Antibacterial protein complexes

Abstract

The present invention relates to protein bacteriocins (PBs) as therapeutic agents, and in particular to protein complexes comprising two or more PB molecules associated with a protein scaffold comprising a homologous immune protein domain against an effector portion of the corresponding PB. In particular, the present invention provides an antibacterial protein complex comprising: (a) a first PB molecule and a second PB molecule; and (b) an immune protein scaffold comprising a first immune protein domain and a second immune protein domain; wherein the first immune protein domain and the second immune protein domain are non-covalently bound to the first PB molecule and the second PB molecule, respectively.

Description

[0001] This application claims priority from GB 2301639.7 filed on 6th February 2023, the contents and elements of which are incorporated herein by reference for all purposes. TECHNICAL FIELD

[0002] The present invention relates to bacteriocins as therapeutic agents, and in particular to protein complexes comprising two or more bacteriocin molecules associated with a protein scaffold comprising a cognate immunoprotein domain to the effector portion of the respective bacteriocin. The complexes of the invention can provide a variety of advantages, including improved cell-killing activity, reduced resistance development, enhanced strain coverage, and improved production and purification efficiency. BACKGROUND

[0003] For Gram-negative pathogens, such as Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli, the efficacy of many classes of antibiotics is severely limited due to horizontal acquisition of antibiotic resistance determinants and the presence of a highly impermeable outer membrane, and treatment options are often limited.

[0004] Accordingly, there is an urgent need to consider alternative strategies to antibiotic development to bolster the development pipeline for antibiotics active against these hard-to-treat bacteria that has produced little in the last few decades.

[0005] An alternative strategy to discover effective antibiotics is to exploit the intraspecies competition by the potent narrow-spectrum antibiotics produced by many bacteria. Protein bacteriocins (PBs) are a class of multi-domain protein antibiotics that include S-type pyocins, klebicins, colicins, cloacins, salmosins and pesticins produced by Pseudomonas aeruginosa, Klebsiella (e.g. Klebsiella pneumoniae), Escherichia coli, Enterobacter cloacae, Salmonella (e.g. Salmonella enterica) and Yersinia pestis, respectively. PBs have evolved to efficiently cross the outer membrane of Gram-negative bacteria by parasitising existing nutrient uptake pathways. The cellular targets of PBs are highly conserved, with the most common forms of cytotoxic activity being enzymatic activity (typically a nuclease or an enzyme that degrades peptidoglycan precursor molecules, thus inhibiting cell wall synthesis) or pore-forming activity targeting the cytoplasmic membrane.

[0006] However, when PBs are active against clinically relevant strains, each bacteriocin tends to target a limited number of strains of a given species. To achieve therapeutic relevant strain coverage, it is therefore desirable to use two or three different bacteriocins together. There are many challenges to producing a mixture of pharmaceutical grade protein bacteriocins, including high purification costs. SUMMARY

[0007] The present invention provides an anti-bacterial protein complex comprising:

[0008] (a) a first protein bacteriocin (PB) molecule and a second PB molecule;

[0009] and

[0010] (b) an immunoprotein scaffold comprising a first immunoprotein domain and a second immunoprotein domain; wherein the first and second immunoprotein domains are non-covalently bound to the respective first and second PB molecules.

[0011] Despite the 3D structure differences, PBs share a characteristic multi-domain structure comprising an effector (or cytotoxicity) portion (usually at the C-terminus) and a cell targeting portion (usually at the N-terminus of the effector domain), as discussed in more detail below. They are produced by Gram-negative bacteria, and often the species in which the bacteriocin functions is the same species. The organism expressing the PB also expresses a cognate immunoprotein that binds to the bacteriocin (usually to the effector portion of the bacteriocin) with ultra-high affinity (~fM) and thus prevents the bacteriocin from killing the host. Some examples of species-specific PBs and their targets include colicin (E. coli), S-type pyocin (P. aeruginosa), klebicin (Klebsiella, e.g., K. pneumoniae), enterocin (E. cloacae), salmonicin (Salmonella, e.g., S. enterica), and yersiniocin (Y. pestis). Thus, each bacteriocin generally comprises a cell targeting portion and an effector portion, and binds to the corresponding immunoprotein domain of the immunoprotein scaffold through its effector portion.

[0012] The complexes of the present invention utilize an immunoprotein scaffold containing multiple immunoprotein domains. This configuration allows a single complex to contain two or more bacteriocin molecules that are delivered to the target cell surface in close physical proximity to one another. The interaction between the bacteriocin molecule and its cognate receptor on the surface of the target bacteria generally results in dissociation of the bacteriocin from the immunoprotein scaffold. As described in more detail below, the present inventors have discovered that such multivalent complexes (i.e., complexes containing multiple bacteriocin molecules) provide a number of advantages over the corresponding single bacteriocins, including ease of manufacture and purification, increased microbial killing activity, and decreased resistance development.

[0013] The immunoprotein scaffold comprises a plurality of immunoprotein domains coupled to one another. The immunoprotein scaffold can contain any number of immunoprotein domains, for example two, three, four, five immunoprotein domains or even more. Thus, when fully occupied, the anti-bacterial protein complex contains the same number of bacteriocin molecules, each bacteriocin being non-covalently associated with a corresponding immunoprotein domain of the immunoprotein scaffold via its effector moiety. (It will be appreciated, however, that any population of such complexes can inevitably contain some with less than fully occupied complexes.) Two or three immunoprotein domains can be preferred, such that the anti-bacterial protein complex contains two or three bacteriocin molecules, respectively. Thus, the immunoprotein scaffold can comprise a third immunoprotein domain, and the anti-bacterial protein complex can comprise a third bacteriocin molecule non-covalently bound to the third immunoprotein domain.

[0014] For the avoidance of doubt, it will be appreciated that the immunoprotein scaffold and the corresponding bacteriocin molecules are separate molecules, associated by non-covalent interactions between the immunoprotein domains and the effector moieties of the bacteriocin molecules. These can be substantially the only interactions between the multiple components of the complex.

[0015] The scaffold can comprise multiple (two or more) repeats of the same immunoprotein domain. For example, all of the immunoprotein domains can be the same. Thus, the complex will contain multiple copies of the same bacteriocin. (Or at least, multiple bacteriocins each having the same effector moiety).

[0016] However, typically the scaffold contains two or more different immunoprotein domains. Thus, the complex contains bacteriocin molecules having two or more different effector moieties. The scaffold can contain two, three, four or five different immunoprotein domains or even more. In some embodiments, each component immunoprotein domain in a given scaffold molecule is different from each other component immunoprotein domain. Two or three different immunoprotein domains can be preferred, such that the anti-bacterial protein complex contains bacteriocin molecules having two or three different effector moieties, respectively.

[0017] The immunoprotein scaffold is typically a fusion protein, i.e. a single peptide chain comprising the relevant immunoprotein domains (optionally separated by linker peptides).

[0018] The immunoprotein scaffold can also comprise a heterologous moiety, i.e. a moiety that is neither an immunoprotein domain nor a linker peptide. In some embodiments, the heterologous moiety can comprise a cytotoxic domain, such that the immunoprotein scaffold itself is an additional toxin in addition to its associated PBs. The heterologous moiety can comprise a cytotoxic domain and a moiety capable of mediating transport across the outer membrane of a target cell, such as a protein bacteriocin targeting moiety. In some preferred embodiments, the heterologous moiety is an M-type bacteriocin, such as colicin M (ColM), KpneM, KpneM2, KvarM, PaeMl or PaeM4, or a functional variant thereof. In such embodiments, the M-type bacteriocin is typically located at the N-terminus of the scaffold molecule, with the immunoprotein domains located C-terminal to the M-type bacteriocin. A suitable linker is typically present between the M-type bacteriocin and the first immunoprotein domain.

[0019] The effector moiety is typically an enzymatic effector moiety. The enzymatic effector moiety typically has nuclease activity. The nuclease can be, for example, a DNAse (capable of degrading DNA) or an RNAse (capable of degrading RNA). The RNAse can be, for example, an rRNAse (having activity against ribosomal RNA) or a tRNAse (having activity against transfer RNA).

[0020] Thus, within a given complex, each bacteriocin typically has an enzymatic effector moiety having nuclease activity. The effector moieties of the bacteriocins can be the same or different. Thus, a complex can comprise two or more bacteriocins having the same effector moiety. For example, all of the bacteriocins within a complex can have the same effector moiety. Alternatively, a complex can comprise two or more bacteriocins having different enzymatic effector moieties. For example, all of the bacteriocins within a complex can have different effector moieties. When the effector moieties are different, they can still have the same enzymatic activity, e.g. DNAse or RNAse (which can be rRNAse or tRNAse). Thus, a complex can comprise two bacteriocins having different effector moieties but in which those effector moieties have the same activity. Alternatively, some or all of the effector moieties can have different enzymatic activities. In some embodiments, each effector moiety in a given complex is an enzymatic effector moiety, and each of those effector moieties can have a different enzymatic activity. Some examples of suitable combinations include:

[0021] - an effector moiety having DNAse activity and an effector moiety having RNAse activity (e.g. rRNAse or tRNAse);

[0022] - an effector moiety having rRNAse activity and an effector moiety having tRNAse activity;

[0023] - effector moieties having DNase activity, effector moieties having rRNAse activity, and effector moieties having tRNAse activity.

[0024] Independently, the complex can comprise bacteriocins all having the same targeting moiety or bacteriocins having two or more different targeting moieties. In some embodiments, each bacteriocin in the complex has a different targeting moiety.

[0025] It can be desirable for all of the bacteriocins within a complex to have targeting moieties that are specific for bacteria of the same species or strain. However, they can bind to two or more different receptors (e.g., each targeting moiety binds to a different receptor) or use two or more different transport portals (e.g., each targeting moiety uses a different transport portal).

[0026] However, it can also be desirable for two or more bacteriocins in a given complex to each have a targeting moiety that is specific for a different species or strain, such that a single complex has activity against two or more bacterial species or strains. Thus, the activity profile of a given complex can be modulated by appropriate selection of the targeting moieties of the bacteriocins present in the complex.

[0027] A bacteriocin molecule comprises an effector (or cytotoxicity) moiety and a targeting moiety. Within a given bacteriocin molecule, the effector domain and the targeting domain can be derived from the same wild-type bacteriocin. Alternatively, the bacteriocin can be chimeric, comprising an effector moiety and a targeting moiety from different wild-type bacteriocins. It will be appreciated that the present application is not limited to the use of wild-type bacteriocin sequences. Modified or engineered bacteriocin sequences can also be used, whether in their effector moiety, targeting moiety, or both.

[0028] One illustrative example of a complex of the present application is a complex comprising:

[0029] a first PB comprising an E. coli bacteriocin E9 effector moiety;

[0030] a second PB comprising an E. coli bacteriocin D effector moiety; and

[0031] an immunoprotein scaffold comprising an E. coli bacteriocin E9 immunoprotein domain ("Im9") and an E. coli bacteriocin D immunoprotein domain ("ImD") (collectively designated "Im9-ImD").

[0032] The first PB can be E. coli bacteriocin E9 (i.e., the first PB also comprises an E. coli bacteriocin E9 targeting moiety). The second PB can be E. coli bacteriocin D (i.e., the second PB also comprises an E. coli bacteriocin D targeting moiety). Alternatively, either or both can be a chimeric PB, comprising a targeting moiety from a different PB than the PB of its effector moiety.

[0033] Another illustrative example of a complex of the application is a complex comprising:

[0034] a first PB comprising a colicin E9 effector moiety;

[0035] a second PB comprising a colicin E3 effector moiety;

[0036] a third PB comprising a colicin D effector moiety; and

[0037] an immunoprotein scaffold comprising a colicin E9 immunoprotein domain ("Im9"), a colicin E3 immunoprotein domain ("Im3"), and a colicin D immunoprotein domain ("ImD") (collectively designated "Im9-Im3-ImD").

[0038] The first PB can be colicin E9 (i.e., the first PB also comprises a colicin E9 targeting moiety). The second PB can be colicin E3 (i.e., the second PB also comprises a colicin E3 targeting moiety). The third PB can be colicin D (i.e., the third PB also comprises a colicin D targeting moiety).

[0039] Alternatively, one, two, or all three of these PBs can be chimeric PBs comprising a targeting moiety from a different PB than the PB of its effector moiety.

[0040] For example, independently:

[0041] The first PB can be a chimeric PB comprising a KlebC targeting moiety and a colicin E9 effector moiety ("KlebC-E9");

[0042] The second PB can be a second chimeric PB comprising a CloDF13 targeting moiety and a colicin E3 effector moiety ("CloDF13-E3"); and

[0043] The third PB can be a chimeric PB comprising a KlebG targeting moiety and a colicin D effector moiety (designated "KlebG-D").

[0044] The application also provides an anti-bacterial method comprising contacting a bacterium or a population of bacteria with an anti-bacterial complex as described herein.

[0045] The application also provides an anti-bacterial complex as described herein for use in a method of medical treatment, for example, for preventing or treating a bacterial infection or a condition caused by or associated with a bacterial infection.

[0046] The present application also provides the use of an antibacterial complex as described herein in the manufacture of a medicament for the prevention or treatment of a bacterial infection or a condition caused by a bacterial infection or a condition associated with a bacterial infection.

[0047] The present application also provides a method of preventing or treating a bacterial infection or a condition caused by a bacterial infection or a condition associated with a bacterial infection, comprising administering to a subject in need thereof an antibacterial complex as described herein.

[0048] The bacterial infection is typically an infection with a Gram-negative bacterium.

[0049] The present application also provides a host cell comprising:

[0050] (i) a nucleic acid encoding an immunoprotein scaffold, the immunoprotein scaffold comprising a first immunoprotein domain and a second immunoprotein domain;

[0051] (ii) a nucleic acid encoding at least one bacteriocin, the or each of the bacteriocins having an effector domain capable of binding to at least one of the immunoprotein domains;

[0052] wherein the host cell is capable of expressing the immunoprotein scaffold and the at least one bacteriocin.

[0053] Upon expression of the scaffold and bacteriocin components, an antibacterial complex of the application can be formed. Where the antibacterial complex contains an immunoprotein scaffold having only one type of immunoprotein domain, and the complex contains only one type of bacteriocin, the cell can encode and express only that single cognate bacteriocin.

[0054] Where the antibacterial complex contains at least two different types of bacteriocin (e.g. the immunoprotein scaffold contains at least two different immunoprotein domains), the cell comprises nucleic acid encoding and is capable of expressing at least two bacteriocins, each of the bacteriocins having an effector capable of binding to at least one of the immunoprotein domains.

[0055] Preferably, the host cell comprises nucleic acid encoding and is capable of expressing a bacteriocin having an effector domain capable of binding to each of the immunoprotein domains of the immunoprotein scaffold.

[0056] Thus, for example, the cell can comprise:

[0057] (i) a nucleic acid encoding an immunoprotein scaffold, the immunoprotein scaffold comprising a first immunoprotein domain and a second immunoprotein domain;

[0058] (ii) a first and second nucleic acid encoding a respective first and second bacteriocin, each bacteriocin having an effector domain capable of binding to a respective one of the immunoprotein domains;

[0059] wherein the host cell is capable of expressing the immunoprotein scaffold and the bacteriocins.

[0060] Further, the cell can comprise:

[0061] (i) a nucleic acid encoding an immunoprotein scaffold, the immunoprotein scaffold comprising a first, second and third immunoprotein domain;

[0062] (ii) a first, second and third nucleic acid encoding a respective first, second and third bacteriocin, each bacteriocin having an effector domain capable of binding to a respective one of the immunoprotein domains;

[0063] wherein the host cell is capable of expressing the immunoprotein scaffold and the bacteriocins.

[0064] As discussed above, it will be apparent that the immunoprotein scaffold can comprise more immunoprotein domains, if desired, in which case the host cell can encode (and express) a corresponding number of bacteriocins, as desired.

[0065] The present application also provides a method of producing an anti-bacterial complex, comprising providing the host cell described above, and culturing the cell under conditions suitable for expression of the immunoprotein scaffold and bacteriocin molecules. The method can further comprise the step of isolating the anti-bacterial complex, and optionally further purification steps.

[0066] Alternatively, it can be desirable not to express all components of the anti-bacterial complex in the same host cell. Rather, the components can be expressed in two or more different host cells, each host cell comprising nucleic acid encoding one or more individual components, and being capable of expressing those components. For example, the immunoprotein scaffold can be expressed in one host cell, and one or more bacteriocin molecules expressed in one or more other host cells.

[0067] Accordingly, the present application also provides a method of producing an anti-bacterial complex, the method comprising contacting an immunoprotein scaffold comprising a first and second immunoprotein domain with a first and second bacteriocin molecule, each having an effector moiety capable of binding to a respective one of the immunoprotein domains, to form a complex of the application.

[0068] In such a case, if the bacteriocin molecule is expressed in a host cell that is susceptible to the bacteriocin and does not also express an immune protein scaffold, the host cell will typically also express a cognate immune protein for the relevant bacteriocin. In such a case, the bacteriocin can be dissociated from the immune protein and isolated therefrom prior to contacting the bacteriocin with the immune protein scaffold.

[0069] The application includes combinations of the described aspects and preferred features, except where such combinations are clearly inadmissible or explicitly avoided. BRIEF DESCRIPTION OF DRAWINGS

[0070] Some embodiments and experiments illustrating the principles of the application will now be discussed with reference to the accompanying drawings, in which:

[0071] Figure 1 (a) Schematic of a hetero-trimeric complex (2 bacteriocin molecules) and (b) a hetero-tetrameric complex (3 bacteriocin molecules). Immune proteins are denoted as “Im” (Im9, Im3 and ImD). Cognate cytotoxic domains are denoted as E9, E3 and D. The remainder of the bacteriocin molecules are simply shown as “bacteriocin”. (c) Sketch structure of Im9, Im3 and ImD linked together with flexible linkers denoted by dashed lines. (d) Sketch structure of Im9-Im3-ImD fusion protein associated with its cognate bacteriocins.

[0072] Figure 2 . Transport pathways of colicin E9 (Francis et al., 2021) and colicin D across the cell envelope. Colicin E9 binds to BtuB and OmpF in the outer membrane, then interacts with TolB in the periplasm, leading to FtsH-dependent transport of the C-terminal DNase domain to the cytoplasm. Colicin D crosses the outer membrane by interacting with FepA, interacts with TonB in the periplasm, leading to FtsH-dependent transport of the C-terminal tRNAse to the cytoplasm (Chauleau et al., 2011).

[0073] Figure 3 . Enterobactin DF13 binds to the ferric aerobactin receptor IutA, then threads its unstructured N-terminus through the trimeric porin IutA to bind to TolB in the periplasm (unpublished). Klebocin G binds to the trimeric porin OmpK35, allowing its N-terminus to cross the outer membrane and bind to TolA. KlebC binds to the efflux pump TolC, with the N-terminus of the klebocin crossing the TolC lumen to interact with TonB in the periplasm.

[0074] Figure 4(i) Im9-ImD + ColE9 + ColD, (ii) Im9-ImD + ColD, (iii) Im9-ImD + ColE9 and (iv) Im9-ImD Superdex 200 increase 10 / 300 GL elution profiles. The center 0.5 ml of the elution peak of each complex was collected for analysis in a killing assay.

[0075] Figure 5 A-C: Killing activity of ColD, ColE9 and [ColE9:Im9-ImD:ColD] against BL21(DE3) (btuB-), fepA- BW25113, tolA- BW25113 and tonB- BW25113 E. coli cells. D: Killing activity of ColE9, ColD, ColE9 + ColD and [ColE9:Im9-ImD:ColD] against tolA- BW25113 E. coli cells.

[0076] Figure 6 Growth inhibition of [ColE9:Im9-ImD:ColD], [Im9-ImD:ColD] and [ColE9:Im9-ImD] on E. coli MG1655. Liquid cultures of E. coli MG1655 were monitored by OD 630nm after 6 hours (top panel) and 24 hours (bottom panel) of incubation in the presence of [ColE9:Im9-ImD:ColD], [Im9-ImD:ColD] or [ColE9:Im9-ImD]. Concentrations of 5.6 pM to 1 μΜ of complex were used.

[0077] Figure 7 Nickel affinity purification of [ColE9:Im9-ImD:ColD] expressed in BL21(DE3) cells with cloning of Colicin E9, Im9-ImD, Colicin D into pET21a and an additional copy of Colicin D cloned into pACYCDuet1. The protein was eluted from the column with a gradient of 0 to 500 mM imidazole over 10 column volumes and fractions (fractions 18 to 23 shown) were analyzed on a 12% SDS-PAGE gel.

[0078] Figure 8 26 / 60 S200 purification of [ColE9:Im9-ImD:ColD] complex. [ColE9:Im9-ImD:ColD] was purified on a 26 / 60 S200 column equilibrated in 25 mM Tris-HCl, pH 7.5, 150 mM NaCl and eluted fractions were analyzed on a 12% SDS-PAGE gel.

[0079] Figure 9Activity of in vivo assembled trimeric [ColE9:Im9-ImD:ColD] complex against soft agar lawns of E. coli. Three-fold serial dilutions of [ColE9:Im9-ImD:ColD] were prepared in a concentration range of 100 nM to 137 pM. 5 μl of each dilution was spotted on soft agar lawns inoculated with E. coli BW25113, btuB-BL21(DE3), fepA-BW25113, tolA-BW25113 or tonB-BW25113. After overnight incubation at 37°C, clearance areas in the bacterial lawn showed bacteriocin activity.

[0080] Figure 10 Purification of assembled tetrameric complex [Im9-Im3-ImD:KlebC-E9:CloDF13-E3:KlebG-D] on a Superdex 200 increase 10 / 300 GL analytical gel filtration column (Cytiva). 12% SDS-PAGE analysis of elution fractions from the peak is shown.

[0081] Figure 11 Killing activity of tetrameric complex [Im9-Im3-ImD:KlebC-E9:CloDF13-E3:KlebG-D] against K. pneumoniae cells of strains SG62, SR3 and SR6, in comparison to previously observed activity of KlebC-E9, CloDF13-E3 and KlebG-D against the same strains.

[0082] Figure 12 Schematic representation of KvarM immunoprotein scaffolds and demonstration of cytotoxic activity. [A] Schematic representation of KvarM-Im9-ImD protein scaffold. [B] Schematic representation of KvarM-Im9-Im7-ImD protein scaffold. [C] Cytotoxic activity of wild-type KvarM and KvarM-immunoprotein scaffolds against K. pneumoniae SG96 cells in nutrient broth.

[0083] Figure 13 Schematic representation of KvarM-immunoprotein scaffold fusions with homologous protein bacteriocin complexes.

[0084] Figure 14 Assembly and activity of trimeric complex [S5E9:Im9-Im7:S5E7] and activity against P. aeruginosa. (A) Schematic representation of the composition of the three-component [S5E9:Im9-Im7:S5E7] complex. Chimeric pyocins S5E9 and S5E7 consist of the outer membrane transport (T om ) and receptor binding (R) domains of pyocin S5, followed by the inner membrane transport domain (TIM ) and DNase domains from colicins E9 and E7, respectively. These chimeric pyocins form complexes with the immunoprotein scaffold through the interaction between the E7 and E9 DNase domains of the chimeric pyocins and the cognate E7 and E9 immunoprotein domains of the scaffold. (B) SDS PAGE of purified proteins. Free S5E9 and S5E7, and S5E9 and S5E7 immunoprotein complex forms, as well as the [S5E9:Im9-Im7:S5E7] complex were loaded onto a 16% SDS PAGE gel. In addition, samples of refolded monomeric pyocin S5E9 and S5E7 showed bands indicative of the presence of multimeric pyocins. (C) Activity of the [S5E9:Im9-Im7:S5E7] complex and component proteins. Three-fold dilutions of each protein or protein complex from an initial concentration of 0.14 mg / ml were spotted onto growing lawns of P. aeruginosa P8. The empty areas indicate killing of P. aeruginosa. DETAILED DESCRIPTION

[0085] Some aspects and embodiments of the present application will now be discussed with reference to the drawings. Other aspects and embodiments will be apparent to the skilled person. All documents mentioned herein are incorporated by reference.

[0086] While protein bacteriocins are active against clinically relevant strains, each bacteriocin tends to target a limited number of strains of a given species. In order to achieve therapeutic relevant strain coverage, it can therefore be desirable to use two or three different bacteriocins together. There are many challenges to producing a mixture of pharmaceutical grade protein bacteriocins, including high purification costs.

[0087] The complexes of the present application provide a number of advantages, including the possibility of expressing all components (bacteriocin proteins and immunoprotein scaffold) in a single culture, enabling purification of a single multifunctional protein complex. Even if the individual components are expressed in two or more cultures, the affinity of the scaffold for the bacteriocin molecules can facilitate purification of a single stoichiometrically defined complex from the relatively crude mixtures of the different cultures, without the need to purify each component separately.

[0088] The complexes described can also provide functional advantages, for example in terms of cell killing and reduction of resistance development. The complexes of the present application are believed to induce higher levels of cell killing than formulations of the same individual bacteriocins, possibly due to an avidity effect provided by the physical association of multiple receptor binding domains. This phenomenon takes advantage of the fact that outer membrane proteins of bacteria tend to associate in clusters.

[0089] Complexes containing different bacteriocins associated with a single immunoprotein scaffold ("hetero" complexes) also present as exhibiting improved cell killing compared to the corresponding individual bacteriocins, even in bacteria lacking components normally required for uptake of one of these bacteriocins, such as one or more outer or inner membrane proteins involved in membrane transport (herein referred to as "transport portals"). Without wishing to be bound by theory, it is thought that the close spatial positioning of different bacteriocin components can enable them to share components of each other's uptake systems in ways that are not possible for individual bacteriocins. This can be facilitated by the aggregation of different bacterial outer membrane proteins into large clusters ("Lipids Mediate Supramolecular Outer Membrane Protein Assembly in Bacteria". Webby M.N. et al., Sci. Adv. 8, eadc9566 (2022); doi: 10.1126 / sciadv.adc9566 ).

[0090] The complexes of the present application can also be readily adapted to target bacteria of different species or strains. Typically, the cytotoxic domain is effective in many types of bacteria and can be readily exchanged between bacteriocin molecules. Strain specificity is determined primarily by the receptor binding and / or transport domains. Thus, the strain specificity of a complex can be simply tailored by exchanging one set of receptor binding and / or transport domains of a bacteriocin molecule for another, enabling the same set of cytotoxic domains (and thus the same immunoprotein scaffold) to be used against a wide range of bacterial types.

[0091] The use of complexes comprising two or more different bacteriocin molecules can also reduce the likelihood of resistance developing to related bacteriocins. For example, a single complex can contain two or more different receptor binding bacteriocin molecules that use different transport pathways, and / or that have different cytotoxic activities, all of which reduce the chance of resistance developing. (It will also be appreciated that a similar effect can be achieved by using a population or "cocktail" comprising multiple different complexes, wherein each given complex carries multiple copies of the same bacteriocin molecule, and the population comprises at least two different bacteriocin molecules, such as three, four, five or even more different bacteriocin molecules.

[0092] Bacteriocins suitable for use in the context of the present application are proteinaceous antimicrobial toxins produced by and effective against Gram-negative bacterial species, designated "Protein Bacteriocins" (PBs).

[0093] Other structurally different types of bacteriocins are known, but are not included within the definition of "Protein Bacteriocins".

[0094] Thus, both granular bacteriocins, such as R-type pyocins (rod-shaped) and F-type pyocins (flexible and non-contractile), are associated with phage tail proteins (from P2 phage and lambda phage, respectively) and are sometimes also referred to as “tailocins” or “high molecular weight bacteriocins”.

[0095] Peptide-like bacteriocins (sometimes referred to as microcins) are anti-bacterial peptides of typically less than 10 kDa in size, which are secreted by bacteria, mainly by the Enterobacteriacea family. They can be divided into class I (less than 5 kDa) and class II (5 to 10 kDa) microcins and show a variety of mechanisms of action, including pore formation in the bacterial membrane (MccV, MccE492 and MccL), inhibition of aspartyl-tRNA synthetase (MccC), inhibition of DNA gyrase GyrB, causing double-stranded DNA breaks (MccB17), inhibition of transcription, and inhibition of cellular respiration through cytochromes (MccJ25), cellular proton channels (MccH47, and possibly MccM and MccI) or ATP synthase (MccH47). For review see Baquero et al., Front. Microbiol., October 2019, Vol. 10, 2019 (doi: 10.3389 / fmicb.2019.02347). 10.3389 / fmicb.2019.02261 ).

[0096] PBs are considered evolutionarily related and share a characteristic multi-domain structure comprising a targeting moiety and an effector (or cytotoxicity) moiety. Typically, the targeting moiety is located at the N-terminus of the molecule and the effector moiety at the C-terminus, especially for those with nuclease activity.

[0097] PBs include colicin (active against E. coli), S-type pyocin (active against P. aeruginosa), K- pyocin (active against Klebsiella species, such as K. pneumoniae, K. quasipneumoniae, K. oxytoca, K. variicola and K. aerogenes), enterocin (active against Enterococcus faecalis), salmonicin (active against Salmonella enterica) and yersiniocin (active against Yersinia pestis). They can also be referred to as “colicin-like bacteriocins”. For review see Behrens et al., Emerging Topics in Life Sciences (2017) 1 :65-74 (doi: 10.1042 / ETLS20160016).

[0098] The effector moiety can constitute a single independently folded domain. The targeting moiety can also constitute a single independently folded domain or can be subdivided into two or more independently folded domains.

[0099] The targeting moiety binds to a receptor at the surface of the target organism (i.e. the outer membrane of a Gram-negative bacterium) and mediates transport of the bacteriocin across the outer membrane. For the avoidance of doubt, the term “receptor” is used merely to denote the molecule on the target organism to which the targeting moiety binds and should not be understood as implying a cognate receptor-ligand interaction in the sense of a pair of molecules typically intended to be expressed by a single organism. The receptor is typically an outer membrane protein but can be any suitable molecule in the outer membrane, such as a lipopolysaccharide (e.g. the common polysaccharide antigen of P. aeruginosa which is thought to act as a receptor for many pyocins, including PyoL1, PyoS2, PyoS3, PyoS5, PyoSD2 and PyoSD3).

[0100] In general, the targeting moiety determines the species and strain specificity (or tropism) of the bacteriocin. In this specification, when a given targeting moiety (or bacteriocin) is described as having “specificity” for a particular target organism (e.g. a bacterial strain or species), this simply means that the targeting moiety is able to deliver the associated effector moiety to the relevant species or strain, typically by binding to a receptor at the surface of the target organism and mediating transport of the effector moiety such that the effector moiety can exert its activity against the target organism. It should be noted that a particular bacteriocin can have activity against more than one bacterial species, e.g. if the receptor and transport pathway used by the targeting moiety of that bacteriocin are sufficiently similar between those different species. For example, Enterobactin DF13 is able to target K. pneumoniae strains (e.g. strain SG62), despite being an enterobactin.

[0101] The targeting moiety of most naturally occurring PBs has a characteristic modular structure comprising up to four recognisable sub-regions, each of which can represent an independently folded domain or can lack recognisable secondary structure, thereby forming a flexible region of the molecule. Sub-region I, located at the N-terminus, is relatively unstructured and contains a Tol or Ton binding epitope. Sub-region II is a transporter binding domain. Sub-region III is a receptor binding domain. Sub-region IV is an inner membrane transport domain. In some PBs, the transport portal also acts as the receptor, in which case sub-regions II and III form a single domain. In other cases, the receptor and transport portal are different molecules, in which case sub-regions II and III are typically separate domains. These sub-regions typically (but not exclusively) occur in that order in an N-terminal to C-terminal direction, although this is not always necessary (provided that sub-region I is always at the N-terminus).

[0102] Without wishing to be bound by any particular theory, it is believed that sub-regions I, II, III, and IV can be at least somewhat interchangeable between molecules.

[0103] Within a given complex, the targeting moieties of the bacteriocin molecules can be the same or different. Thus, a complex can comprise bacteriocins all having the same targeting moiety, or bacteriocins having two or more different targeting moieties. For example, each bacteriocin in a complex can have a different targeting moiety.

[0104] It can be desirable for all of the bacteriocins within a complex to have targeting moieties that are specific for bacteria of the same species or strain. However, they can bind to two or more different receptors on that strain or species. For example, each targeting moiety can bind to a different receptor. In addition or alternatively, the targeting moieties can use two or more different transport portals, such as TolC, trimeric porins, or TonB-dependent transporters.

[0105] For example, a complex can comprise two or more bacteriocins having targeting moieties that are specific for E. coli, such as two or more bacteriocins having different targeting moieties that are specific for E. coli, e.g., derived from colicin. For example, all of the bacteriocins in a complex can have different targeting moieties that are specific for E. coli, e.g., derived from colicin. Colicins include colicin A, El, E2, E3, E4, E6, E7, E8, E9, K, M, N, U, B, la, lb, 5, 10, S4, and Y.

[0106] A complex can comprise two or more bacteriocins having targeting moieties that are specific for Pseudomonas (e.g., P. aeruginosa), such as two or more bacteriocins having different targeting moieties that are specific for Pseudomonas (e.g., P. aeruginosa), e.g., derived from S-type pyocin. For example, all of the bacteriocins in a complex can have different targeting moieties that are specific for Pseudomonas (e.g., P. aeruginosa), e.g., derived from S-type pyocin. S-type pyocins include pyocin G, LI, L2, L3, Ml, M2, M4, SI, S2, S3, S4, S5, S6, S8, SDl, SD2, SD3, AP41, Sn, SXl, and SX2.

[0107] The complex can comprise two or more bacteriocins with targeting moieties specific for Klebsiella (e.g., Klebsiella pneumoniae, K. quasipneumoniae, K. oxytoca, K. planticola, and K. aerogenes), e.g., two or more bacteriocins with different targeting moieties specific for Klebsiella (e.g., Klebsiella pneumoniae, K. quasipneumoniae, K. oxytoca, K. planticola, and K. aerogenes), e.g., derived from klebicin. For example, all of the bacteriocins in the complex can have different targeting moieties specific for Klebsiella (e.g., Klebsiella pneumoniae, K. quasipneumoniae, K. oxytoca, K. planticola, and K. aerogenes), e.g., derived from klebicin. Klebicins include klebicin C, D, G, KpneA, KaerA, Kpnela, Kvarla, Koxyl, KpneM, KpneM2, and KvarM.

[0108] The complex can comprise two or more bacteriocins with targeting moieties specific for Enterobacter cloacae, e.g., two or more bacteriocins with different targeting moieties specific for Enterobacter cloacae, e.g., derived from cloacin. For example, all of the bacteriocins in the complex can have different targeting moieties specific for Enterobacter cloacae, e.g., derived from cloacin. Cloacins include cloacin DF13.

[0109] The complex can comprise two or more bacteriocins with targeting moieties specific for Salmonella enterica, e.g., two or more bacteriocins with different targeting moieties specific for Salmonella enterica, e.g., derived from salmonicin. For example, all of the bacteriocins in the complex can have different targeting moieties specific for Salmonella enterica, e.g., derived from salmonicin. Salmonicins include SalEl a, SalEl b, SalE2, SalE3, and SalE7.

[0110] The complex can comprise two or more bacteriocins with targeting moieties specific for Yersinia pestis, e.g., two or more bacteriocins with different targeting moieties specific for Yersinia pestis, e.g., derived from yersinicin. For example, all of the bacteriocins in the complex can have different targeting moieties specific for Yersinia pestis, e.g., derived from yersinicin. Yersinicins include yersinicin 1.

[0111] In other embodiments, it can be desirable for each of the two or more bacteriocins in a given complex to have targeting moieties specific for different bacterial species or strains, to provide a single complex with activity against two or more species or strains of bacteria. It will be understood that the bacteria in question are Gram-negative bacteria.

[0112] For example, the complex may comprise a bacteriocin having a targeting portion specific to Escherichia coli (e.g., an escherichiacin targeting portion) and one or more bacteriocins having a targeting portion specific to one or more of the following: Pseudomonas spp. (e.g., Pseudomonas aeruginosa) (e.g., an S-type pyocytin targeting portion), Klebsiella spp. (e.g., Klebsiella pneumoniae, Klebsiella pneumoniae-like bacteria, Klebsiella oxytocin, Klebsiella heterotrophus, and Klebsiella aerogenes) (e.g., a klebsicin targeting portion), Enterobacter cloacae (e.g., an enterobacter cloacae targeting portion), Salmonella enterica (e.g., a salmonid targeting portion), or Yersinia pestis (e.g., a plague targeting portion).

[0113] The complex may contain a bacteriocin having a targeting portion (e.g., an S-type pyocytin targeting portion) that is specific to the genus Pseudomonas (e.g., Pseudomonas aeruginosa) and one or more bacteriocins having a targeting portion that is specific to one or more of the following: Escherichia coli (e.g., an escherichia coli targeting portion), Klebsiella pneumoniae (e.g., Klebsiella pneumoniae targeting portion), Enterobacter cloacae (e.g., an enterobacter cloacae targeting portion), Salmonella enterica (e.g., a salmonella targeting portion), or Yersinia pestis (e.g., a plague targeting portion).

[0114] The complex may comprise a bacteriocin having a targeting portion (e.g., a klebsiella pneumoniae targeting portion) that is specific to Klebsiella spp. (e.g., Klebsiella pneumoniae, Klebsiella pneumoniae-like bacteria, Klebsiella acidogenic bacteria, Klebsiella heterotropha, and Klebsiella aerogenes) and one or more bacteriocins having a targeting portion that is specific to one or more of the following: Escherichia coli (e.g., a coli-targeting portion), Pseudomonas spp. (e.g., Pseudomonas aeruginosa) (e.g., a pyocytin-targeting portion), Enterobacter cloacae (e.g., an enterobacter cloacae targeting portion), Salmonella enterica (e.g., a salmonid targeting portion), or Yersinia pestis (e.g., a plague-targeting portion).

[0115] The complex may comprise a bacteriocin having a targeting portion specific to Enterobacter cloacae (e.g., an enterobacteriocin targeting portion) and one or more bacteriocins having targeting portions specific to one or more of the following: Escherichia coli (e.g., an escherichiacin targeting portion), Pseudomonas spp. (e.g., Pseudomonas aeruginosa) (e.g., an S-type pyocytin targeting portion), Klebsiella spp. (e.g., Klebsiella pneumoniae, Klebsiella pneumoniae-like bacteria, Klebsiella acidogenic bacteria, Klebsiella heterotrophus, and Klebsiella aerogenes) (e.g., a klebsicin targeting portion), Salmonella enterica (e.g., a salmonid targeting portion), or Yersinia pestis (e.g., a plague targeting portion).

[0116] The complex can comprise a bacteriocin having a targeting moiety specific for Salmonella enterica (e.g., a salmonellicin targeting moiety) and one or more bacteriocins having a targeting moiety specific for one or more of: E. coli (e.g., a colicin targeting moiety), Pseudomonas (e.g., Pseudomonas aeruginosa) (e.g., a pyocin S-type targeting moiety), Klebsiella (e.g., Klebsiella pneumoniae, K. oxytoca, K. planticola, K. quasipneumoniae, and K. aerobacter) (e.g., a klebicin targeting moiety), Enterobacter cloacae (e.g., an enterocin targeting moiety), or Yersinia pestis (e.g., a pesticin targeting moiety).

[0117] The complex can comprise a bacteriocin having a targeting moiety specific for Yersinia pestis (e.g., a pesticin targeting moiety) and one or more bacteriocins having a targeting moiety specific for one or more of: E. coli (e.g., a colicin targeting moiety), Pseudomonas (e.g., Pseudomonas aeruginosa) (e.g., a pyocin S-type targeting moiety), Klebsiella (e.g., Klebsiella pneumoniae, K. oxytoca, K. planticola, K. quasipneumoniae, and K. aerobacter) (e.g., a klebicin targeting moiety), Enterobacter cloacae (e.g., an enterocin targeting moiety), or Salmonella enterica (e.g., a salmonellicin targeting moiety).

[0118] As mentioned previously, the effector moieties used in the bacteriocins of the application are enzymatic effector moieties, and are typically nucleases, as only these typically interact with cognate immunoprotein domains.

[0119] However, targeting moieties from any PB can be used, regardless of what effector domain they are typically associated with in the corresponding wild-type PB.

[0120] Pore-forming bacteriocins kill target cells by depolarization of the cytoplasmic membrane. These include pyocins S5, colicins A, El, K, N, U, B, la, lb, 5, 10, S4, and Y, and klebicins KpneA, KaerA, Kpnela, Kvarla, and Koxyl (see Denkovaite et al., 2019).

[0121] Enzymatic PB effector moieties can have a variety of activities.

[0122] As mentioned previously, many act as nucleases.

[0123] Some nuclease effector moieties have DNase activity, including pyocins G, SI, S2, SD2, S3, and AP41, and colicins E2, E7, E8, and E9. Klebicin G is also considered a DNase.

[0124] Some nuclease effector moieties have RNase activity, e.g. rRNAse or tRNAse activity. Those with rRNAse activity include colicin E3, E4 and E6, klebicin C and enterobactin DF13. Those with tRNAse activity include pyocin S4, colicin E5 and D and klebicin D).

[0125] Other enzymatic effector moieties have different modes of activity, including the degradation of peptidoglycan or precursors thereof, leading to inhibition of cell wall synthesis. These include colicin M, PaeMl, PaeM4 and klebicin KpneM, KpneM2 and KvarM.

[0126] The targets on which the effector moieties act tend to be highly conserved across the bacterial kingdom, and so a given effector moiety will typically have activity against a broad spectrum of organisms. Thus, species specificity is determined primarily by the targeting moiety of the bacteriocin molecule. Thus, effector moieties can be relatively freely exchanged between PBs to produce chimeric bacteriocins. For example, chimeric pyocins have been shown to retain Pseudomonas killing activity containing the targeting moiety of S1 or S2 pyocin linked to the effector moiety of E2 or E3 colicin (Kageyama et al., 1996). Chimeric bacteriocins are also used in the examples below.

[0127] Thus, the bacteriocins can comprise any suitable effector moiety having a cognate immunoprotein that can be incorporated into an immunoprotein scaffold. As already noted, these are typically enzymatic (nuclease) effector moieties, which can be combined with a targeting moiety from any suitable PB.

[0128] Thus, within a given complex, each bacteriocin typically has an enzymatic effector moiety having nuclease activity. The effector moieties of the bacteriocins can be the same or different. Thus, a complex can comprise two or more bacteriocins having the same effector moiety. For example, all of the bacteriocins within a complex can have the same effector moiety. Alternatively, a complex can comprise two or more bacteriocins having different enzymatic effector moieties. For example, all of the bacteriocins within a complex can have different effector moieties. When the effector moieties are different, they can still have the same enzymatic activity, e.g. DNase or RNase (which can be rRNAse or tRNAse). Thus, a complex can comprise two bacteriocins having different effector moieties but in which those effector moieties have the same activity, or, some or all of the effector moieties can have different enzymatic activities. In some embodiments, each effector moiety in a given complex is an enzymatic effector moiety, and each of those effector moieties can have a different enzymatic activity. Some examples of suitable combinations include:

[0129] - an effector moiety having DNase activity and an effector moiety having RNase activity (e.g. rRNAse or tRNAse);

[0130] - an effector moiety having rRNAse activity and an effector moiety having tRNAse activity;

[0131] - an effector moiety having DNase activity, an effector moiety having rRNAse activity and an effector moiety having tRNAse activity.

[0132] From the above discussion it will be appreciated that within a given bacteriocin molecule, the effector domain and the targeting domain can be derived from the same wild-type bacteriocin. Alternatively, the bacteriocin can be chimeric, comprising effector and targeting portions from different bacteriocins. For example, the effector and targeting portions can be from different wild-type bacteriocins, although it will be appreciated that the application is not limited to wild-type bacteriocin sequences, and that modified or engineered bacteriocin sequences can also be used. They can be modified in their effector portion, in their targeting portion, or both.

[0133] The effector portion of the bacteriocin used according to the application can have the amino acid sequence of a wild-type PB effector portion, or a functional fragment thereof, or can have at least 70% sequence identity with a wild-type effector portion sequence, or a functional fragment thereof, e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a wild-type effector portion. Typically, the effector portion (or fragment thereof) has the same effector activity (i.e. enzymatic activity, typically a nuclease) as the wild-type effector portion. The effector portion or fragment thereof is also capable of binding to a cognate immunoprotein domain of an associated immunoprotein scaffold. For example, it can be capable of binding to a cognate wild-type immunoprotein.

[0134] Suitable effector portions include the effector portions of:

[0135] Pyocin G, S1, S2, SD2, S3 and AP41, colicin E2, E7, E8 and E9, and Klebicin G (thought to be a DNase), colicin E3, E4 and E6, Klebicin C, and Enterocin DF13 (thought to be a rRNAse); and

[0136] Pyocin S4, colicin E5 and D, and Klebicin D (thought to be a tRNAse).

[0137] The targeting moiety of the bacteriocin used according to the application can have the amino acid sequence of the wild-type PB targeting moiety or a functional fragment thereof (i.e. a fragment capable of binding to the appropriate receptor on the target cell and mediating transport of the effector moiety into the cytosol of the target cell). Alternatively, it has at least 70% sequence identity to the wild-type targeting moiety sequence or a functional fragment thereof, for example at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the wild-type targeting moiety or a functional fragment thereof. Typically, the targeting moiety binds to the same receptor as the wild-type targeting moiety and uses the same transport portal as the wild-type targeting moiety.

[0138] Suitable targeting moieties include those from:

[0139] Colicin A, El, E2, E3, E4, E6, E7, E8, E9, K, M, N, U, B, la, lb, 5, 10, S4 and Y;

[0140] Pyocin G, LI, L2, L3, Ml, M2, M4, SI, S2, S3, S4, S5, S6, S8, SDl, SD2, SD3, AP41, Sn, SXl and SX2;

[0141] Klebicin C, D, G, KpneA, KaerA, Kpnela, Kvari a, KoxY, KpneM, KpneM2 and KvarM;

[0142] CloDF13;

[0143] Salmonicin SalEla, SalElb, SalE2, SalE3 and SalE7; and

[0144] Yersiniocin 1.

[0145] Some illustrative examples of chimeric bacteriocins include:

[0146] a KlebC targeting moiety and a Colicin E9 effector moiety (designated "KlebC-E9");

[0147] a CloDF13 targeting moiety and a Colicin E3 effector moiety (designated "CloDF13-E3"); and

[0148] a KlebG targeting moiety and a Colicin D effector moiety (designated "KlebG-D").

[0149] These will be described in more detail below.

[0150] The broad spectrum effectiveness of the effector moieties makes it possible to readily adapt the complexes of the application to target different bacterial strains or species by retaining the immunoprotein scaffold and the bacteriocin effector moieties and simply changing the targeting moieties. Thus, a host cell has been constructed which is capable of expressing an immunoprotein scaffold and one or more cognate bacteriocins, the targeting specificity of the complex produced by that cell can be changed by simply exchanging one targeting moiety (or more targeting moieties) of one bacteriocin (or more bacteriocins) for other targeting moieties which are specific for one or more different bacterial strains or species. There is no need to construct a whole new host cell with a new immunoprotein scaffold and cognate bacteriocins.

[0151] immunoprotein scaffold

[0152] The immunoprotein scaffold comprises a plurality (i.e. two or more) of immunoprotein domains, each of which is capable of binding a cognate bacteriocin effector moiety.

[0153] In their natural environment, immunoproteins are single domain proteins. However, it will be appreciated that the term "immunoprotein domain" is used in this specification to refer to a functional domain of an immunoprotein scaffold. Thus, an immunoprotein scaffold comprises a plurality of immunoprotein domains, each of which corresponds to a single individual immunoprotein. Each domain can be referred to by the name of the corresponding immunoprotein (e.g. "E. coli bacteriocin E9 immunoprotein domain") or by an appropriate abbreviation or shorthand (e.g. "Im9 domain").

[0154] The immunoprotein domains in a given scaffold molecule can be the same or different. Where an immunoprotein scaffold contains multiple copies of the same immunoprotein domain, the complex can still contain bacteriocins with two or more different targeting domains, but they will typically have the same effector moiety, or at least be sufficiently similar that they can bind to the same immunoprotein domain. However, if it is desired that the complex contains multiple different bacteriocins, then this can be controlled more directly by using an immunoprotein scaffold with multiple different immunoprotein domains.

[0155] The immunoprotein domains for use in an immunoprotein scaffold according to the application can have the amino acid sequence of a wild-type PB immunoprotein or a functional fragment thereof, or can have at least 70% sequence identity to a wild-type immunoprotein sequence or a functional fragment thereof, for example at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a wild-type immunoprotein sequence or a functional fragment thereof. Each immunoprotein domain (or fragment thereof) is capable of binding to a cognate effector moiety of one of the bacteriocins which together with the immunoprotein scaffold make up the complex. For example, it can be capable of binding to a cognate wild-type effector moiety.

[0156] Suitable immunoprotein domains include immunoprotein domains of:

[0157] Pyocin G, S1, S2, SD2, S3 and AP41, colicin E2, E7, E8 and E9, and klebicin G, colicin E3, E4 and E6; klebicin C, and cloacin DF13; and

[0158] Pyocin S4, colicin E5 and D and klebicin D;

[0159] As noted above, it also provides suitable cognate effector domains.

[0160] Some illustrative examples of immunoprotein scaffolds include:

[0161] A colicin E9 immunoprotein domain (“Im9”) and a colicin D immunoprotein domain (“ImD”) (collectively named “Im9-ImD”);

[0162] A colicin E9 immunoprotein domain (“Im9”), a colicin E3 immunoprotein domain (“Im3”), and a colicin D immunoprotein domain (“ImD”) (collectively named “Im9-Im3-ImD”).

[0163] It will be appreciated that “Im9-ImD” will form a complex with a PB having effector domains from colicin E9 and colicin D. “Im9-Im3-ImD” will form a complex with a PB having effector domains from colicin E9, colicin E3 and colicin D. The PB can be chimeric, with targeting moieties from other PBs, as described elsewhere in this specification.

[0164] Typically, the immunoprotein domain binds to its cognate bacteriocin effector moiety with very high affinity. For example, the Kdof an RNase-Im complex can be 10 d M or less, for example Kdof an RNase-Im complex can be 10 -10 M or less, for example Kdof an RNase-Im complex can be 10 d M or less, for example Kdof an RNase-Im complex can be 10 -10 M or less, for example Kdof an RNase-Im complex can be 10 -11 M or less, or 10 - 12 M or less (for example as measured at pH 7 and 25°C, for example by stopped-flow fluorescence, for example as described in Walker et al. (2003) Biochemistry 42, 4161 ). The Kdof a DNase-Im complex can be 10 d M or less, for example Kdof an RNase-Im complex can be 10 -10 M or less, for example Kdof an RNase-Im complex can be 10d may be 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -13 M or less, or 10 -14 M or less (e.g. as measured for RNase-Im complexes, or as described in Wallis et al. (1995) Biochemistry 34, 13743-13750).

[0165] Within a given scaffold, the immunoprotein domains are covalently coupled to one another. In some embodiments, they can be expressed separately and conjugated together, e.g. by a chemical linker. However, it is often more convenient to express the immunoprotein scaffold as a fusion protein.

[0166] To produce an immunoprotein scaffold as a fusion protein, a nucleic acid expression vector is constructed which comprises the coding sequences for each immunoprotein component in one continuous open reading frame, such that the individual immunoprotein components can be translated as part of the same polypeptide chain.

[0167] Typically, a peptide linker is included between each component to allow them to interact freely with their respective bacteriocin cytotoxic domain without steric hindrance. The skilled person is fully able to design suitable linkers. Conventionally, such linkers are 10 to 20 amino acids in length, and have a high proportion of small and hydrophilic amino acid residues (e.g. glycine and serine) to provide the required flexibility without compromising the water solubility of the molecule. In some embodiments, it can be desirable to use a more rigid linker, including other residues such as proline and asparagine, to provide more separation between the individual domains. In some embodiments, the linker can have a propensity to form a defined secondary structure (e.g. an alpha-helix). Sequences which use such structures are well known. For example, the repeating unit of the sequence EAAAK (SEQ ID NO: 1) can be used to form a helical linker, but the skilled person will be well aware of others.

[0168] For example, the immunoprotein scaffolds described in the examples below employ the linker sequences NINGGPTGIGVS (SEQ ID NO: 2) and NGGGNGNSGGGS (SEQ ID NO: 3) between the immunoprotein domains, both of which are intrinsic unstructured sequences derived from the N-terminal subdomain of colicin E9. A helical linker ((EAAAK)3EL) (SEQ ID NO: 4) is used between the M-type bacteriocin (KvarM) and the immunoprotein domains. The skilled person will be able to design other suitable linkers as required.

[0169] The immunoprotein scaffold can also comprise a heterologous moiety, i.e. a moiety that is neither an immunoprotein domain nor a linker peptide.

[0170] The heterologous moiety can be a peptide or a non-peptide. When the heterologous moiety is a peptide, it can be part of the same fusion protein as the rest of the immunoprotein scaffold. Alternatively, it can be attached to the immunoprotein scaffold by chemical conjugation.

[0171] For example, the heterologous moiety can serve to increase solubility and / or half-life and / or bioavailability in vivo (e.g. in plasma) in a subject compared to a corresponding scaffold (or complex) that lacks such a moiety, otherwise being identical. Such modifications are also known to decrease clearance (e.g. renal clearance) of therapeutic proteins and peptides. Suitable peptide moieties include immunoglobulin Fc domains. Suitable non-peptide moieties include polymeric moieties. The polymeric moiety is preferably water-soluble (amphipathic or hydrophilic), non-toxic and pharmaceutically inert. Some examples include polyethyleneglycol (PEG), homo- or co-polymers of PEG, monomethyl substituted polymers of PEG (mPEG), or polyoxyethylene glycerol (POG). See, for example, Francis et al., (1998), Int. J. Hematology 68: 1-18; Zalipsky (1995), Bioconjugate Chem. 6: 150-165; and Delgado et al. (1992), Crit. Rev. Therap. Drug Carrier Syst. 9: 249-304.

[0172] Other suitable polymeric moieties include polyamino acids, such as polylysine, polyaspartic acid and polyglutamic acid (see, for example, Gombotz, et al. (1995), Bioconjugate Chem., vol. 6: 332-351; Hudecz, et al. (1992), Bioconjugate Chem., vol. 3, 49-57; Tsukada, et al. (1984), J. Natl. Cancer Inst., vol 73,: 721-729; and Pratesi, et al. (1985), Br. J. Cancer, vol. 52: 841-848).

[0173] The polymeric moiety can be linear or branched. It can have a molecular weight of 500 to 40,000 Da, for example 500 to 10,000 Da, 1000 to 5000 Da, 10,000 to 20,000 Da, or 20,000 to 40,000 Da.

[0174] Alternatively, the heterologous moiety can comprise a cytotoxic domain (e.g. a protein bacteriocin cytotoxic domain), such that the immunoprotein scaffold itself is an additional toxin in addition to the PB with which it is associated. It can also comprise a moiety capable of mediating transport across the outer membrane of a target cell, e.g. a protein bacteriocin targeting moiety. In some preferred embodiments, the heterologous moiety is an M-type bacteriocin, e.g. colicin M (ColM), KpneM, KpneM2, KvarM, PaeM1 or PaeM4, or a functional variant thereof.

[0175] M-type bacteriocins act by degrading peptidoglycan precursors in the periplasm, leading to cell lysis (Schaller et al., 1982). They are found in a range of different species, including E. coli, Pseudomonas, Pectobacterium, Klebsiella and Burkholderia (Chérier et al., 2021). Colicin M from E. coli is the most fully studied example of this class of bacteriocins. KvarM (see Dekovskiené et al., 2019) is a 30.8 kDa protein that binds to and is transported through the outer membrane iron siderophore receptor FhuA into the periplasm of the cell in a process driven by the Ton system, and KvarM is of particular interest because it shows activity against a broad spectrum of Klebsiella strains, including those with multi-drug resistance in plate, liquid and biofilm killing assays.

[0176] KvarM and colicin M are organised into three characteristic regions of bacteriocins: an N-terminal unstructured transport region (Pilsl et al., 1993), a central globular portion that interacts with its outer membrane receptor FhuA, and a C-terminal catalytic region that hydrolyses the lipid II precursor (Sham et al., 2014). However, their relatively small size and compact folding means that these regions do not form independently folded domains, and attempts to truncate these molecules often result in misfolded proteins (Barreteau et al., 2010).

[0177] When the immunoprotein scaffold comprises an M-type bacteriocin, the M-type bacteriocin is typically located at the N-terminus of the scaffold molecule, with the immunoprotein domain located C-terminal to the M-type bacteriocin. A suitable linker is typically present between the M-type bacteriocin and the first immunoprotein domain. It can be desirable to have a rigid linker between the M-type bacteriocin and the first immunoprotein domain, for example a linker having an alpha-helical secondary structure, for example as exemplified in the Examples below.

[0178] The incorporation of an M-type bacteriocin into an immunoprotein scaffold has the potential to increase the receptor binding ability and cytotoxic activity of the complex as a whole. The resulting complex is able to bind to the receptors for the M-type bacteriocin as well as those targeted by the PB component of the complex (in non-covalent association with the immunoprotein domain), thereby further increasing its potential affinity for interaction with the surface of a target cell, and being cytotoxic to bacteria susceptible to the M-type bacteriocin.

[0179] Host cell

[0180] The present application extends to a host cell capable of expressing an immunoprotein scaffold.

[0181] The present application therefore provides a host cell comprising a nucleic acid encoding an immunoprotein scaffold, the immunoprotein scaffold comprising a first immunoprotein domain and a second immunoprotein domain, and wherein the host cell is capable of expressing the immunoprotein scaffold.

[0182] One advantage of the present application is that the host cell can also express the PB component, enabling the production of the complete antibacterial complex with the correct stoichiometry from a single cell, thereby minimising production and purification costs.

[0183] The present application therefore also provides a host cell comprising:

[0184] (i) a nucleic acid encoding an immunoprotein scaffold, the immunoprotein scaffold comprising at least a first immunoprotein domain and a second immunoprotein domain;

[0185] (ii) a nucleic acid encoding at least one bacteriocin, the or each of the bacteriocins having an effector domain capable of binding to at least one of the immunoprotein domains;

[0186] wherein the host cell is capable of expressing the immunoprotein scaffold and the at least one bacteriocin.

[0187] When the immunoprotein scaffold contains only one type of immunoprotein domain, and thus the complex contains only one type of bacteriocin, the cell can encode and express only that single homologous bacteriocin. More commonly, the immunoprotein scaffold contains at least two different immunoprotein domains, and the cell comprises nucleic acid encoding at least two bacteriocins, each having an effector capable of binding to at least one of the immunoprotein domains, thereby producing an anti-bacterial complex containing at least two different types of bacteriocin.

[0188] Thus, the host cell comprises nucleic acid encoding each respective bacteriocin. The nucleic acid encoding the immunoprotein scaffold and the bacteriocins is typically provided as part of one or more nucleic acid expression constructs or vectors. Thus, they can be provided on a single vector or on two or more separate vectors.

[0189] The skilled person is able to design suitable nucleic acid expression constructs or vectors as required to obtain expression of the immunoprotein scaffold and the bacteriocin molecules. Typically, such vectors comprise suitable transcriptional and translational regulatory sequences operably linked to the sequences encoding the desired proteins, such that the proteins are capable of being transcribed and translated by the host cell. The vectors can contain other sequences as required, for example a selectable marker gene, depending on the particular host cell. The vectors can be designed to integrate into the host cell chromosome, or can exist and replicate independently of the host chromosome (e.g. plasmids).

[0190] Since the immunoprotein scaffold and the respective bacteriocin molecules are separate molecules in the final complex, which associate through non-covalent interactions between the immunoprotein domains and the effector portions of the bacteriocin molecules, it will be appreciated that they are typically expressed as separate molecules within the host cell.

[0191] The host cell is typically a bacterial host cell, for example an E. coli host cell.

[0192] As noted above, the specificity of a PB is determined by its targeting portion. Thus, by simply changing the targeting portion of the component PBs while leaving the immunoprotein scaffold and PB effector portions unchanged, the anti-bacterial complex can be adapted for use against different bacterial strains or species. This can be readily achieved by making modifications to an existing host cell. There is no need to construct a whole new host cell with a new immunoprotein scaffold and homologous bacteriocins.

[0193] Thus, the nucleic acid or expression construct encoding a given PB can be designed to facilitate exchange of the targeting portion. For example, suitable restriction sites can be provided between the sequence encoding the targeting portion and the sequence encoding the effector portion. Another restriction site can be provided at the opposite end of the sequence encoding the targeting portion, for example 5’ of the start of the open reading frame of the PB.

[0194] The application also provides a method of producing an anti-bacterial complex, comprising providing a host cell as described, and culturing the cell under conditions suitable for expression of the immunoprotein scaffold and the bacteriocin molecule. The method can further comprise a step of isolating the anti-bacterial complex, and optionally further purification steps.

[0195] Alternatively, it can be desirable not to express all components of the anti-bacterial complex in the same host cell. Rather, the components can be expressed in two or more different host cells, each comprising nucleic acid encoding one or more individual components, and capable of expressing those components. For example, the immunoprotein scaffold can be expressed in one host cell, and one or more bacteriocin molecules can be expressed in one or more further host cells.

[0196] The application therefore also provides a method of producing an anti-bacterial complex, the method comprising contacting an immunoprotein scaffold comprising a first immunoprotein domain and a second immunoprotein domain with a first bacteriocin molecule and a second bacteriocin molecule, each having an effector moiety capable of binding to a respective one of the immunoprotein domains, to form a complex of the application.

[0197] In such cases, if the bacteriocin molecule is expressed in a host cell susceptible to that bacteriocin and which also does not express the immunoprotein scaffold, the host cell will typically also express a cognate immunoprotein for the relevant bacteriocin. In such cases, the bacteriocin can be dissociated from the immunoprotein and isolated therefrom prior to contacting the bacteriocin with the immunoprotein scaffold.

[0198] Subject and treatment conditions

[0199] The complexes and methods of the application are suitable for use in the prevention and / or treatment of bacterial infections, typically Gram-negative bacterial infections, especially infections with E. coli, Pseudomonas spp. (especially P. aeruginosa), Klebsiella spp. (e.g. K. pneumoniae), Enterobacter cloacae, Salmonella spp. (e.g. S. enterica) and Yersinia pestis, and conditions caused by or associated with such infections.

[0200] As described above, the strain specificity of any given bacteriocin is typically determined by its targeting moiety. Thus, to treat an infection caused by or a condition associated with a given bacterial species or strain, a complex comprising at least one bacteriocin having a targeting moiety specific for that species or strain will typically be used.

[0201] The infection can be acute or chronic.

[0202] For example, P. aeruginosa infection of the lower respiratory tract is particularly common in patients with cystic fibrosis (where it represents a major cause of mortality) and chronic obstructive pulmonary disease (COPD). Other patients with impaired respiratory function and / or impaired immune function can also be susceptible to infection, including patients with congestive heart failure, AIDS patients, and patients taking immunosuppressive drugs or undergoing other immunosuppressive therapy (e.g., for cancer (especially chemotherapy), rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, sarcoidosis, focal segmental glomerulosclerosis, Crohn's disease, Behcet's Disease, pemphigus, ulcerative colitis, etc.).

[0203] Acute conditions associated with or caused by Pseudomonas infection include community-acquired pneumonia and nosocomial infections, such as ventilator-associated pneumonia and hospital-acquired pneumonia.

[0204] Thus, for treating infection by Pseudomonas (e.g., P. aeruginosa) or conditions associated therewith, it is generally desirable to use a complex comprising at least one such bacteriocin having a targeting moiety specific for Pseudomonas (e.g., P. aeruginosa), such as the targeting moiety of an S-type pyocin. As discussed elsewhere in this specification, it can be desirable to use a complex having at least two such bacteriocins, or a complex containing only such bacteriocins.

[0205] E. coli infection is associated with a variety of conditions. Pathogenic, adherent-invasive E. coli (AIEC) that are capable of forming biofilms and of invading and replicating within host cells can be particularly important. For example, abnormal colonization of the ileal mucosa by AIEC is thought to be a factor in the development of Crohn's disease. E. coli is also commonly associated with urinary tract infections and other conditions.

[0206] For treating infection by E. coli or conditions associated therewith, it is generally desirable to use a complex comprising at least one such bacteriocin having a targeting moiety specific for E. coli, such as the targeting moiety of colicin. As discussed elsewhere in this specification, it can be desirable to use a complex having at least two such bacteriocins, or a complex containing only such bacteriocins.

[0207] K. pneumoniae infection can be associated with, for example, sepsis, or bronchitis or pneumonia (often in the form of bronchopneumonia). Affected patients are prone to developing lung abscesses, cavitation, empyema, or pleural adhesions.

[0208] For the treatment of an infection with or a condition associated with K. pneumoniae, it is generally desirable to use a complex comprising at least one bacteriocin having a targeting moiety specific for K. pneumoniae, such as a targeting moiety of a klebicin. As discussed elsewhere in this specification, it can be desirable to use a complex having at least two such bacteriocins, or a complex containing only such bacteriocins.

[0209] Similarly, for the treatment of an infection with or a condition associated with other bacterial types, such as S. enterica, E. cloacae or Y. pestis, it is generally desirable to use a complex comprising at least one bacteriocin having a targeting moiety specific for that bacterial species or strain, such as a targeting moiety of a salmonicin, cloacacin or pesticin, as the case can be. As discussed elsewhere in this specification, it can be desirable to use a complex having at least two such bacteriocins, or a complex containing only such bacteriocins.

[0210] Typically, the subject to be treated is a mammal. The subject is typically a human, but can also be any other primate (great ape, old world monkey or new world monkey), or a domesticated, laboratory or farm animal, such as a mouse, rat, guinea pig, lagomorph (e.g. a rabbit), cat, dog, pig, cow, horse, sheep or goat.

[0211] Pharmaceutical compositions

[0212] The complexes described in this specification can be formulated into pharmaceutical compositions. These compositions can further comprise a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art in addition to one of the above. Such materials should be nontoxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can depend on the route of administration, e.g. oral, intravenous, transdermal or subcutaneous, pulmonary, intramuscular, intraperitoneal route or topical administration. Oral, intravenous or pulmonary routes can be preferred.

[0213] Pharmaceutical compositions for oral administration can be in tablet, capsule, powder or liquid form. A tablet can comprise a solid carrier, such as gelatin or an excipient. Liquid pharmaceutical compositions generally comprise a liquid carrier, such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. A saline solution, dextrose or other saccharide solution, or glycols, such as ethylene glycol, propylene glycol or polyethylene glycol, can be included.

[0214] For intravenous, transdermal or subcutaneous injection, or injection at the site of pain, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitably a suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to use, for example, isotonic vehicles (such as sodium chloride injection, Ringer's Injection, Lactated Ringer's Injection) to prepare suitably solutions. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included, as required.

[0215] The term "pulmonary administration" is intended to encompass any suitable delivery method of the active agents to the lungs. The most common pulmonary administration methods are oral and / or nasal inhalation.

[0216] The complexes of the application can be formulated for pulmonary administration in any suitable way, for example in liquid or solid (typically powder) form. The formulation can be delivered by any suitable mechanism or delivery device, including inhalers (such as metered dose inhalers, dry powder inhalers) nebulizers (such as ultrasonic nebulizers, jet nebulizers, vibrating mesh nebulizers) and the like.

[0217] The application therefore also provides a device for pulmonary administration of a therapeutic composition to a subject, the composition comprising an S-type pyocin as described elsewhere in this specification. The device can be an inhaler (such as a metered dose inhaler, a dry powder inhaler) or a nebulizer (such as an ultrasonic nebulizer, a jet nebulizer, a vibrating mesh nebulizer).

[0218] Liquid compositions will generally include an aqueous carrier, such as water or a physiologically salt solution. Dextrose or other sugar solutions, or glycols, such as ethylene glycol, propylene glycol or polyethylene glycol, can be included.

[0219] Emulsions and nanoparticle encapsulation, both of which employ lipids, can also be used.

[0220] Solid (e.g. powder) formulations can utilise carriers such as sugars, cyclodextrins and the like. They can be prepared by any suitable method, including spray-drying, spray-freeze-drying, solvent precipitation, jet-milling and the like.

[0221] Preferably, administration is in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case can be, although prophylaxis can be considered therapy) sufficient to show benefit to the individual. Actual amounts and rates and time course of administration depend on the nature of the condition being treated and its severity. Treatment protocols (e.g. dosage determination etc.) are within the skill of a general practitioner and other medical practitioners and typically take into account the condition to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to the practitioner. Suitable carriers, adjuvants, excipients etc. can be found in standard pharmaceutical texts, such as Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins; and Handbook of Pharmaceutical Excipients, 2nd Edition, 1994.

[0222] Sequence

[0223] The following sequences were used in the examples below. Deviations from the corresponding wild-type sequence are shown, which deviations are generally due to the incorporation of suitable restriction sites to facilitate exchange of targeting moieties. It will be appreciated that wild-type or variant sequences (or further variants) can be used in the context of the present application.

[0224] ColE9: Amino acid sequence

[0225] (E9 DNase domain underlined )

[0226]

[0227] [In the wild-type sequence, the last residue of the targeting moiety is D (not A), and the first residue of the effector moiety is K (not M).]

[0228] ColE9 targeting moiety: Amino acid sequence

[0229]

[0230] [In the wild-type sequence, the last residue of the targeting moiety is D (not A).]

[0231] ColE9 effector moiety (DNase): Amino acid sequence

[0232]

[0233] [In the wild-type sequence, the first residue of the effector moiety is K (not M).]

[0234] ColE9: Coding sequence

[0235] (E9 DNAse domain underlined , Ncol site in bold)

[0236]

[0237] ColE9 immune protein ("Im9"): amino acid sequence

[0238]

[0239] ColE9 immune protein ("Im9") with C-terminal LE(His)6 tag: amino acid sequence

[0240] (C-terminal LE(His)6 tag underlined )

[0241]

[0242] ColE9 immune protein ("Im9") with C-terminal LE(His6) tag: coding sequence

[0243] (C-terminal LE(His)6 tag underlined , Xhol site in bold)

[0244]

[0245] ColD: amino acid sequence

[0246] (tRNase domain underlined )

[0247]

[0248] [In the wild-type sequence, the first two residues of the effector portion are VY (not MD).]

[0249] ColD targeting portion: amino acid sequence

[0250]

[0251] ColD effector portion: amino acid sequence

[0252]

[0253] [In the wild-type sequence, the first two residues of the effector portion are VY (not MD).]

[0254] ColD: coding sequence

[0255] (tRNase domain underlined , Ncol site in bold)

[0256]

[0257] ColD immunity protein ("ImD"): amino acid sequence

[0258]

[0259] ColD immunity protein ("ImD") with C-terminal LE(His6) tag: amino acid sequence

[0260] LE(His6) tag underlined )

[0261]

[0262] ColD immunity protein ("ImD") with C-terminal His6 tag: coding sequence

[0263] LE(His6) tag underlined Xho I cloning site is in bold

[0264]

[0265] KlebC targeting moiety: amino acid sequence

[0266]

[0267] KlebC-E9 chimera (KlebC targeting moiety; E9 effector moiety): amino acid sequence

[0268] E9 effector moiety (DNase) underlined )

[0269]

[0270]

[0271] KlebC-E9 chimera: coding sequence

[0272] E9 effector moiety (DNase) underlined )

[0273]

[0274] CloDF13 targeting moiety: amino acid sequence

[0275]

[0276] ColE3 effector moiety (rRNAse): amino acid sequence

[0277]

[0278] ColE3 immunity protein ("Im3"): amino acid sequence

[0279]

[0280] KvarM: amino acid sequence

[0281]

[0282] CloDF13-E3 chimera (CloDF13 targeting moiety; ColE3 effector moiety): amino acid sequence

[0283] (ColE3 effector moiety (rRNAse) underlined )

[0284]

[0285] CloDF13-E3 chimera (CloDF13 targeting moiety; E3 effector moiety): coding sequence

[0286] (E3 effector moiety (rRNAse) underlined )

[0287]

[0288] KlebG targeting moiety: amino acid sequence

[0289]

[0290] KlebG-D chimera (KlebG targeting moiety; ColD effector moiety (tRNAse)): amino acid sequence

[0291] (ColD effector moiety underlined )

[0292]

[0293] KlebG-D chimera (KlebG targeting moiety; ColD effector moiety (tRNAse)): coding sequence

[0294] (ColD effector moiety underlined )

[0295]

[0296] Im9-ImD immunity protein scaffold: amino acid sequence (Im9 underlined , ImD )

[0297]

[0298] Im9-ImD immunoprotein scaffold with C-terminal LE(His6) tag: amino acid sequence (Im9 underlined , ImD LE(His6) tag )

[0299]

[0300] Im9-ImD immunoprotein scaffold with C-terminal LE(His6) tag: coding sequence (Im9 underlined , ImD Xho I cloning site in bold, LE(His6) tag band )

[0301]

[0302] Im9-Im3-ImD immunoprotein scaffold: amino acid sequence (Im9 underlined , Im3 ImD )

[0303]

[0304] Im9-Im3-ImD immunoprotein scaffold with C-terminal LE(His6) tag: amino acid sequence (Im9 underlined , Im3 ImD )

[0305]

[0306] Im9-Im3-ImD immunoprotein scaffold with C-terminal LE(His6) tag: coding sequence (Im9 underlined , Im3 ImD )

[0307]

[0308] KvarM-Im9-ImD immunoprotein scaffold with C-terminal LE(His6) tag: amino acid sequence (KvarM italic, Im9 underlined , ImD LE(His6) tag )

[0309]

[0310] KvarM-Im9-ImD immunoprotein scaffold with C-terminal LE(His6) tag: amino acid sequence (KvarM italic, Im9 underlined , ImD XhoI cloning site in bold, LE(His6) tag )

[0311]

[0312] KvarM-Im9-Im7-ImD immunoprotein scaffold with C-terminal LE(His6) tag: amino acid sequence (KvarM italic, Im9 underlined , Im7 ImD )

[0313]

[0314] KvarM-Im9-Im7-ImD immunoprotein scaffold with C-terminal LE(His6) tag: amino acid sequence (KvarM italic, Im9 underlined , Im7 ImD )

[0315]

[0316]

[0317] Im97 immunoprotein scaffold with C-terminal LE(His6) tag: amino acid sequence (Im9 underlined , Im7 LE(His6) )

[0318]

[0319] S5E9 chimera: amino acid sequence

[0320] Laminarin S5 outer membrane translocon domain italic; Laminarin S5 receptor binding domain underlined ; Laminarin G inner membrane translocon domain Colicin E9 DNase domain )

[0321]

[0322] S5E7 chimera: amino acid sequence

[0323] Italicized version of the pyogenic S5 outer membrane transport domain; pyogenic S5 receptor-binding domain underlined ; Pyruvicin G inner membrane transport domain E. coli E7 DNA enzyme domain )

[0324]

[0325] ColE7 immune protein with C-terminal LE (His6) tag (“Im7”): amino acid sequence

[0326] (LE(His6) tag) underlined )

[0327]

[0328] Example

[0329] To illustrate the flexibility and applicability of the method, the inventors constructed a heterotrimeric complex (containing two bacteriocinolone molecules and a homodivalent immunoglobulin scaffold, as described in Example 1) and a heterotetrameric complex (containing three bacteriocinolone molecules and a homotrivalent immunoglobulin scaffold, as described in Example 2). These complexes in Figure 1 As shown in the image.

[0330] Example 1

[0331] Components of the trimer complex

[0332] The bacteriocin components of the trimeric complex are colicin E9 (ColE9) and colicin D (ColD). The immunoprotein scaffold is a fusion protein containing its homologous immunoproteins "Im9" and "ImD" separated by a flexible linker peptide. The scaffold is represented by the name "Im9-ImD". The scaffold with ColE9, ColD, or complexes of both are represented by the following abbreviations:

[0333] [ColE9:Im9-ImD];

[0334] [ColE9:Im9-ImD:ColD]; and

[0335] [Im9-ImD:ColD].

[0336] Escherichia coli E9 and E3 are group A bacteriocins that bind to vitamin B with nanomolar affinity. 12The receptor BtuB binds and then passes its intrinsic unstructured N-terminus through the trimeric porin in the outer membrane, e.g. OmpF. This intrinsic unstructured N-terminus contains a TolB box that binds to TolB, which is part of the transperiplasmic primed Tol-Pal complex. Parasitism of the Tol-Pal system leads to the release of the immunizing protein and transport of the cytotoxic C-terminal domain into the cytoplasm. Colicins E9 and E3 are highly conserved except for their cytotoxic domains, which are a DNAse in E9 and an rRNAse in E3.

[0337] Colicin D is a group B bacteriocin that binds with high affinity to the iron-colicin receptor FepA. Colicin D passes through FepA and interacts with TonB of the Ton system in the periplasm, which leads to the release of the immunizing protein and transport of the C-terminal tRNAse domain into the cytoplasm. The uptake pathways of Colicins E9 and D are summarized in Figure 2

[0338] In vitro assembly of trimeric complexes

[0339] Individual bacteriocins were expressed in E. coli BL21(DE3) as heterodimeric complexes with their respective immunizing proteins. The immunizing proteins were engineered to carry a His6 tag at their C-terminus. Cell pellets were lysed by sonication, clarified by centrifugation and loaded onto 5 ml HisTrap HP columns. In each case, the free bacteriocins were eluted from the nickel affinity columns using 6 M guanidinium hydrochloride. Eluted bacteriocins were refolded by dialysis into 25 mM Tris-HCl, pH 7.5, 150 mM NaCl and purified by gel filtration over a 26 / 60 S200 column equilibrated in the same buffer. The immunizing protein scaffold Im9-ImD, which also carries a C-terminal His6 tag, was expressed separately and purified by nickel affinity chromatography (eluted with imidazole), followed by gel filtration over a 26 / 60 S200 column equilibrated in 25 mM Tris-HCl, pH 7.5.

[0340] Samples of 500 μl or less were prepared in 25 mM Tris-HCl, pH 7.5, 150 mM NaCl: (i) 20 μΜ Im9-ImD + 40 μΜ ColE9 + 40 μΜ ColD, 20 μΜ Im9-ImD + 40 μΜ ColD, 20 μΜ Im9-ImD + 40 μΜ ColE9 and 20 μΜ Im9-ImD and purified on a Superdex 200 increase 10 / 300 GL analytical gel filtration column (Cytiva) equilibrated in the same buffer.

[0341] ​A good baseline separation between ColE9:Im9-ImD:ColD and free colicin was observed, which is necessary to ensure that any killing activity observed is due to the heterotrimeric complex and not contaminating free colicin. Figure 4 )

[0342] Cell killing by the trimeric complex

[0343] LB-0.7% agar plates. Serial dilutions of ColD, ColE9, and [ColE9:Im9-ImD:ColD] were prepared over a concentration range of 100 nM to 137 pM, and 3 μΐ of each dilution was spotted onto each inoculated agar plate. Plates were grown overnight at 37°C, and colicin activity was visualized as clear zones in the bacterial lawn. Results are shown in Figures A to C. In a similar experiment, serial dilutions of ColD, ColE9, a mixture of ColE9 + ColD, and [ColE9:Im9-ImD:ColD] were spotted onto lawns of tolA-BW25113. Results are shown in Figure D. Figure 5 A to C. In a similar experiment, serial dilutions of ColD, ColE9, a mixture of ColE9 + ColD, and [ColE9:Im9-ImD:ColD] were spotted onto lawns of tolA-BW25113. Results are shown in Figure D. Figure 5 D. In a similar experiment, serial dilutions of ColD, ColE9, a mixture of ColE9 + ColD, and [ColE9:Im9-ImD:ColD] were spotted onto lawns of tolA-BW25113. Results are shown in Figure D.

[0344] As expected, ColD had no activity against tonB- or fepA- cells, while ColE9 had no activity against tolA- or btuB- cells. The heterotrimer [ColE9:Im9-ImD:ColD] retained activity against all strains tested, indicating that both ColE9 and ColD are active within the complex. Both ColD and ColE9 were tested as complexes with the Im9-ImD fusion protein ([ColE9:Im9-ImD] and [Im9-ImD:ColD]), which had no negative effect on the activity of either ColD or ColE9 alone (not shown).

[0345] Interestingly, ColD activity was impaired against tolA- cells, indicating lower levels of FepA expression in these cells. However, [ColE9:Im9-ImD:ColD] showed better activity against tolA- cells than either ColD or the mixture of ColE9 + ColD alone, indicating that while ColE9 cannot enter and kill, the presence of the ColE9 receptor binding domain can allow the heterotrimeric complex to bind to the cell surface and aid in the FepA-dependent activity of the ColD portion.

[0346] Trimer complexes avoid the development of bacterial resistance

[0347] In addition to increasing strain coverage, the combination of multiple bacteriocins targeting independent uptake pathways also significantly reduces the chance of resistance developing in cells susceptible to more than one bacteriocin component. Resistance requires simultaneous mutation of multiple components to affect both uptake pathways. In vitro, resistant mutants can be much more prevalent than in vivo, as the influence of nutrient receptors is minimal when grown on rich media. Bacteriocin receptors are often virulence factors, upregulated during infection, and their loss affects the ability of the bacteria to colonize.

[0348] In 96-well plates, three-fold serial dilutions of [ColE9:Im9-ImD:ColD], [Im9-ImD:ColD], and [ColE9:Im9-ImD] were prepared in LB from 1 μΜ to 5.6 pM. Overnight MG1655 E. coli was used to inoculate each well of the plate at a 1 to 125 dilution. Cultures were grown at 37 °C with shaking at 190 rpm, and OD660nmwas measured after 6 hours and 24 hours. Figure 6 ).

[0349] After 6 hours, [ColE9:Im9-ImD:ColD], [ColE9:Im9-ImD], and [Im9-ImD:ColD] of 460 pM, 460 pM, and 37 nM, respectively, were sufficient to inhibit growth. However, after 24 hours, 1.4 nM, 1 μΜ, or 0.33 μΜ of [ColE9:Im9-ImD:ColD], [ColE9:Im9-ImD], and [Im9-ImD:ColD], respectively, were required to prevent growth. This can reflect the dominance of resistant mutants in overnight cultures of ColE9- or ColD-treated cultures. To test this, the frequency of resistant mutants was calculated by plating serial dilutions of MG1655 overnight cultures onto plates containing 25 nM [ColE9:Im9-ImD:ColD], 25 nM [ColE9:Im9-ImD], 50 nM [Im9-ImD:ColD], or no bacteriocin. The original culture contained 1.8 x 10 9 c.f.u. / ml, reduced to 775 c.f.u. / ml by 50 nM [Im9-ImD:ColD], to 5750 c.f.u. / ml by 25 nM [ColE9:Im9-ImD], and no detectable colonies for the sample plated on 25 nM [ColE9:Im9-ImD:ColD].

[0350] In an attempt to determine the value of [ColE9:Im9-ImD:ColD], 5 ml of 1.75 x 10 10 c.f.u. of MG1655 E. coli overnight culture were treated with 25 nM [ColE9:Im9-ImD:ColD] and all samples were plated and incubated overnight at 37°C. No colonies were obtained. The frequency of resistant mutants for [ColE9:Im9-ImD] and [Im9-ImD:ColD] were 1 in 3.0 x 10 5 and 1 in 2.3 x 10 6 Since resistance to [ColE9:Im9-ImD:ColD] requires mutations in both the ColE9 and ColD uptake pathways, the frequency of resistant mutants as the product of the frequencies of resistance to the individual components (1 in 6.9 x 10 11 ) is meaningful, which is consistent with the observation that no resistant mutants were observed in the experiment.

[0351] In vivo expression of the trimeric complex

[0352] The genes encoding colicin E9, Im9-ImD, and colicin D were cloned into pET21a and transformed into E. coli BL21(DE3). After expression and purification, only colicin E9 and Im9-ImD were apparent. A second copy of the colicin D gene was cloned into pACYCDuet1, which was co-transformed into BL21(DE3) cells with colicin E9, Im9-ImD, and colicin D cloned into pET21a. The expressed proteins were initially purified using the His tag on the C-terminus of Im9-ImD, and colicin D, colicin E9, and Im9-ImD were apparent in the nickel column elution Figure 7 ).

[0353] The [ColE9:Im9-ImD:ColD] complex was further purified on a 26 / 60 S200 gel filtration column, separating the intact complex from the [ColE9:Im9-ImD] and [Im9-ImD:ColD] subcomplexes. The initial elution peak at 110 ml (fractions Al to A5) corresponded to aggregated material eluting in the void volume of the column. The second elution peak at 145 ml (fractions Al l to B2) was the [ColE9:Im9-ImD:ColD] complex. The third elution peak at 174 ml was a mixture of [ColE9:Im9-ImD] and [Im9-ImD:ColD]. SDS-PAGE analysis of representative fractions is shown in Figure 8 The initial purification of [ColE9:Im9-ImD:ColD] resulted in a yield of approximately 10 mg per liter of culture.

[0354] Purified [ColE9:Im9-ImD:ColD] was tested for activity against E. coli BW25113, btuB-BL21(DE3), fepA-BW25113, tolA-BW25113 and tonB-BW25113 over a concentration range of 100 nM down to 137 pM. Like [ColE9:Im9-ImD:ColD] assembled in vitro, the complex assembled in vivo was active against E. coli strains lacking components of the colicin E9 or colicin D uptake pathway Figure 9 ).

[0355] Example 2

[0356] Components of the tetrameric complex

[0357] The bacteriocin components of the tetrameric complex are chimeric proteins containing the targeting part (receptor binding and transport domain) of KlebC, CloDF13 and KlebG linked to the effector part (cytotoxic domain) of colicin E9, colicin E3 and colicin D, respectively. The resulting chimeric proteins are named KlebC-E9, CloDF13-E3 and KlebG-D.

[0358] The immunoprotein scaffold is a fusion protein containing its cognate immunoproteins ColE9 (“Im9”), ColE3 (“Im3”) and ColD (“ImD”) separated by flexible linker peptides. The scaffold is denoted by the name “Im9-Im3-ImD”. The complex between the scaffold and the three chimeric bacteriocins is denoted by the short-hand notation [Im9-Im3-ImD:KlebC-E9:CloDF13-E3:KlebG-D].

[0359] Protein-protein interactions involved in the uptake of cloacin DF13 (Krone et al., 1983; Thomas & Valvano, 1993; Wooldridge & Williams, 1991), KlebC (Housden et al., 2021) and KlebG (unpublished) are summarized in Figure 3 .

[0360] In vitro assembly of the tetrameric complex

[0361] Chimeric bacteriocins were expressed in E. coli BL21(DE3) as heterodimeric complexes with their respective immunoprotein (Im9, Im3 and ImD) of the cytotoxic domain. The immunoproteins were engineered to carry a His6 tag at their C-terminus. Cell pellets were lysed by sonication, clarified by centrifugation and loaded onto 5 ml HisTrap HP columns. In each case, free bacteriocin was eluted from the nickel affinity column using 6 M guanidinium hydrochloride. Eluted bacteriocins were refolded by dialysis into 25 mM Tris-HCl, pH 7.5, 150 mM NaCl and purified by gel filtration over a 26 / 60 S200 column equilibrated in the same buffer. The immunoprotein scaffold Im9-Im3-ImD, which also carries a C-terminal His6 tag, was expressed separately and purified by nickel affinity chromatography (elution with imidazole) followed by gel filtration over a 26 / 60 S200 column equilibrated in 25 mM Tris-HCl, pH 7.5.

[0362] A 500 μl sample containing 20 μM Im9-Im3-ImD, 30 μM KlebC-E9, 30 μM CloDF13-E3 and 30 μM KlebG-D was prepared in 25 mM Tris-HCl, pH 7.5, 150 mM NaCl and purified over a Superdex 200 increase 10 / 300 GL analytical gel filtration column (Cytiva) equilibrated in the same buffer. SDS-PAGE analysis of representative fractions is shown in Figure 10

[0363] Microbicidal activity of the tetrameric complex

[0364] Serial dilutions of the tetrameric complex [Im9-Im3-ImD:KlebC-E9:CloDF13-E3:KlebG-D] were prepared at concentrations ranging from 6.1 μM to 25 nM. 5 μl of the dots were applied to nutrient broth (Merck) soft agar lawns inoculated with K. pneumoniae SG62, SR3 or SR6. The plates were incubated at 37 °C overnight and bacteriocin activity was visualized as areas of clearing in the bacterial lawn. Figure 11

[0365] ​​The tetramer [Im9-Im3-ImD:KlebC-E9:CloDF13-E3:KlebG-D] complex retained activity of KlebC-E9 against SR3, CloDF13-E3 against SG62, and KlebG-D against SR6. In addition, the tetramer [Im9-Im3-ImD:KlebC-E9:CloDF13-E3:KlebG-D] complex showed enhanced activity against SR3 relative to the individual component bacteriocins, suggesting that increased receptor avidity due to multiple targeting moieties contributes to the killing activity of the complex.

[0366] Example 3

[0367] Immunoprotein scaffolds incorporating the M-type bacteriocin KvarM were designed. The first contained KvarM linked to two immunoprotein domains, Im9 and ImD (KvarM-Im9-ImD), and the second contained KvarM linked to three immunoprotein domains, Im9, Im7 and ImD (KvarM-Im9-Im7-ImD). In both scaffolds, KvarM (at the N-terminal end of the fusion protein) was linked to Im9 using a rigid helical linker ((EAAAK)3EL), and the immunoprotein domains were linked by intrinsic unstructured sequences derived from the N-terminal subregion of colicin E9. A His6 tag for nickel affinity purification was added to the C-terminal end of the fusion proteins (after the last immunodomain). The scaffolds were expressed in E. coli BL21 (DE3) cells and purified by nickel affinity chromatography followed by gel filtration. Serial dilutions of wild-type KvarM, KvarM-Im9-ImD and KvarM-Im9-Im7-ImD spanning 2.5 μΜ to 4.9 nM in concentration were spotted onto soft agar lawns of K. pneumoniae SG96 in nutrient broth. Once dry, the plates were incubated overnight at 37 °C, with clearance zones indicating bacteriocin-mediated killing. Figure 12 [A] and [B] are shown schematically.

[0368] KvarM-Im9-ImD and KvarM-Im9-Im7-ImD were expressed in E. coli BL21 (DE3) cells and purified by nickel affinity chromatography followed by gel filtration. Serial dilutions of wild-type KvarM, KvarM-Im9-ImD and KvarM-Im9-Im7-ImD spanning 2.5 μΜ to 4.9 nM in concentration were spotted onto soft agar lawns of K. pneumoniae SG96 in nutrient broth. Once dry, the plates were incubated overnight at 37 °C, with clearance zones indicating bacteriocin-mediated killing.

[0369] Both KvarM-immunoprotein scaffolds showed cytotoxic activity against K. pneumoniae SG96 cells, with KvarM-Im9-ImD showing clearance zones over a concentration range of 2.5 μΜ to 156 nM, and KvarM-Im9-ImD killing occurring at as low as 312 nM ( Figure 12[C]). This indicates that the KvarM-Immunoprotein scaffold can bind to the outer membrane receptor FhuA of KvarM and that covalent fusion of the C-terminus of the KvarM cytotoxic region to two and three immunoprotein domains does not abolish its enzymatic murein precursor degradation activity. Interestingly, these results indicate that the immunoprotein scaffold, which normally dissociates from the complexed nuclease bacteriocin upon binding to the target cell surface receptor, is transported into the periplasm of the target cell.

[0370] As with the other immunoprotein scaffolds described herein, the KvarM-Immunoprotein scaffold fusion can complex with a protein bacteriocin having an effector moiety (cytotoxic domain) homologous to the immunoprotein domain, which in this case is the DNAse domain of colicin E9 or E7. This is schematically shown in Figure 13 The addition of KvarM improves the receptor binding and cytotoxic capacity of the complex, as the KvarM component provides binding activity to its own receptor (in addition to those recognized by the other PB components of the complex) and cytotoxic activity against KvarM susceptible strains.

[0371] Example 4

[0372] Components of the trimeric complex

[0373] The bacteriocin component of the complex is a chimeric protein based on the pore-forming pyocin S5. These chimeric pyocins are constructed by replacing the pore-forming cytotoxic domain of pyocin S5 with a fusion of the inner membrane translocation domain of pyocin G and the DNAse type cytotoxic domain of colicin E9 or E7. Thus, the chimeric pyocins contain the outer membrane translocation (T om ) and receptor binding (R) domains of pyocin S5, followed by the inner membrane translocation domain (T IM ) of pyocin G, and the DNAse domain of colicin E9 or E7. The resulting chimeric proteins are designated S5E9 and S5E7, respectively.

[0374] The immunoprotein scaffold is a fusion protein of the immunoproteins of colicin E9 and E7 (Im9 and Im7, respectively) connected by a 7 amino acid linker having the sequence SASGSAS. The scaffold is denoted by the name “Im97” (but can also be named “Im9-Im7”), and the trimeric complex is denoted by the shorthand notation “S5E9-Im97-S5E7” (equivalent to “[S5E9:Im9-Im7:S5E7]”). The scaffold and chimeric pyocins are schematically shown in Figure 15A.

[0375] In vitro assembly of the trimeric complex

[0376] Proteins were expressed in E. coli BL21(DE3) and purified by nickel affinity chromatography. The immunoprotein scaffold Im97 was isolated by nickel affinity chromatography with the aid of a C-terminal His6 tag and further purified by gel filtration (Superdex 75). The chimeric pyocins S5E9 and S5E7 were co-expressed with their cognate immunoproteins Im9 and Im7, respectively. The pyocin-immunoprotein complexes were isolated by nickel affinity chromatography with the aid of a C-terminal His6 tag on the immunoprotein and further purified by gel filtration (Superdex S200). To prepare uncomplexed pyocins, purified pyocin-immunoprotein complexes were loaded onto a nickel affinity column and pyocins were eluted with 6 M guanidinium hydrochloride. The application of guanidinium hydrochloride causes the proteins to unfold, thereby causing the pyocins to dissociate, while the immunoprotein remains bound to the column. Pyocins were refolded by dialysis into 50 mM Tris, 200 mM NaCl pH 7.5.

[0377] To construct and isolate the trimeric complex scaffold Im97 as well as the chimeric pyocins S5E9 and S5E7, the component proteins were mixed in a 1 :4:4 ratio in 50 mM Tris, 200 mM NaCl pH 7.5 and incubated for 1 hour at room temperature. The complex was isolated by nickel affinity chromatography and eluted with imidazole. The complex was further purified by gel filtration (Superdex S200) to remove aggregated proteins, resulting in a monodisperse S5E9-Im97-S5E7 complex.

[0378] SDS PAGE was performed to demonstrate the purity and integrity of the component pyocins and S5E9-Im97-S5E7 complex in free form and in immunoprotein complex form (Figure 15B). All proteins showed high purity with little signs of degradation. However, multimeric pyocins were observed in samples where uncomplexed pyocins S5E9 and S5E7 were refolded.

[0379] Cell killing by the trimeric complex

[0380] The activity of the S5E9-Im97-S5E7 trimeric complex and the chimeric pyocins alone, in their free form and in immunoprotein complex form, against P. aeruginosa is shown in Figure 15C.

[0381] Conclusion

[0382] Production of the Im9-ImD fusion protein enables the in vitro assembly of a hetero-trimeric [ColE9:Im9-ImD:ColD] complex. This complex retains all the activities of its component protein bacteriocins. The components of the trimeric [ColE9:Im9-ImD:ColD] complex have also been successfully co-expressed in E. coli, allowing purification of the assembled complex, thus simplifying the production and purification process.

[0383] A hetero-tetrameric complex [Im9-Im3-ImD:KlebC-E9:CloDF13-E3:KlebG-D] has been assembled in vitro and retains the activities of the three component protein bacteriocins.

[0384] It has further been shown that a hetero-trimeric complex containing a chimeric pyocin is effective against P. aeruginosa. This construct also shows that targeting moieties from pore-forming PBs can be combined with nuclease effector moieties from other PBs in the context of the complexes described in this specification.

[0385] ***

[0386] The features disclosed in the foregoing description, or the accompanying claims, or the accompanying drawings, may, alone or in any combination(s), be material for realizing the application in its diverse forms, suitably expressed in its specific form or in terms of means for performing disclosed functions or in terms of methods or processes for obtaining disclosed results.

[0387] While the application has been described in connection with the above exemplary embodiments, it will be evident for a person skilled in the art that many modifications and variations are possible without departing from the spirit and scope of the application. Thus, the exemplary embodiments of the application set forth above are considered to be illustrative and not restrictive in character.

[0388] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

[0389] Any section headings herein are used for organizational purposes only and are not to be construed as limiting the subject matter described.

[0390] Throughout this specification (including the claims), unless the context requires otherwise, the word "comprise" and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0391] It must be noted that, as used herein, the singular articles "a", "an" and "the" can be read to include the plural unless the context clearly indicates otherwise. Ranges can be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is recited, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about", it will be understood that the particular value forms another embodiment. The term "about" in relation to a numerical value is optional and means, for example, + / - 10%.

[0392] References

[0393] Numerous publications are cited above to more fully describe and disclose the application and the state of the art to which the application pertains. Full citations for these references are provided below. The entirety of each of these references is herein incorporated by reference.

[0394] Barreteau, H., Bouhss, A., Gerard, F., Duché, D., Boussaid, B., Blanot, D., Llubes, R., Mengin-Lecreulx, D., Touzé, T., 2010. J Biol Chem 285, 12378-12389.

[0395] Chauleau, M., Mora, L., Serba, J., & de Zamaroczy, M. (2011). FtsH-dependent processing of RNase colicins d and e3 means that only the cytotoxic domains are imported into the cytoplasm. Journal of Biological Chemistry, 286(33). https: / / doi.org / 10.1074 / jbc.M111.242354

[0396] Chérier, D., Patin, D., Blanot, D., Touzé, T., Barreteau, H., 2021. Antibiotics 10(9): 1109

[0397] Denkovskiene, E. et al. (2019). Broad and Efficient Control of Klebsiella Pathogens by Peptidoglycan-Degrading and Pore-Forming Bacteriocins Klebicins. Scientific Reports 9: 15422 https: / / doi.org / 10.1038 / s41598-019-51969-1

[0398] Francis, M. R., Webby, M. N., Housden, N. G., Kaminska, R., Elliston, E., Chinthammit, B., Lukoyanova, N., & Kleanthous, C. (2021). Porin threading drives receptor disengagement and establishes active colicin transport through Escherichia coli OmpF. The EMBO Journal, 40(21). https: / / doi.org / 10.15252 / embj.2021108610

[0399] Housden, N. G., Webby, M. N., Lowe, E. D., El-Baba, T. J., Kaminska, R., Redfield, C., Robinson, C. v., & Kleanthous, C. (2021) Toxin import through the antibiotic efflux channel TolC. Nature Communications, 12(1). https: / / doi.org / 10.1038 / s41467-021-24930-y

[0400] Kageyama M, Kobayashi M, Sano Y, Masaki H. (1996) Construction and characterization of pyocin-colicin chimeric proteins. J Bacteriol. 178(1), 103-10.

[0401] Kleanthous, C., & Walker, D. (2001). Immunity proteins: Enzyme inhibitors that avoid the active site. In Trends in Biochemical Sciences (Vol. 26, Issue 10). https: / / doi.org / 10.1016 / S0968-0004(01)01941-7

[0402] Krone, W. J. A., Luirink, J., & Koningstein, G. (1983). Subcloning of the cloacin DF13 / Aerobactin receptor protein and identification of a pColV-K30-determined polypeptide involved in ferric-aerobactin uptake. Journal of Bacteriology, 156(2). https: / / doi.org / 10.1128 / jb.156.2.945-948.1983

[0403] Pilsl, H., Glaser, C., Gross, P., Killmann, H., T., Braun, V., 1993. Mol Gen Genet 240, 103-112.

[0404] Schaller, K, J., Bacteriology, V. B.-J. of, 1982, undefined, 1982. Am Soc Microbiol 152, 994-1000.

[0405] Sham, L. T., Butler, E. K., Lebar, M. D., Kahne, D., Bernhardt, T. G., Ruiz, N., 2014. Science 345, 220-222.

[0406] Thomas, J. A., & Valvano, M. A. (1993). Role of tol genes in cloacin DF13 susceptibility of Escherichia coli K-12 strains expressing the cloacin DF13-aerobactin receptor lutA. In Journal of Bacteriology (Vol. 175, Issue 2). https: / / doi.org / 10.1128 / jb.175.2.548-552.1993

[0407] Wooldridge, K. G., & Williams, P. H. (1991). Sensitivity of Escherichia coli to cloacin DF13 involves the major outer membrane protein OmpF. Journal of Bacteriology, 173(8). https: / / doi.org / 10.1128 / jb.173.8.2420-2424.1991

[0408] For standard molecular biology techniques, see Sambrook, J., Russel, D. W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

Claims

1. An antibacterial protein complex comprising: (a) a first protein bacteriocin (PB) molecule and a second PB molecule; and (b) an immunoprotein scaffold comprising a first immunoprotein domain and a second immunoprotein domain; wherein the first and second immunoprotein domains are non-covalently bound to the respective first and second PB molecules.

2. The antibacterial complex of claim 1, wherein each bacteriocin comprises a cell targeting moiety and an effector moiety, and binds to the respective immunoprotein domain via its effector moiety.

3. The antibacterial complex of claim 1 or claim 2, wherein the immunoprotein scaffold comprises a third immunoprotein domain, and the complex comprises a third bacteriocin molecule non-covalently bound to the third immunoprotein domain.

4. The antibacterial complex of any one of the preceding claims, wherein the immunoprotein scaffold comprises two or more repeats of the same immunoprotein domain, for example wherein each of the immunoprotein domains is the same.

5. The antibacterial complex of any one of claims 1 to 4, wherein the immunoprotein scaffold contains two or more different immunoprotein domains, for example wherein each of the immunoprotein domains is different from each of the other component immunoprotein domains.

6. The antibacterial complex of any one of the preceding claims, wherein the immunoprotein scaffold is a fusion protein.

7. The antibacterial complex of claim 6, wherein the immunoprotein scaffold fusion protein further comprises an M-type bacteriocin moiety, for example colicin M (ColM), KpneM, KpneM2, KvarM, PaeMl or PaeM4.

8. The antibacterial complex of any one of the preceding claims, comprising bacteriocins having two or more different effector moieties, for example wherein the different effector moieties have different enzymatic activities.

9. The antibacterial complex of claim 8, wherein each effector moiety has a different enzymatic activity.

10. The antibacterial complex of any one of the preceding claims, comprising bacteriocins having two or more different targeting moieties, for example wherein each of the bacteriocins has a different targeting moiety.

11. The antibacterial complex of claim 10, wherein each bacteriocin has a targeting moiety specific for bacteria of the same species or strain.

12. The antibacterial complex of claim 10 or claim 11, wherein the targeting moieties bind to two or more different receptors or use two or more different transport portals.

13. The antibacterial complex of claim 12, wherein each targeting moiety binds to a different receptor and / or each targeting moiety uses a different transport portal.

14. The antibacterial complex of claim 13, comprising bacteriocins having targeting moieties specific for bacteria of two or more different species or strains. ​ 15. A method of combating bacteria comprising contacting a bacterium or a population of bacteria with an antibacterial complex according to any one of the preceding claims.

16. An antibacterial complex according to any one of claims 1 to 14 for use in a method of medical treatment.

17. An antibacterial complex according to any one of claims 1 to 14 for use in the prophylaxis or treatment of a bacterial infection or a condition caused by or associated with a bacterial infection.

18. A host cell comprising: (i) a nucleic acid encoding an immunoprotein scaffold comprising a first immunoprotein domain and a second immunoprotein domain; (ii) a nucleic acid encoding at least one bacteriocin, the or each of which has an effector domain capable of binding to at least one of the immunoprotein domains; wherein the host cell is capable of expressing the immunoprotein scaffold and the bacteriocin.

19. The host cell of claim 18, wherein the cell comprises a first nucleic acid and a second nucleic acid encoding respective first and second bacteriocins, each having an effector domain capable of binding to a respective one of the immunoprotein domains; wherein the host cell is capable of expressing the immunoprotein scaffold and the bacteriocins.

20. The host cell of claim 18 or claim 19, comprising: (i) a nucleic acid encoding an immunoprotein scaffold comprising a first immunoprotein domain, a second immunoprotein domain and a third immunoprotein domain; (ii) a first nucleic acid, a second nucleic acid and a third nucleic acid encoding respective first, second and third bacteriocins, each having an effector domain capable of binding to a respective one of the immunoprotein domains; wherein the host cell is capable of expressing the immunoprotein scaffold and the bacteriocins.

21. A method of producing an antibacterial complex comprising providing a host cell according to any one of claims 18 to 20, culturing the cell under conditions suitable for expression of the immunoprotein scaffold and bacteriocin molecules, and optionally further comprising isolating the antibacterial complex.

22. A method of producing an antibacterial complex, the method comprising contacting an immunoprotein scaffold comprising a first immunoprotein domain and a second immunoprotein domain with a first bacteriocin molecule and a second bacteriocin molecule, each having an effector moiety capable of binding to a respective one of the immunoprotein domains to form an antibacterial complex.

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