taxonomic classification is used. Reference: Zheng, J., Wittouck, S., Salvetti, E., Franz, C. M. A. P., Harris, H. M. B., Mattarelli, P., et al. (2020). A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, amended description of the genus Lactobacillus Beijerinck 1901 , and union of Lactobacillaceae and Leuconostocaceae. International Journal of Systematic and Evolutionary Microbiology, 70(4), 2782-2858. http://doi.Org/10.1099/ijsem.0.004107).

The protein may be a native or natural protein (i.e., a protein having an amino acid sequence as found in nature) and may be from any species in which these proteins occur. Generally, the protein will be a mammalian protein and as indicated above human and murine proteins are preferred.

In an embodiment, the recombinant protein is a chemokine.

In another embodiment, the recombinant protein is not a protein selected from one or more or any combination of: an antibody, a growth factor, a vaccine, or a cytokine.

In another embodiment, the recombinant protein is not involved in the production of adenosine, for example it is not a substrate for the production of adenosine.

The protein may be selected from IL4, IL13, IL22, IL23, IL27, CSF-1 , TGF , CXCL10, CXCL11, CXCL12, CXCL17, Ym1 , CCL22 and CCL2. In one particular embodiment, the protein is selected from CXCL12 and CXCL17. In another particular embodiment, the protein is selected from CXCL12.

However, the native protein sequences may be modified, for example by one or more amino acid additions, insertions, deletions and/or substitutions, as long as the function or activity of the protein is not substantially or significantly altered, e.g., as long as the activity of the protein is substantially retained. The protein may be fragment or truncated variant of a natural protein. For example, a sequence-modified variant protein may exhibit at least 80, 85, 90 or 95% of the activity of the parent protein from which it is derived. This may be assessed according to tests known in the art for activity of the protein in question. Activity may for example be measured in in vitro chemokine activity tests or in systems of receptor phosphorylation or calcium flux upon ligation in cultured cells treated with the protein, in systems of cell chemotaxis in vitro or in vivo in models of cell recruitment to the injected protein. As an illustration, the terms “CXCL12” and “CXCL17” thus include not only the native proteins but also functionally equivalent variants or derivatives thereof. The proteins may thus be synthetic or sequence- modified variants, or may comprise one or more other modifications, e.g., post- translational modifications, etc.

As mentioned above, the recombinant proteins may have the amino acid sequences indicated above for the native human or murine proteins, namely SEQ ID NOS. 1, 2, 3, and 4 for murine and human CXCL12 respectively, 5, 6, 7, and 8 for murine and human CXCL17 respectively, and 9, 10, 11 , and 12 for murine and human Ym1 respectively, or an amino acid sequence having at least 80% sequence identity to any aforesaid sequence. Thus, the recombinant protein may have an amino acid sequence as shown in SEQ ID NOS. 2 and 5 for murine and human CXCL12 respectively, 8 and 11 for murine and human CXCL17 respectively, or an amino acid sequence having at least 80% sequence identity to any aforesaid sequence. In other embodiments the recombinant protein(s) may have an amino acid sequence which has at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% 90%, 91% 92% 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any aforesaid amino acid sequence.

Sequence identity may readily be determined by methods and software known and readily available in the art. Thus, sequence identity may be assessed by any convenient method. However, for determining the degree of sequence identity between sequences, computer programs that make multiple alignments of sequences are useful, for instance Clustal W (Thompson et al., (1994) Nucleic Acids Res., 22: 4673- 4680). Programs that compare and align pairs of sequences, like ALIGN (Myers et al., (1988) CABIOS, 4: 11-17), FASTA (Pearson et al., (1988) PNAS, 85:2444-2448; Pearson (1990), Methods Enzymol., 183: 63-98), BLAST and gapped BLAST (Altschul et al., (1997) Nucleic Acids Res., 25: 3389-3402) are also useful for this purpose, and may be used using default settings. Furthermore, the Dali server at the European Bioinformatics institute offers structure-based alignments of protein sequences (Holm (1993) J. Mol. Biol., 233: 123-38; Holm (1995) Trends Biochem. Sci., 20: 478-480; Holm (1998) Nucleic Acid Res., 26: 316-9). Multiple sequence alignments and percent identity calculations may be determined using the standard BLAST parameters, (e.g., using sequences from all organisms available, matrix Blosum 62, gap costs: existence 11, extension 1). Alternatively, the following program and parameters may be used: Program: Align Plus 4, version 4.10 (Sci Ed Central Clone Manager Professional Suite). DNA comparison: Global comparison, Standard Linear Scoring matrix, Mismatch penalty = 2, Open gap penalty = 4, Extend gap penalty = 1. Amino acid comparison: Global comparison, BLOSUM 62 Scoring matrix.

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

Derivatives of the proteins as defined herein may also be conceived. By derivative is meant a protein as described above or a variant thereof in which the amino acid is chemically modified, e.g., by glycosylation and the like, etc.

Where a protein comprises an amino acid substitution relative to the sequence of the native protein, the substitution may preferably be a conservative substitution. The term “a conservative amino acid substitution” refers to any amino acid substitution in which an amino acid is replaced (substituted) with an amino acid having similar physicochemical properties, i.e., an amino acid of the same class/group. For instance, small residues Glycine (G), Alanine (A) Serine (S) or Threonine (T); hydrophobic or aliphatic residues Leucine (L), Isoleucine (I); Valine (V) or Methionine (M); hydrophilic residues Asparagine (N) and Glutamine (Q); acidic residues Aspartic acid (D) and Glutamic acid (E); positively-charged (basic) residues Arginine (R), Lysine (K) or Histidine (H); or aromatic residues Phenylalanine (F), Tyrosine (Y) and Tryptophan (W), may be substituted interchangeably without substantially altering the function or activity of the protein.

The said bacterial strain is preferably a lactic acid bacteria strain such as a Lactobacillus strain or a Lactococcus (e.g., Lactococcus lactis) strain. More preferably, the bacterial strain is a Lactobacillus reuteri strain such as Lactobacillus reuteri R2LC or Lactobacillus reuteri DSM20016. The said strains (Lactobacillus reuteri R2LC/DSM20016 and Lactococcus lactis) are not found on human skin as determined by phylogenetic analysis of the forearm skin biota of six subjects.

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

As well as the pharmaceutical compositions and kits comprising the recombinant protein and live lactic acid bacteria, further products of the invention include pharmaceutical products, dressings and medical devices containing said recombinant protein and bacteria. Such compositions and devices may include in particular wound dressings, packing materials, swabs, implants etc., or indeed any wholly or partially in-dwelling medical device which may be introduced or present at the site of a wound (e.g., at a surgical wound site), for example a line or catheter. Also included are probiotic products, that is products containing the bacteria for administration to a subject, e.g., for oral administration, for example for consumption or ingestion (for example a capsule or a tablet comprising freeze-dried recombinant protein and bacteria), or for topical application to a wound or direct administration to a wound site, e.g., during surgery, or rectally, vaginally, etc.

Accordingly, the products (e.g., pharmaceutical compositions, kits, medical devices, and wound dressings, etc.) according to the invention are useful in medical therapy, in particular for promoting wound healing in human or animal subjects. As used herein, the term “promoting wound healing” means augmenting, improving, increasing, or inducing closure, healing, or repair of a wound. In preferred aspects of

RECTIFIED SHEET (RULE 91) ISA/EP the invention, the human or animal subject is in need of wound healing due to an underlying medical condition leading to impaired wound healing, such as reduced peripheral blood perfusion (peripheral artery disease), hyperglycemia or neuropathy, or the subject may be immunocompromised for any reason, e.g., due to an underlying disease (whether acquired or inherited) or as an effect of medical treatment. In particular the subject may be suffering from diabetes.

The wound to be healed can include any injury, trauma or damage to any portion of the body of a subject. Examples of wounds that can be treated by the method include acute conditions or wounds; such as thermal burns (hot or cold), chemical burns, radiation burns, electrical burns, burns caused by excess exposure to ultraviolet radiation (e.g., sunburn); damage to bodily tissues, such as the perineum as a result of labor and childbirth; injuries sustained during medical procedures, such as episiotomies, trauma- induced injuries including cuts, incisions, excoriations; injuries sustained from accidents; post-surgical injuries, as well as chronic conditions; such as pressure sores, bedsores, ulcers, conditions related to diabetes and poor circulation, and all types of acne. In addition, the wound can include dermatitis, wounds following dental surgery; periodontal disease; wounds following trauma; and tumor associated wounds. Further examples are gastrointestinal wounds occurring during for instance gastritis or inflammatory bowel disease.

Thus, the term “wound” is used broadly herein to include any breach of the integrity of a tissue, namely any damage, trauma or injury to tissue or any lesion, howsoever caused (e.g., due to accidental injury or trauma, surgical or other intended or purposeful injury or disease). The trauma may include any physical or mechanical injury or any damage caused by an external agent including pathogens or biological or chemical agents. Wounds may include any type of burn. The wound may be acute or chronic. A chronic wound may be described as any wound stalled in a healing stage, e.g., in the inflammatory phase, or any wound that has not healed in 30, 40, 50 or 60 days or more. The wound may be present in or on an internal or external surface or tissue of the body.

For some conditions, the term “ulcer” is used alternative to the term “wound”. Ulcers falling within the same type of description as wounds are also covered by this application.

In a particular embodiment the wound is on an external surface or tissue of the body, e.g., it is a skin (i.e., cutaneous) wound or a mucosal wound, in particular a wound in an external mucosal tissue or surface of the body (e.g., in the eye, ear or nose, etc.) The recombinant protein and bacteria may be administered in any convenient or desired way, e.g., orally, or topically, or by direct administration to a wound site e.g., by direct infusion or application or introduction of a pharmaceutical composition or dressing or device containing the recombinant protein bacteria and bacteria. In other embodiments it may be administered to the oral cavity, or intranasally or by inhalation, rectally or vaginally. The recombinant protein and bacteria may thus be administered to, or via, any orifice of the body.

The recombinant protein and the bacteria may be formulated or prepared in any convenient or desired way for administration by any of the above routes, according to procedures and using means well known and routine in the art. Thus, as well as pharmaceutical compositions, kits, medical devices and dressings, etc.

Oral and rectal administration forms include powders, tablets, capsules and liquids etc. For topical administration, the product may be formulated as a liquid e.g., a suspension, freeze-dried cake, or a spray or aerosol (powder or liquid), gel, cream, lotion, paste, ointment or salve, etc. or as any form of dressing, e.g., bandage, plaster, pad, strip, swab, sponge, mat, etc., with or without a solid support or substrate. Further the bacteria and recombinant protein may be provided on (e.g., coated on) the surface of a medical device such as an implant (e.g., a prosthetic implant), tube, line or catheter, etc.

The bacteria may be provided in any convenient or desired form, e.g., as an active or growing culture or in lyophilized or freeze-dried form.

The recombinant protein and bacterial strains according to the invention can be formulated for topical or oral administration to treat surface wounds of skin or mucosa. Alternatively, for topical application, the product is e.g., a lotion or a lotion- soaked wound dressing, comprising recombinant protein and a bacterial strain according to the invention.

The product of the invention (i.e., the pharmaceutical composition or device or dressing, etc.) may comprise further pharmaceutically suitable excipients, for example a liquid, buffers, etc. This may be provided as part of the product (e.g., incorporated into or included in a dressing) or separately, e.g., as part of a kit or combination product, as defined above. When co-formulated together in a product (e.g., a dressing or device) the recombinant protein and bacteria may be provided in a format in which the recombinant protein is separated from the bacteria and are brought together (or contacted) in use, For example, the recombinant protein and bacteria may be in separate compartments which are brought together (e.g., contacted or mixed), or may be separated by a barrier (e.g., a membrane or other partition) which may be broken or disrupted or opened in use). Alternatively, the recombinant protein may be formulated and provided separately (e.g., in a kit also containing the bacteria, or a product containing the bacteria), and may be brought together (e.g., contacted) with the bacteria during use. This may be before, during or after administration to the subject. For example, a product comprising the bacteria may be administered first and then the recombinant protein may be added or applied to the bacteria. In another embodiment the recombinant protein and bacteria may be premixed before administration, e.g., just before or immediately before, or during administration.

Where bacteria are provided in lyophilized or freeze-dried form, it may be desirable to reconstitute, or resuspend, them prior to administration e.g., prior to or during use. This may depend on the wound and the format of the product which is used. For example, in the case of some wounds there may be sufficient liquid present to allow for the bacteria to be reconstituted/resuspended and become active. However, in other embodiments it may be desirable to provide a liquid for reconstitution (or alternatively expressed, for suspension or resuspension) of the bacteria. This may be provided in a separate vessel or container (e.g., as part of a kit or combination product) or in a separate compartment of a container, or vessel or device). The liquid may comprise or contain the recombinant protein, or the liquid, when present, may be provided in a separate vessel or container or compartment. The liquid may be any suitable liquid for reconstitution or suspension of freeze-dried bacteria, e.g., water, or an aqueous solution, or buffer or growth or culture medium.

Thus, by way of example a two-compartment system (e.g., in a dressing or device or container, or capsule or vessel (e.g., a bottle)) may comprise freeze-dried bacteria in one compartment and a liquid in another. In use, or prior to use, the two compartments may be mixed or brought into contact, and applied to the wound. In a more particular embodiment, the bacteria may be administered to a wound in liquid form, and a separate dressing may then be applied. It will be seen therefore that in one simple embodiment, a kit may simply contain a first vessel or container comprising the freeze-dried bacteria and a second vessel or container containing a liquid for reconstitution of the bacteria.

For cutaneous wounds, the said wound dressing can comprise freeze- dried recombinant protein in one compartment and bacteria in another compartment. When applied to the wound, the two compartments are brought together so that the recombinant protein and bacteria are brought into contact with. Alternatively, the bacteria can be contained in a gel-like structure on the adhesive side of a waterproof plaster or the side of the dressing in contact with the exudate. At the time of use, the recombinant protein is manually applied to the bacteria and the plaster or dressing is applied to the wound area.

Viable bacteria may also be comprised in a hydrocolloid, for example a natural gelatin. The bacteria can be incorporated by crosslinking into hydrocolloid e.g., gelatin films, plasticized and dried, retaining viability during storage until hydration. Viable bacteria may also be encapsulated within cross-linked electrospun hydrogel fibers. In this format the bacteria need not be freeze-dried.

For wounds in the mouth (e.g., on the gums), the recombinant protein and bacteria according to the invention can be administered in a high viscous paste.

Specifically, formulations for topical administration to the skin can include ointments, creams, gels, and pastes to be administered in a pharmaceutically acceptable carrier. Topical formulations can be prepared using oleaginous or water- soluble ointment bases, as is well known to those in the art. For example, these formulations may include vegetable oils, animal fats, and more preferably semisolid hydrocarbons obtained from petroleum. Particular components used may include white ointment, yellow ointment, acetyl esters wax, oleic acid, olive oil, paraffin, petrolatum, white petrolatum, spermaceti, starch glycerite, white wax, yellow wax, lanolin, anhydrous lanolin, and glyceryl monostearate. Various water-soluble ointment bases may also be used including, for example, glycol ethers and derivatives, polyethylene glycols, polyoxyl 40 stearate, and polysorbates.

The recombinant protein and bacteria can be provided in and/or on a substrate, solid support, and/or wound dressing for delivery of active substances to the wound. The solid support or substrate may be a medical device or a part thereof. As used herein, the term “substrate” or “solid support” and “wound dressing” refer broadly to any substrate when prepared for, and applied to, a wound for protection, absorbance, drainage, etc. In an embodiment the invention provides a wound healing material or dressing attached to or comprising the recombinant protein and bacteria, i.e. , the dressing is a vehicle for the recombinant protein and bacteria of the invention. Alternatively, the vehicle may be a plaster or bandage. The present invention may include any one of the numerous types of substrates and/or backings that are commercially available, the choice of wound healing material will depend on the nature of the wound to be treated. The most commonly used wound dressings are described briefly below.

Transparent film dressings are made of (e.g., made of polyurethane, polyamide, or gelatin. These synthetic films, permeable to water vapor oxygen and other gases but impermeable to water and bacteria, have low absorbency and are suitable for wounds with low exudate), hydrocolloids (hydrophilic colloidal particles bound to polyurethane foam), hydrogels (cross-linked polymers containing about at least 60% water have higher absorbency and eliminate toxic components from the wound bed and maintain the moisture level and temperature in the wound area), foams (hydrophilic or hydrophobic e.g., polymeric foam dressings produced through the modification of polyurethane foam have good absorbency and are permeable to water vapour), calcium alginates (non-woven composites of fibers from calcium alginate from the phycocolloid group, alginates have a very high absorbent capacity. They also promote autolytic debridement because ion-exchange between the alginate and the exudate converts the insoluble calcium alginate into soluble sodium alginate, providing the wound bed with a moist, intact surface ideal for wound healing)), and cellophane (cellulose with a plasticizer). The shape and size of a wound may be determined and the wound dressing customized for the exact site based on the measurements provided for the wound. As wound sites can vary in terms of mechanical strength, thickness, sensitivity, etc., the substrate can be molded to specifically address the mechanical and/or other needs of the site. For example, the thickness of the substrate may be minimized for locations that are highly innervated, e.g., the fingertips. Other wound sites, e.g., fingers, ankles, knees, elbows and the like, may be exposed to higher mechanical stress and require multiple layers of the substrate.

In yet a further aspect, the invention provides a method for wound healing in a human or animal subject, comprising administering to a human or animal subject in need thereof a therapeutic recombinant protein and an excipient lactic acid bacteria strain according to the invention. The said bacterial strain is preferably comprised in a pharmaceutical composition or wound dressing as hereinbefore described. In such methods, the human or animal subject is preferably in need of wound healing due to an underlying medical condition leading to impaired wound healing, such as reduced peripheral blood perfusion (peripheral artery disease), hyperglycemia or neuropathy.

Results obtained and included in the Examples below demonstrate the advantages of the invention. In particular, improved wound healing (e.g., in terms of better or faster wound closure) may be obtained by using the therapeutic recombinant protein and the excipient lactic acid bacteria of the invention, as compared to, for example, a protein preparation directly (i.e., just the protein, no bacteria) or just bacteria alone. It is thus advantageous to co-deliver the protein and live bacteria to the wound. It is believed that there may be synergistic effect. In other words, there may be a synergy between the effect of the recombinant protein and lactic acid bacteria and the effect on wound healing.

It is believed in this respect that the effect of the bacteria in lowering pH the site of (e.g., in) the wound may assist in augmenting or enhancing or promoting the activity of the protein. Further effects, as higher tissue bioavailability and prolonging the half-live of the recombinant protein, may or may not be promoted by the lowering of pH. In a further experiment, the addition of lactic acid (having a pH adjusted to 6.35) to the wound when administrating only recombinant protein did not provide any significant wound-healing effect (effect similar to lowering the pH to 6.35 according as shown in Example 4). Thus, the wound healing effect obtained by administering lactic acid bacteria and recombinant protein as shown in Example 3, cannot be attributed solely to co-administration of lactic acid and or additional lowering of pH in the wound (Example 4). This leads to the belief that the use of the live lactic acid bacteria creates a synergy between the lactic acid bacteria itself and the therapeutic protein, which increases the wound healing effect.

The effect of the therapeutic protein on wound healing may or may not be immediate, and may take some time to be seen (e.g., 1 , 2, 3, 4, 5 or 6 or more days to be seen, or longer, e.g., 8, 10, 12, 15, 18, 20 or 24 days or more before improved wound healing can be observed).

A particular and important utility of the present invention lies in the treatment of chronic wounds, particularly ulcers and in particular in the treatment of diabetic foot ulcers.

The prevalence of chronic foot ulcers in persons with diabetes is about 18 %. In 2013, the European population reached 742.5 million, which translates into 32.7 million with diabetes, of which 2.9-5.8 million have chronic foot ulcers. Mean duration of an ulcer of this type is in the range of months where less than 25 % of the wounds are healed within 12 weeks when standard care is given. The end stage of this condition is amputation of the affected limb. Today the treatment of people having chronic foot ulcers is divided between primary care, home care, nursing homes, relatives, selfmanagement and visits to hospital wound clinics. The current treatment relies on offloading, removal of dead tissue using surgical debridement, repeated changes of wound dressings, systemic antibiotics and in special cases treatment with living larvae or collagenase and at a few locations in Sweden hyperbaric oxygen treatment can be offered. If an underlying cause also includes obstructions of larger arteries, this can be corrected surgically by bypassing vein graft. Today the wounds are treated every second to third day. Treatment with the suggested recombinant protein and lactic acid bacteria in any of the suggested forms or formulations would not disrupt this practical routine. Improved healing of such wounds by the treatments of the present invention would thus be of considerable economic benefit, as well as of personal benefit to the patient. The bacteria are active at the wound surface for a period of time (e.g., about one hour) in vivo. They may then become inactive and die. Slow or dead lactic acid bacteria can with no risk be in the wound/dressing environment until the dressing is changed as normal.

The pharmaceutical composition, according to the present invention will have lower production cost compared to traditional biologies being protein-based drug compounds.

Open wounds such as diabetic foot ulcers, together with loss of function in the foot, cause considerable discomfort, and even disability to the patient, and can have a significant negative impact on quality of life, including significant risk or infection and therefore prolonged use of antibiotics, and ultimately amputation. Improved healing would thus be of tremendous personal benefit to the patient and would also have the benefit of reducing antibiotic use (and consequently the spread of antibiotic resistance). It is believed that treating such chronic wounds according to the invention may amplify endogenous alarm signals in the wound, and kick start the healing process in stalled or chronic wounds, and accelerate healing time.

Further, the invention may have advantages in flexibility and ease of use by medical staff.

Representative methods and preferred embodiments according to the present invention will be further described with reference to the following non-limiting Examples:

EXAMPLES

MATERIALS AND METHODS

Wounds were in all examples induced according to a standard protocol, including hair removal by shaver and Veet hair removal cream. A 5mm biopsy punch was used to induce the wounds, whereafter Emla cream was applied to the wounds and they were documented by taking a photograph. The immune-effective protein used was recombinant murine CXCL12 1a, from RnD Systems. The lactic acid bacteria used was Lactobacillus reuteri R2LC Luc (having a luciferase insert for monitoring purposes).

EXAMPLE 1 (Previous proprietary studies): Dose escalation study of administration of recombinant CXCL12 alone to a skin wound

For a dose escalation study of recombinant CXCL12 (0,2 pg, 0,6 pg, or 1 pg) was added to the wound in 10 pl saline at one time point once per day. Recombinant CXCL12 1a (RnD System) was diluted with saline such that 10 l saline solution contained the respective amounts of the recombinant CXCL12.

Results:

The higher dosages led to worsened treatment effect than the control (no treatment), but the 0,2 pg showed a slight, but not significant improvement. Thus, no accelerated wound healing could be shown.

EXAMPLE 2 (Previous proprietary studies): Administration of recombinant CXCL12 alone to a skin wound in various forms and concentrations

It is known that CXCL12 accelerates wound healing. However, in Example 1 , when the protein was administered (as a recombinant protein) directly to the wound surface in saline buffer there was no effect when administered once daily for two days.

In the present experiment, recombinant CXCL12 was diluted with saline such that 10 pl saline solution contained 0,2 pg, 0,6 pg, or 1 pg recombinant CXCL12 (murine CXCL12 1a, RnD Systems). Fresh supernatants from modified CXCL12- producing L. reuteri or the above-mentioned different concentrations of recombinant CXCL12 were applied topically to wounds once daily, wound closure was not accelerated. However, in the same experiment, CXCL12 (0.2 pg) was given every 10th minute for 1 h once daily to mimic continuous delivery accelerated wound closure. This continuous provision of the protein did enhance the wound healing, demonstrating that sustained CXCL12 delivery was needed to promote wound healing. The present inventors have also proven this by the studies of wound treatment by administration of modified CXCL12-producing L. reuteri, as described in reference 5 in the reference list.

EXAMPLE 3 (current studies): Administration of a combination of recombinant CXCL12 (0.2 pg and 0.02 pg) and live bacteria to a skin wound

In this experiment, the theory of synergistic relationship between live bacteria and recombinant protein was tested.

Methods:

Wound induction and wound monitoring:

Wounds (16-25 mm²) were induced on the right leg of mice using 5 mm biopsy punch. The wounds were photographed together with a scale before treatment and daily until the end of experiment. The wound size was measured from photographs using Image J software.

Treatment:

The wounds were then treated with 10 pl of Lactobacillus reuteri R2LC with a luciferase insert (R2LC LUC) in MRS broth and 10 pl saline containing 0.2 and 0.02 pg CXCL12 of recombinant mouse CXCL12 (RnD systems). Recombinant CXCL12 was prepared by resuspending recombinant mouse CXCL12 (RnD systems) resuspended in PBS (as described in the product sheet) to a concentration of 100 pg/ml and diluting with saline such that 10 pl saline solution contained 0.2 pg and 0.02 recombinant CXCL12, respectively. The mice were followed and treated for 8 days before the experiment was terminated and the wound tissue harvested. The tissue was snap frozen in optimal cutting temperature compound (OCT, 10.24 % polyvinyl alcohol, 4.26 % polyethylene glycol, 85.5 % non-reactive ingredients) using liquid nitrogen.

Culturing bacteria:

Frozen bacteria was added to 10 ml of MRS with 10 pl of erythromycin and grown overnight. 1 ml from the overnight culture was added to 10 ml of MRS with 10 pl erythromycin and grown for approximately 4 hours. The tube was centrifuged and most of the media was discarded (leaving approximately 1 ml). Bacteria was resuspended before being ready for wound administration.

Results:

Overall, a significantly quicker wound closure was seen during the first 48 hours for the treated wounds (there is a difference in the AUC at day 2 (t-test, 0.01) but not at day 8 (see Table 1 below).

However, early reduction of wound area is important for avoiding and limiting risk for infections (less space for pathogenic or opportunistic bacteria). Less risk of infection leads to less use of antibiotics, thereby lowering the risk of antibiotic resistance and the total bodily damage that can cause). EXAMPLE 4 (current studies): Administration of recombinant CXCL12 (0,2 pg) while lowering the pH

This experiment was made to determine the pH effect on wound healing. Wounds were induced and monitored as described in Example 3.

Methods:

Wounds were induced as described in example 3, photographed and then treated with of 0.2 pg recombinant CXCL12 in a solption with a pH valpe set to 6.35 by pse of acid. The wopnds were followed for 48 hoprs post wopnd indpction, with daily treatment and photographs of the wopnds.

Resplts:

The wopnds of the recombinant CXCL12 had a smaller start volpme, and the wopnds remained smaller thropghopt opt the two days. No treatment difference copld be observed (see Table 1 below). Thps, a decreased pH valpe did not seem to affect the wopnd healing ability of the recombinant protein.

Table 1 - Summary of results from Examples 3 and 4

% healed from baseline at wound induction REFERENCES

1. Demidova-Rice TN, Hamblin MR and Herman IM. Acute and impaired wound healing: pathophysiology and current methods for drug delivery, part 1: normal and chronic wounds: biology, causes, and approaches to care. Advances in skin & wound care. 2012;25:304-14.

2. Demidova-Rice TN, Hamblin MR and Herman IM. Acute and impaired wound healing: pathophysiology and current methods for drug delivery, part 2: role of growth factors in normal and pathological wound healing: therapeutic potential and methods of delivery. Advances in skin & wound care. 2012;25:349-70.

3. Poutahidis T, Kearney SM, Levkovich T, Qi P, Varian BJ, Lakritz JR, Ibrahim YM, Chatzigiagkos A, Alm EJ and Erdman SE. Microbial symbionts accelerate wound healing via the neuropeptide hormone oxytocin. PLoS One. 2013;8:e78898.

4. Ramos AN, Cabral ME, Noseda D, Bosch A, Yantorno OM and Valdez JC. Antipathogenic properties of Lactobacillus plantarum on Pseudomonas aeruginosa: the potential use of its supernatants in the treatment of infected chronic wounds. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society. 2012;20:552- 62.

5. WQ2016102660 A1 , ILYA PHARMA AB, Vagesjd et al

6. Gao Z, Tseng C-h, Pei Z and Blaser MJ. Molecular analysis of human forearm superficial skin bacterial biota. Proceedings of the National Academy of Sorvig E, Mathiesen G, Naterstad K, Eijsink VGH and Axelsson L. High-level, inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors. Microbiology. 2005; 151 :2439-2449. Sciences. 2007;104:2927-2932.

7. Salcedo R, Wasserman K, Young HA, Grimm MC, Howard OMZ, Anver MR, Kleinman HK, Murphy WJ and Oppenheim JJ. Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor Induce Expression of CXCR4 on Human Endothelial Cells: In Vivo Neovascularization Induced by Stromal-Derived Factor-1a. The American Journal of Pathology. 1999;154:1125-1135.

8. Hattermann K, Sebens S, Helm O, Schmitt AD, Mentlein R, Mehdorn HM and Held-Feindt J. Chemokine expression profile of freshly isolated human glioblastoma-associated macrophages/microglia. Oncology reports. 2014;32:270- 6. 9. Badillo AT, Chung S, Zhang L, Zoltick P and Liechty KW. Lentiviral gene transfer of SDF-1 alpha to wounds improves diabetic wound healing. The Journal of surgical research. 2007;143:35-42.

SEQUENCES

Table 2: Summary of sequences

(Original in Electronic Form)

(This sheet is not part of and does not count as a sheet of the international application)

FOR RECEIVING OFFICE USE ONLY

FOR INTERNATIONAL BUREAU USE ONLY

Claims

1. A pharmaceutical composition comprising a recombinant protein and live wildtype or non-wild type bacteria, wherein the bacteria do not express the recombinant protein.

2. The pharmaceutical composition of claim 1, wherein the bacteria are live wildtype or non-wild type lactic acid bacteria.

3. The pharmaceutical composition of any of claims 1 or 2, wherein the recombinant protein is selected from an interleukin, a cytokine or a chemokine, preferably a CXC protein, more preferably selected from the group consisting of CXCL12 and CXCL17.

4. The pharmaceutical composition of any of claims 1 to 3, wherein the bacteria are a Lactobacillus strain.

5. The pharmaceutical composition of claim 4, wherein the bacteria are Lactobacillus reuteri.

6. The pharmaceutical composition of claim 5, wherein the bacteria are Lactobacillus reuteri R2LC.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the recombinant protein has an amino acid sequence selected from:

(i) murine CXCL12-1a having an amino acid sequence as shown in SEQ ID NO: 3 or 2, or an amino acid sequence with at least 80% sequence identity thereto;

(ii) human CXCL12-1a having an amino acid sequence as shown in SEQ ID NO: 6 or 5, or an amino acid sequence with at least 80% sequence identity thereto;

(iii) murine CXCL17 having an amino acid sequence as shown in SEQ ID NO: 9 or 8, or an amino acid sequence with at least 80% sequence identity thereto; and (iv) human CXCL17 having an amino acid sequence as shown in SEQ ID NO: 12 or 11, or an amino acid sequence with at least 80% sequence identity thereto.

8. The pharmaceutical composition of any one of claims 1 to 7 for use in wound healing in a human or animal subject.

9. The pharmaceutical composition of any one of claims 1 to 7 for use in cutaneous or mucosal wound healing in a human or animal subject.

10. The pharmaceutical composition of claim 8, for use in cutaneous treatment of skin wounds in a human or animal subject.

11. The pharmaceutical composition of claim 8, for use in treatment of gastrointestinal wounds in a human or animal subject.

12. A wound dressing comprising the pharmaceutical composition according to any one of claims 1 to 7.

13. A medical device comprising the pharmaceutical composition according to any one of claims 1 to 7.

14. A kit for healing wounds, said kit comprising a recombinant protein, preferably selected from a recombinant protein, more preferably selected from CXC proteins, and most preferably selected from the group consisting of CXCL12 and CXCL17, and live wild-type or non-wild type bacteria, preferably live wildtype or non-wild type lactic acid bacteria, wherein the bacteria do not express the recombinant protein.

15. The kit of claim 14, being a pharmaceutical product comprising the recombinant protein and live wild-type or non-wild type lactic acid bacteria, as a combined preparation for separate, sequential or simultaneous use in wound healing.

16. The kit of claim 14, wherein the kit further comprises a wound dressing.

17. The kit of claim 16, wherein the kit comprises a wound dressing comprising the recombinant protein and live wild-type or non-wild type bacteria.

18. A method of treating a subject to heal a wound, said method comprising administering to said subject, or to the wound in said subject, an amount of a recombinant protein, more preferably selected from CXC proteins, and most preferably selected from the group consisting of CXCL12 and CXCL17, effective to promote healing of the wound, and live wild-type or non-wild type bacteria, preferably live wild-type or non-wild type lactic acid bacteria, wherein the bacteria do not express the recombinant protein.

19. The method of claim 18, wherein the bacteria are a Lactobacillus strain.

20. The method of claim 18, wherein the strain is Lactobacillus reuteri.

21. The method of claim 18, wherein the strain is Lactobacillus reuteri R2LC.

22. The method of any one of claims 18 to 20, wherein the recombinant protein has an amino acid sequence selected from:

(i) murine CXCL12-1a having an amino acid sequence as shown in SEQ ID NO: 1 or 2, or an amino acid sequence with at least 80% sequence identity thereto;

(ii) human CXCL12-1a having an amino acid sequence as shown in SEQ ID NO: 3 or 4, or an amino acid sequence with at least 80% sequence identity thereto;

(iii) murine CXCL17 having an amino acid sequence as shown in SEQ ID NO: 5 or 6, or an amino acid sequence with at least 80% sequence identity thereto; and

(iv) human CXCL17 having an amino acid sequence as shown in SEQ ID NO: 7 or 8, or an amino acid sequence with at least 80% sequence identity thereto.