Chimera Invasion System

Abstract

A cross-kingdom platform for the delivery of therapeutic agents to target cells. The system maintains the transport and uptake functions of the Inv while altering targeting from β1 integrin to other proteins expressed on the surface of target eukaryotic organisms (i.e., cell surface proteins) or chemical moieties expressed on the surface of target eukaryotic cells (i.e., cell surface chemical moieties) by genetically replacing D4 and D5 of the Inv with binding domains from heterologous proteins. These heterologous proteins can be derived from bacterial, fungal, animal, or viral genomes. This engineering results in the construction of chimeric Inv proteins in which D1-D3 (i.e., non-binding domains) are fused in frame to alternative binding domains from heterologous proteins. The alternative binding domains interact with different cell surface proteins or chemical moieties, which can sometimes be referred to as receptors, on the surface of eukaryotic cells, thereby allowing specific targeting of the Inv to cells independent of endogenous β1 integrin binding.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 363,536, filed April 25, 2022, and U.S. Provisional Application No. 63 / 367,518, filed July 1, 2022.

[0002] FIELD OF THEINVENTION The present invention relates to bacterial delivery vehicles for therapeutic applications. More specifically, the present invention relates to bacterial vehicles capable of targeting specific types of eukaryotic cells. [Background technology]

[0003] 2. Background of the Invention To date, precise in vivo delivery of therapeutic modalities to specifically targeted cells remains challenging. Drug delivery involves two key components: the vehicle itself and the mechanism by which the vehicle specifically reaches the desired cell type with minimal off-target delivery. Current delivery strategies are primarily based on mechanical techniques (e.g., electroporation, hydrodynamic injection, and microinjection) and viral vector delivery (e.g., lentivirus, adenovirus, and adeno-associated virus). Non-viral delivery methods such as liposomes and nanoparticles are also used, but the size and number of cargo moieties they can carry are quite limited. While useful in vitro, many of these methods cannot be readily translated clinically to animal or human patients. Bacterial delivery vehicles offer many advantages. One particular advantage is their ability to deliver therapeutic moieties into cells via invasion. Currently, bacterial delivery vehicles reach their targets via passive mechanisms, often relying on niche-specific biological features (e.g., hypoxic tumor microenvironment). Alternatively, bacteria are specifically targeted through ligand-receptor interactions with factors on the target cell surface.To date, even the most specific targeting methods may have off-target effects due to the expression of targeting ligands across multiple cell types in multiple tissues and organs.To realize the full potential of bacterial delivery vehicles, more specific targeting mechanisms are needed to reduce off-target effects (i.e., delivery to unwanted or undesired tissues and cell types). Summary of the Invention [Problem to be solved by the invention]

[0004] Means for solving the problem The present invention provides systems and methods for specific targeting to cells in eukaryotic hosts using highly specific targeting of invasive, non-pathogenic bacterial delivery vehicles, where the bacterial delivery vehicles are engineered to generate chimeric invasin (Inv) polypeptides with altered binding domains. In its non-chimeric form, the Inv polypeptide has five domains, designated D1, D2, D3, D4, and D5, along with a β-barrel that spans the bacterial outer membrane. Effect of the Invention

[0005] In certain aspects, the invention provides systems and methods for maintaining the transport (i.e., transport to the surface of bacterial cells) and uptake functions of the Inv protein from Yersinia pseudotuberculosis while altering its targeting from β1 integrin to other protein domains expressed on the surface of target eukaryotic cells (i.e., cell surface proteins) or chemical moieties expressed on the surface of target eukaryotic cells (i.e., cell surface chemical moieties) by genetically replacing D4 and D5 of Inv with binding domains from heterologous proteins. These heterologous proteins can be derived from bacterial, fungal, animal, or viral genomes. This engineering results in the construction of chimeric Inv proteins in which D1-D3 (i.e., non-binding domains) from Inv are fused in frame to alternative binding domains from heterologous proteins or synthetic binding domains. The alternative binding domains interact with different cell surface proteins or chemical moieties, which may in some cases be referred to as receptors, on the surface of eukaryotic cells, thereby allowing specific targeting of Inv to cells independent of endogenous β1 integrin binding. In some cases, the binding domain of the heterologous protein may be referred to as a ligand binding domain.

[0006] It is contemplated that one can take a non-binding domain from Inv (e.g., D1, D2 and D3) or a full-length Inv, add a linker sequence such as those described immediately below, and then add a binding domain from one of the proteins listed in Tables 1, 2, or 3. Thus, a sequence from an Inv can be utilized, such as SEQ ID NO:1, from about amino acid 1 to about amino acid 795, or from about amino acid 1 to about amino acid 986, as provided below, or one that is 95% or 90% identical thereto.

[0007] In certain aspects, the invention provides a non-pathogenic bacterium engineered to express a chimeric targeting ligand. The bacterium is engineered to have a sequence encoding a non-binding domain (e.g., D1, D2 and / or D3) of an Inv protein fused to a sequence encoding a heterologous binding domain (e.g., see Tables 1-3 below for proteins that have available binding sites from that protein). Thus, a chimeric Inv protein with an altered binding domain can be generated upon expression of the sequence. This can allow an interkingdom delivery vehicle to be directed to other tissues than can be achieved using a binding domain that targets β1 integrin. SEQ ID NO:1 below discloses the amino acid sequence of an Inv polypeptide. It is contemplated that the D1, D2 and / or D3 regions of that sequence can be used to construct chimeric proteins, such as by conversion to a sequence having 99%, 95% or 90% homology to the corresponding nucleic acid sequence or the nucleic acid sequence of the D1, D2 and / or D3 regions (see SEQ ID NO:38). Because D1-D3 promote targeting to target cells rather than invasion, bacteria expressing chimeric targeting ligands comprising D1-D3 fused to heterologous binding domains that target specific factors on the surface of target cells can be used to label or detect cells.

[0008] In a further embodiment, the invention provides a non-pathogenic bacterium engineered to express a chimeric targeting ligand engineered to have a sequence encoding a complete Inv protein (i.e., D1, D2, D3, D4, D5) fused to a sequence encoding a binding domain from a heterologous protein or a synthetic binding domain (see, e.g., Tables 1-3 below for proteins that have available binding sites from that protein). Thus, a chimeric Inv protein with an altered binding domain can be generated upon expression of the sequence. This can allow the interkingdom delivery vehicle to be directed to other cell types and tissues than can be achieved using a binding domain that targets β1 integrin. SEQ ID NO:1 below discloses the amino acid sequence of an Inv polypeptide. It is contemplated that the D1, D2, D3, D4, and D5 regions of that sequence can be used to construct chimeric proteins, such as by conversion to a corresponding nucleic acid sequence or sequences having 95% or 90% homology to the nucleic acid sequence encoding the D1, D2, D3, D4, and D5 regions of Inv.

[0009] In constructing fusion (i.e., chimeric) proteins, various linker sequences, specific sequences of amino acids, can be used to connect protein domains (i.e., amino acid sequences that fold independently). Linker sequences are usually classified as rigid or flexible linkers, and some may contain cleavage sites as described herein. The physical characteristics of the linker can affect important properties of the fusion protein, including expression levels, biological activity, or other in vivo behavior. Linker sequences containing glycine and serine are generally flexible, allowing the domains to move independently of each other. Proline-containing linker sequences tend to be more rigid and limit the relative motion of the domains [see generally Chen X, Zaro JL, Shen WC. Fusion protein linkers: property, design and functionality Adv Drug Deliv Rev. 2013 Oct;65(10):1357-69. doi:10.1016 / j.addr.2012.09.039. Epub 2012 Sep 29. PMID:23026637; PMCID:PMC3726540].

[0010] In a further aspect, the chimeric polypeptide according to the invention utilizes a sequence comprising a linker sequence for linking the non-binding domain of an Inv fused to a sequence encoding a heterologous binding domain. The linker sequence may be a sequence selected from SEQ ID NOs: 2-20 disclosed below. In a particular embodiment, the binding domain sequence is a sequence encoding a binding domain selected from any one of the polypeptides mentioned in Tables 1-3.

[0011] In an advantageous embodiment, the chimeric targeting ligand utilizes a sequence that includes a composite linker sequence to link the non-binding domain of Inv or the full-length Inv to a sequence encoding a heterologous binding domain (BD), including a synthetic binding domain. The first part (N-terminus) of the linker sequence may be a sequence selected from SEQ ID NOs: 2-20 disclosed below. The second part of the linker may include one or more peptidase or protease cleavage sites or multiple cleavage sites provided in Table 4. The use of a composite linker helps to ensure that the correct target cell type has been reached, since it requires the presence of peptidase(s) or protease(s) on the target cell surface to allow entry via removal of the heterologous BD. In a particular embodiment, the BD sequence is a sequence encoding a BD selected from any one of the polypeptides mentioned in Tables 1-3 or a synthetic BD.

[0012] Non-pathogenic bacteria engineered to express chimeric Inv polypeptides according to various embodiments can utilize sequences encoding the non-binding domain of an Inv protein that encodes a polypeptide that is 90% (or 95% or even 99%) identical to amino acids 1 to 794 of SEQ ID NO:1, as shown below.

[0013] Non-pathogenic bacteria engineered to express a chimeric targeting ligand can alternatively utilize a sequence encoding all domains of the Inv protein that are 90% (or 95% or 99%) identical to amino acids 1 to 986 of SEQ ID NO:1.

[0014] Non-pathogenic bacteria engineered to express chimeric Inv polypeptides may be further engineered to express therapeutic nucleic acids, proteins, antibodies, antibody derivatives, polypeptides, gene editing systems (CRISPR and other gene editing nucleases), eukaryotically translatable mRNAs, or combinations thereof. Therapeutic nucleic acids, proteins, antibodies, antibody derivatives, polypeptides, gene editing systems (CRISPR and other gene editing nucleases), eukaryotically translatable mRNAs, or combinations thereof may be expressed from sequences on the bacterial chromosome or plasmids.

[0015] In a further aspect, the invention provides a non-pathogenic bacterium engineered to express a chimeric Inv polypeptide. The expressed chimeric Inv polypeptide may have a non-binding domain of an Inv protein (e.g., D1, D2 and / or D3) or a full-length Inv (D1-D5) fused to a heterologous binding domain to generate a chimeric Inv protein. The chimeric targeting ligand produced by the bacteria may be used to alter the target cell or tissue type of the bacterial delivery vehicle.

[0016] In a further aspect, the invention provides a bacterium for nucleic acid delivery or delivery of another molecule (e.g., a protein, an antibody, an antibody derivative, a polypeptide, a gene editing system (CRISPR and other gene editing nucleases), a eukaryotically translatable mRNA) to a eukaryotic cell, including a non-pathogenic bacterium, wherein the bacterium is engineered to express at least one entry factor, and the entry factor has a non-binding domain of an Inv protein or a full-length Inv fused to a heterologous binding domain to generate a chimeric Inv protein. The heterologous protein can be a protein that binds to a cell surface protein or a cell surface chemical moiety on a target eukaryotic cell. The binding domain can be a fragment of a heterologous protein, such as one that is made without including the non-binding region of the heterologous protein. In an advantageous embodiment, the binding domain of the heterologous protein is translated from a sequence encoded in the genome of a bacterium, fungus, virus, or animal, but is engineered to be encoded and translated as part of the chimeric Inv protein by the bacterial delivery vehicle. Alternatively, the binding domain can be any synthetic (i.e., non-naturally occurring) protein that facilitates binding to a target cell surface.

[0017] The chimeric Inv targeting ligand may be expressed from a sequence on the chromosome of the engineered bacterial delivery vehicle or from a plasmid carried by the engineered bacterial delivery vehicle.

[0018] In an advantageous embodiment, the chimeric Inv targeting ligand has a peptide linker fused between the non-binding domain of the Inv protein and the binding domain from a heterologous protein. The peptide linker may have one or more amino acids fused in frame to the non-binding domain of the Inv protein and the binding domain from a heterologous protein. The non-binding domain of the Inv protein may be the D1, D2, and D3 domains of Inv, or a combination or subset thereof, or the full-length Inv.

[0019] In an advantageous embodiment, the chimeric Inv protein has a peptide linker fused between the non-binding domain of the Inv protein and the binding domain from a heterologous protein. The peptide linker may have one or more amino acids fused in-frame to the full-length Inv protein and the heterologous binding domain.

[0020] Bacteria for nucleic acid delivery to eukaryotic cells can further be engineered to express therapeutic nucleic acids from sequences on the bacterial chromosome or from plasmids.

[0021] Thus, in certain aspects, the invention provides an expression cassette for the production of a chimeric invasin (Inv) polypeptide. The expression cassette may comprise a prokaryotic promoter and a nucleic acid sequence encoding an Inv polypeptide fused to a linker polypeptide at the carboxy terminus of the Inv polypeptide. Expression of the nucleic acid encoding the chimeric invasin polypeptide is controlled by the prokaryotic promoter. In certain advantageous embodiments, the expression cassette for the production of a chimeric invasin polypeptide comprises a sequence encoding a binding domain for binding to a surface moiety on a target cell. The binding domain is fused to the amine terminus of the linker polypeptide. The binding domain may be a binding domain derived from a protein listed in Tables 1-3, or the binding domain may be a synthetic binding domain. The nucleic acid sequence of the invasin region of the chimeric polypeptide may be 90% identical, 95% identical or even 99% identical to nucleic acids 1-2958 of SEQ ID NO: 37. Similarly, the nucleic acid sequence of the invasin region of the chimeric polypeptide is 90% identical, 95% identical or even 99% identical to nucleic acids 1 to 2382 of SEQ ID NO:37 or SEQ ID NO:38.

[0022] An expression cassette for the production of a chimeric invasin polypeptide according to the first aspect may further comprise a nucleic acid sequence encoding a linker polypeptide and / or a protease cleavage site. Exemplary linkers include SEQ ID NO:2 to SEQ ID NO:20. Exemplary protease cleavage sites include SEQ ID NO:22 to SEQ ID NO:36.

[0023] A sequence encoding a protease cleavage site that is cleaved by a peptidase or protease can be located between the sequence encoding the invasin and the sequence encoding the binding domain.

[0024] Expression cassettes for production of chimeric invasin polypeptides can use prokaryotic promoters such as the T7, lacUV5, gapA, T5, recA, Ptac, Patac, pAl, lac, Sp6, araBad, and trp promoters. Prokaryotic promoters can also be hybrid or synthetic prokaryotic promoters.

[0025] Bacteria expressing the chimeric invasin polypeptide may be constructed, the bacteria being engineered to contain an expression cassette for production of the chimeric invasin polypeptide. The bacteria may be selected from the group consisting of Clostridium difficile, Escherichia coli, Clostridium tetani, Helicobacter pylori, Fusobacterium nucleatum, Gardnerella vaginalis, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Listeria monocytogenes, Staphylococcus aureus, Campylobacter jejuni, Vibrio vulnificus, Salmonella typhi, Clostridium botulinum, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium lepromatosis, Corynebacterium diphtheriae, Klebsiella pneumoniae, Acinetobacter baumannii, Streptococcus mutans, and group B bacteria. The bacterium may be selected from the group consisting of Streptococcus, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecalis, Listeria, Yersinia, Rickettsia, Shigella, Salmonella, Legionella, Chlamydia, Brucella, Neisseria, Burkholderia, Bordetella, Borrelia, Coxiella, Mycobacterium, Helicobacter, Staphylococcus, Streptococcus, Porphyromonas, Vibrio, Treponema, Lactobacillus, and Bifidobacterium. In an advantageous embodiment, the bacterium expressing the chimeric invasin polypeptide is Escherichia coli.

[0026] In yet another aspect, the invention provides a method of treating or preventing disease in a subject by administering a bacterium expressing a chimeric invasin polypeptide that is further engineered to express a therapeutic nucleic acid produced by the bacterium. Similarly, in addition to expressing a chimeric invasin polypeptide, the bacterium can be engineered to express therapeutic nucleic acids, proteins, antibodies, antibody derivatives, polypeptides, gene editing systems (CRISPR and other gene editing nucleases), eukaryotically translatable mRNA, or combinations thereof from sequences on the bacterial chromosome or plasmid.

[0027] The present invention also provides chimeric invasin polypeptides. The chimeric invasin polypeptide may comprise an Inv polypeptide and a linker polypeptide, the linker polypeptide having a first end (N-terminus) and a second end (C-terminus), the first end (N-terminus) of the linker polypeptide being attached to the C-terminus of the Inv polypeptide. The chimeric invasin polypeptide may further comprise a peptidase or protease cleavage site.

[0028] In an advantageous embodiment, the chimeric invasin polypeptide further comprises a heterologous protein binding domain or a synthetic binding domain attached to the second end (C-terminus) of the linker polypeptide. Advantageous binding domains can be binding domains derived from the proteins listed in Tables 1-3.

[0029] The chimeric invasin Inv polypeptide amino acid sequence may have a sequence that is 90% (or 95% or 99%) identical to amino acids 1 to 986 of SEQ ID NO:1, or 90% (or 95% or 99%) identical to amino acids 1 to 985 of SEQ ID NO:39.

[0030] The chimeric invasin Inv polypeptide amino acid sequence may be 90% (or 95% or 99%) identical to amino acids 1 to 794 of SEQ ID NO:1, or may have a sequence that is 90% (or 95% or 99%) identical to amino acids 1 to 794 of SEQ ID NO:39.

[0031] In a further aspect, the invention provides a composition for selective binding of a substrate to a target molecule. The composition can utilize a chimeric invasin polypeptide conjugated at the amino terminus of an Inv polypeptide to a biological and synthetic substrate surface, the substrate being selected from the group consisting of beads, viruses, exosomes, rigid substrates (e.g., for the manufacture of lateral flow strips), paper-based biosensors, plastic substrates (e.g., for the manufacture of plastic-based biosensors), graphene-based substrates, or nanomaterials (e.g., lipid nanoparticles, metal nanoparticles, mesoporous silica nanoparticles, nanowires, ITO, organic polymers).

[0032] In a further aspect, the invention provides a chimeric invasin polypeptide comprising the D1-D3 domains of an Inv polypeptide attached to a heterologous protein binding domain or a synthetic binding domain. The chimeric invasin polypeptide may comprise a linker, the linker polypeptide having a first end (N-terminus) and a second end (C-terminus), the first end of the linker polypeptide being attached to the C-terminal amino acid of the Inv polypeptide and the second end of the linker polypeptide being attached to the amino terminus of the heterologous protein binding domain or the amino terminus of the synthetic binding protein.

[0033] For a more complete understanding of the present invention, reference should be made to the following detailed description taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0034] [Figure 1] Figure 1 is a series of four diagrams (labeled (A)-(D)) of the structure of a full-length Inv (A) and the structures of various versions of the chimeric Inv proteins described herein ((B)-(D)). The chimeric Inv proteins include D1-D3 of an Inv fused to a binding domain of a heterologous protein (Figure 2B) or a full-length Inv fused to a linker sequence that may or may not contain a peptidase or protease cleavage site (Figures 2C and 2D). [Diagram 2]FIG. 2 is a diagram illustrating the three-step chimeric ligand targeting and entry paradigm described herein. [Diagram 3] Figure 3 is a micrograph of A549 cells treated with FEC19 bacteria carrying pSi_1fHER2-scr.c. The image (A549 cells) confirms that the bacterial delivery vehicle can specifically target and then enter HER2-positive furin-positive A549 cells, but not HER2-negative HeLa cells. [Figure 4] Figure 4 is a diagram (A) and schematic (B) showing a two-factor chimeric Inv protein used in the following examples. Figure 4A shows a schematic of a chimeric Inv protein comprising Inv D1-D5 linked in frame to a synthetic nanobody that binds HER2. The linker contains a cleavage site for the cell surface protease Furin. Figure 4B provides a schematic of the sequence of the chimeric Inv protein shown in (A). Only the C-terminus of Inv D5 is shown for illustrative purposes. [Diagram 5] FIG. 5 is a pair of diagrams displayed in (a) and (b) providing an annotated linear representation of the Inv amino acid sequence, including D1 through D4 / D5, with domain names and functions displayed. The chimeric Inv proteins described herein include D1, D2, and D3, with D4 / D5 replaced with a heterologous binding domain. The diagrams in (a) and (b) are identical, with (b) being a 90 degree rotation of the diagram in (a) to expand the data presented herein. The sequences shown in (A) and (B) are identical and are presented below as SEQ ID NO:1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides a system for targeted intracellular delivery of therapeutic or non-therapeutic moieties to eukaryotic cells using a non-pathogenic bacterial delivery platform that expresses a bifunctional, chimeric targeting-entry factor ("chimeric targeting ligand") that interacts with and binds to a factor (receptor) on the surface of the target cell, and then triggers internalization of the bacterial delivery vehicle by the target cell. The chimeric ligand contains a constant region that includes the Yersinia pseudotuberculosis invasin (Inv) protein (encoded by the inv gene) and a variable region that is customized for targeting purposes. The variable region includes a peptide or protein that binds to a receptor on the target cell. The variable region can include, for example, a single domain antibody, nanobody, camel IgG antibody, llama IgG antibody, peptibody, any other immune polypeptide, or any peptide made up of amino acid residues that specifically bind to a receptor molecule found on the outer membrane of a eukaryotic cell. This chimeric ligand can target the bacteria to a specific cell type or to a class of cell types that express the same receptor.

[0036] The Gram-negative genus Yersinia includes at least 17 species, three of which are human and animal pathogens: Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis. The virulence of these bacteria depends on factors that allow them to adhere to cells and cross the cell membrane to reach the cytoplasm of the target cell. These organisms express a variety of such factors, including invasin (Inv), YadA, YadB, YadC, Ail, Pla, and Ph6 antigens. Together, these various proteins are known as adhesins, and each protein acts at a specific step of the host-pathogen interaction. Importantly, each of these proteins binds to various host factors, including b1 integrin, collagen, fibronectin, laminin, and complement-related factors. Furthermore, all of these proteins are anchored to the bacterial outer membrane (OM), where they form a rod-like structure. Presentation on the OM allows the proteins to mediate interactions with factors on the surface of their target cells. Transport from the bacterial cell cytoplasm to the OM can also occur through various mechanisms. It is contemplated that the YadA, YadB, YadC, Ail, Pla, and Ph6 antigens can be used to construct chimeric polypeptides by replacing the inv nucleic acid sequence for expression with sequences encoding the YadA, YadB, YadC, Ail, Pla, and Ph6 antigens, which can create alternative chimeric targeted bacteria. Thus, for example, a YadA chimeric polypeptide can include a binding domain as taught herein for chimeric invasins, and optionally further includes a linker sequence and / or cleavage site between the YadA and BD amino acids. The sequences of these adhesins are known, such as YadA (UniProt P31489-YADA1_YEREN; UniProt P10858-YADA_YERPS, which are incorporated by reference). Sequences of the respective adhesins that are 90% identical, 95% identical or 99% identical to the consensus sequence of the YadA, YadB, YadC, Ail, Pla or Ph6 antigens may be used.

[0037] Invasin is the first adhesin expressed during invasion by enteropathogenic Yersinia spp. Its primary role is the invasion of epithelial cells via β1 integrin binding, which allows the bacteria to initiate colonization and internalization of host epithelial cells. Invasin has a modular structure that includes several well-defined functional sequences. In the broadest sense, Inv contains structural elements associated with autotransporters: a beta-barrel "transporter" structure at the amino (N) terminus, and an extracellular "passenger" domain at the carboxy (C) terminus. The passenger domain spontaneously passes from the periplasm to the outer membrane (OM) without the need for an energy source (e.g., ATP). In the case of Inv, this transport is thought to be mediated by passage of the protein into the periplasm via an N-terminal signal peptide, followed by insertion of the beta-barrel domain into the OM to form a pore for the passage of the passenger domain. The structure of the passenger domain is highly modular and contains five protein domains (D1-D5). The secondary structure of D1-D4 mainly contains beta sheets, whereas the secondary structure of D5 contains an alpha / beta helix secondary structure. Together, D4 and D5 form a module that binds to integrins with high affinity.

[0038] In a particular aspect, the present invention provides a bacterial-mediated delivery vehicle comprising an invasive, non-pathogenic bacterium that expresses and then transports a chimeric ligand to the outer membrane of the bacterial cell. The bacterium may contain a prokaryotic expression cassette encoding the chimeric ligand under the control of a prokaryotic promoter (synthetic or endogenous). This novel bacterial delivery platform expressing and displaying the ligand can provide cell- and tissue-specific delivery and internalization of the delivery vehicle in any eukaryotic cell at any cell cycle stage (dividing, non-dividing, quiescent) as long as the cell expresses the cognate cell surface receptor. Targeting to the desired eukaryotic cell can be controlled through the selection of a variable region specific for the receptor on the target eukaryotic cell.

[0039] In a further aspect, the present invention advances the delivery of therapeutic nucleic acids, proteins, antibodies, antibody derivatives, polypeptides, gene editing systems (CRISPR and other gene editing nucleases), and eukaryotically translatable mRNAs using E. coli interkingdom delivery vehicles by enabling precise targeting of bacteria to target eukaryotic cells expressing specific surface proteins or chemical moieties. Delivery of therapeutic modalities is discussed, for example, in U.S. Patent No. 11,312,954 B2 to Linke et al. and U.S. Patent Publication No. 2022 / 0364122 A1 to Linke et al., the contents of which are incorporated by reference. Interkingdom bacterial delivery vehicles must target and enter specific cell types for intracellular cargo transport, but targeting and entry are not trivial or passive processes, especially if the target cells do not naturally ingest the bacteria, for example, via phagocytosis.

[0040] Bacteria use various invasion factors to invade non-phagocytic cells, as exemplified by Yersinia pseudotuberculosis (Mikula et al., 2012). These bacteria rely on invasin proteins (Invs), surface-displayed invasion factor proteins that bind to β1 integrins on the surface of target eukaryotic cells. After binding, intrinsic properties of Invs stimulate the uptake of bacteria by another non-phagocytic eukaryotic cell. This uptake process depends on three specific properties of Invs: 1) transport of Invs to the bacterial surface, 2) binding of Invs to β1 integrins on the cell surface, and 3) stimulation of bacterial uptake.

[0041] The Y. pseudotuberculosis Inv protein is a multidomain protein that contains five independently folded domains, D1, D2, D3, D4, and D5. The primary accession number of the Inv protein is UNIPROT P11922 and the inv gene is YPTB1668 (Isberg et al., 1987, Leong et al., 1990, Chain et al., 2004) (the full sequence is shown in Table 5 below). The key entry functions of Inv described above are compartmentalized into these various domains. D1, D2, and D3 are involved in Inv transport to the bacterial surface and stimulation of cellular uptake, while D4 and D5 are required for β1 integrin binding (Figures 7 and 8) (Dersch and Iseberg, 2000). Inv is an autotransporter protein (Leo et al., 2014), meaning that its transport to the bacterial cell surface is an intrinsic property of the protein, i.e., it does not require any separate transport mechanism. Thus, by separating the domains of Inv, its functions of transport, stimulation of uptake, and targeting are separated and can be exploited independently for targeting and entry of interkingdom delivery vehicles.

[0042] The present invention describes a method to maintain the transport and uptake functions of Inv while changing its targeting from β1 integrin to other proteins (i.e., cell surface proteins) or chemical moieties (i.e., cell surface chemical moieties) expressed on the surface of target eukaryotic cells by replacing D4 and D5 of Inv with binding domains from heterologous proteins or synthetic (i.e., non-natural) binding domains or fusing full-length Inv to binding domains from heterologous proteins or synthetic (i.e., non-natural) binding domains via genetic engineering. Heterologous proteins can be derived from bacterial, fungal, animal, or viral genomes. Alternatively, BDs can include synthetic proteins (i.e., proteins that do not occur in nature). The source of synthetic BDs can generally be experimental procedures based on biochemical techniques or computational discovery (e.g., via computer modeling or artificial intelligence). Synthetic BDs can be single domain antibodies, nanobodies, or any other ligands that bind to moieties on the surface of target cells. This manipulation results in the construction of chimeric Inv proteins in which D1-D3 (i.e., non-binding domains) are fused in-frame to alternative heterologous binding domains, or in which Inv D1-D5 (i.e., full-length Inv) are fused in-frame to heterologous binding domains. The alternative binding domains interact with different cell surface proteins or chemical moieties other than the Inv's native binding domain, which in some cases may be referred to as receptors on the surface of eukaryotic cells, thereby allowing specific targeting to cells independent of the Inv's native β1 integrin binding. In some cases, the binding domain of the heterologous protein may be referred to as a ligand binding domain. Examples of bacterial heterologous proteins and their binding partners (proteins or chemicals) are shown in Table 1. Examples of fungal heterologous proteins and their binding partners (proteins or chemicals) are shown in Table 2. Examples of viral heterologous proteins and their binding partners (proteins or chemicals) are shown in Table 3.Examples of animal heterologous proteins that contain binding domains include glycan-binding proteins and cell adhesion proteins (e.g., GalNAc-binding proteins, lectins, the group of cell adhesion molecules (CAMs), the group of sulfated glycosaminoglycans (GAG)-binding proteins, selectins, integrins, laminins, cadherins, fibronectins, collagens, thrombospondin, vitronectin, tenascin, apolipoproteins B, E, and A-V, lipoprotein lipase, hepatic lipase, siglecs, galectins, immunoglobulins, and annexins, among others).

[0043] Entry agents (e.g., SARS-CoV2 viruses) interact with and enter their target cells through a multi-step process in which they first bind to a receptor on the target cell surface via a specific binding moiety, followed by proteolytic processing of the binding moiety to enable and enhance entry. This proteolytic processing occurs when proteases or peptidases cleave the protein at a specific cognate cleavage site (e.g., the SARS-CoV2 spike protein must be cleaved at a furin cleavage site). This strategy may help optimize ligand binding and entry functions [see, e.g., Jackson, CB, Farzan, M., Chen, B. et al. "Mechanisms of SARS-CoV-2 entry into cells". Nat Rev Mol Cell Biol 23, 3-20 (2022); Pager CT, Dutch RE. "Cathepsin L is involved in proteolytic processing of the Hendra virus fusion protein". J Virol. 2005 Oct;79(20):12714-20. doi:10.1128 / JVI.79.20.12714-12720.2005. PMID:16188974; PMCID:PMC1235853; Carruthers See also VB, Blackman MJ. "A new release on life: emerging concepts in proteolysis and parasite invasion." Mol Microbiol. 2005 Mar;55(6):1617-30. doi:10.1111 / j.1365-2958.2005.04483.x. PMID:15752188. Through genetic engineering, a similar paradigm can be applied to the bacterial delivery platform described herein.To construct such a bifunctional entry system, peptidase or protease recognition and cleavage sites (including but not limited to those of the proteins shown in Table 4 below) are placed in frame between the Inv sequence and the heterologous binding domain sequence (see Figure 1). The bacterial delivery vehicle then enters the target cell via a three-step process (Figure 2): (1) targeting: the heterologous binding domain recognizes and binds to a receptor on the target cell surface, thereby targeting the bacterial vehicle to a specific cell type. (2) translocation: the heterologous binding domain is cleaved from the chimeric Inv protein by a specific peptidase or protease found on the target cell surface, activating the entry function of the Inv protein. (3) entry: the activated Inv protein binds to β1 integrin on the target cell surface, facilitating entry of the target cell.

[0044] The function of multidomain proteins such as Invs requires specific topological interactions between their own domains or with other binding partners (e.g., proteins or chemical moieties). One important feature of a protein that can affect these topological interactions is the spacing between its internal domains, which is determined by specific amino acid sequences (i.e., linker peptides) (Chen et al., 2012). In the case of chimeric Invs, this is the spacing between the non-binding and binding domains. Therefore, when engineering a chimeric Inv protein, it is advantageous to modify the amino acid sequence of the linker peptide between the domains to modulate these interactions in order to optimize the binding of the chimeric Inv protein to its binding partner on the surface of a eukaryotic cell. This modification may be done by changing the amino acid sequence of the linker peptide between the domains (i.e., peptide linker) to modulate flexibility and spacing. Examples of peptide linker amino acid sequences that may be useful include: [SEQ ID NO:2] EAAAREAAAR, [SEQ ID NO:3] EAAAREAAAREAAAREAAAR, [SEQ ID NO:4] GSGSGS, [SEQ ID NO:5] GSGSGSGSGS, [SEQ ID NO:6] GGGS, [SEQ ID NO:7] GGGGS, [SEQ ID NO:8] GGGSGGGGSGGGS, [SEQ ID NO:9] GGSG, [SEQ ID NO:10] GGSGGGSG, [SEQ ID NO:11] GGSGGGSGGGSG, [SEQ ID NO:12] GSGGS, [SEQ ID NO:13] GSSGS, [SEQ ID NO:14] ACGSLSCGSF, [SEQ ID NO:15] GENLYFQSGG, [SEQ ID NO:16] SACYCELS, [SEQ ID NO:17] RPACKIPNDLKQKVMNH, [SEQ ID NO:18] PPPYQPLGGGGS, [SEQ ID NO:19] WRKRLRKKRLRKKRRLKKRRRKKQRRKRR, LEGSGQGPGSGQGSGSPGSGQG and [SEQ ID NO:20] GS. It is contemplated that one can take a non-binding domain from an Inv (e.g., D1, D2 and D3) or a full-length Inv, add a linker sequence such as those described immediately above, and then add a binding domain from one of the proteins listed in Tables 1, 2 or 3 or a synthetic binding protein / binding domain.Thus, sequences such as the sequence provided in FIG. 1 from about amino acid 1 to about amino acid 795, or from about amino acid 1 to about amino acid 986, or sequences that are 95% or 90% identical thereto, can be used.

[0045] The present invention advances the delivery of nucleic acids, proteins, antibodies, antibody derivatives, polypeptides, gene editing systems, and eukaryotically translatable mRNAs by providing a bacterial delivery platform, more precisely, the delivery of nucleic acids, proteins, antibodies, antibody derivatives, polypeptides, gene editing systems, and eukaryotically translatable mRNAs, that can be further tailored to target specific cell surface proteins and cell surface chemical moieties. The described transport and uptake domains of Inv fused (or linked) to binding domains of heterologous or synthetic proteins can be encoded in bacterial cells via genomic or plasmid expression. Similarly, it is often advantageous to express nucleic acid coding sequences from bacterial chromosomes rather than from plasmids for multiple reasons, including low metabolic burden on the host cell, stable expression levels, genetic stability, and no need for selective drugs (Ou et al., 2018).

[0046] It is also contemplated that the described transport and uptake domains of Invs fused with binding domains of heterologous proteins (chimeric Invs) or full-length Invs can be expressed, attached, or conjugated to other biological and synthetic surfaces, including beads, viruses, exosomes, rigid substrates (e.g., for the manufacture of lateral flow strips), paper-based biosensors, plastic substrates (e.g., for the manufacture of plastic-based biosensors), graphene-based substrates, or nanomaterials (e.g., lipid nanoparticles, metal nanoparticles, mesoporous silica nanoparticles, nanowires, ITO, organic polymers).

[0047] Exosomes, liposomes and other lipid vesicles have been used as nucleic acid delivery platforms carrying RNA payloads for delivery to distant tissues. Delivery vehicles such as liposomes have drawbacks including leakage of vesicle contents, batch-to-batch variability, high production costs, and limited targeting capabilities. This interkingdom delivery system is based on the use of non-pathogenic bacteria-mediated RNAi delivery vehicles that use receptor-mediated phagocytosis for specific intracellular delivery at the tissue site of action, resulting in accumulation of shRNA in endosomes and efficient release of the shRNA payload into the cytoplasm of target cells for RNAi silencing. These interkingdom vehicles were Escherichia coli (E. coli), engineered to specifically target mucosal epithelial tissues and deliver a payload of constitutively produced shRNA in a sequence-independent manner.

[0048] Example. Bacterial vehicle entry of HER2-positive cancer cells via chimeric Inv-targeted ligands (two-factor entry paradigm).

[0049] Successful entry of HER2-positive cancer cells by a chimeric Inv-targeting ligand comprising D1-D5 of Inv, a linker containing a furin cleavage site, and a nanobody specific for HER2, a cell surface-displayed protein on target cells, was demonstrated via entry assays and laser scanning confocal microscopy.

[0050] A plasmid ("pSi_1fHER2-scr.c") encoding a chimeric Inv protein containing D1-D5 of Inv linked to a synthetic (i.e., non-natural) nanobody via an in-frame Furin protease cleavage site was constructed via molecular cloning, as in Figure 4. In this example, the linker is a compound linker, i.e., a common linker sequence with a fused Furin protease cleavage site. The nanobody specifically binds to HER2, a receptor expressed on the surface of eukaryotic target cells. Bacterial transcription of the chimeric Inv protein is constitutive under the control of a modified lacUV5 promoter, and transcription is terminated via a standard bacterial transcription terminator. Following transcription-translation of the chimeric Inv protein by bacteria, the protein is translocated to the bacterial surface via the autotransport activity of domains D1-D3 of Inv. pSi_1fHER2-scr.c was transformed into E. coli (FEC19), which is not invasive in the absence of the chimeric Inv D1-D5 contained on the pSi_1fHER2-scr.c plasmid. Transformed FEC19 were plated onto brain heart infusion (BHI) agar containing the appropriate antibiotic for selection. Cultures for invasion validation in this study were prepared from each of two isolated colonies of each strain and incubated at 37°C in BHI medium with the appropriate antibiotic to late logarithmic phase (OD 600 The α-Kα ratio was increased to 0.8–1.0.

[0051] A standard invasion assay was also used to demonstrate bacterial invasion of human alveolar basal epithelial cells (A549 cells), which are positive for both the HER2 receptor and Furin.

[0052] Cells were then isolated and transferred to slides for imaging by laser scanning confocal microscopy. Cells were fixed in 10% NBF and mounted under coverslips with Fluoromount-G mounting medium containing DAPI. Slides were imaged with a Zeiss LSM510 Meta microscope, and images were collected at 40x magnification with an excitation wavelength of 488nm.

[0053] The micrograph in Figure 3 shows successful entry of FEC19 / pSi_1fHER2-scr.c into A549 cells, thus demonstrating the functionality of this chimeric entry targeting system. The FEC19 bacteria are visualized as small bacillus-type grey masses near the larger grey mass, which is the nucleus of the eukaryotic cell.

[0054] In addition to natural binding domains, proteins and polypeptides with synthetic (i.e., non-natural) binding domains (e.g., single domain antibodies or nanobodies) can be found using a variety of computational and biochemical approaches. The use of synthetic binding domains designed to target specific surface-displayed binding ligands on the target cell surface offers further opportunities to target specific cell types. For example, as shown in the Examples, a synthetic nanobody that binds to the human protein HER2, found on the surface of many cancer cells, was fused in frame to a full-length Inv sequence (D1-D5) to form a chimeric Inv protein that enters only HER2-positive cells (i.e., cells that have HER2 on their surface); in this Example, A549 cells were used.

[0055] Claim Glossary As used herein, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

[0056] With respect to the compounds of the present invention, the term "administration" and variations thereof (e.g., "administering" a compound) refers to the introduction of the compound into the body of a subject in need of treatment. When the compounds of the present invention are provided in combination with one or more other active agents (e.g., an AIV vaccine, etc.), "administration" and variations thereof are understood to include simultaneous and sequential introduction of the compound or other agents, respectively.

[0057] As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product that results directly or indirectly from combining the specified ingredients in the specified amounts.

[0058] As used herein, the term "therapeutically effective amount" refers to an amount of an active compound or pharmaceutical agent that elicits a biological or medical response in a tissue, system, animal, or human as desired by a researcher, veterinarian, physician, or other clinician. With reference to a viral infection, an effective amount includes an amount sufficient to prevent morbidity or reduce the severity of disease, as evidenced by clinical disease, clinical symptoms, viral titer, or viral shedding from a subject, or by the ability to prevent or reduce transmission between animals. In some embodiments, an effective amount is an amount sufficient to delay the onset of clinical disease and / or symptoms, or to prevent disease. In some embodiments, an effective amount is an amount sufficient to reduce viral titer and / or reduce viral shedding. An effective amount may be administered in one or more doses.

[0059] As used herein, "treatment" refers to obtaining beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviation of one or more symptoms, reduction in the extent of viral infection, stabilization (i.e., not worsening) of the viral infection state, prevention or slowing of spread (e.g., shedding) of viral infection, prevention, slowing or slowing of progression of viral infection, and / or weight maintenance / weight gain. The methods of the present invention contemplate any one or more of these therapeutic aspects.

[0060] A "pharmaceutical acceptable" ingredient is one that is suitable for use in humans and / or animals without undue adverse side effects (such as toxicity, irritation, and allergic reactions) commensurate with a reasonable benefit / risk ratio.

[0061] A "safe and effective amount" refers to the amount of a component, when used in the methods of the invention, sufficient to obtain a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response), commensurate with a reasonable benefit / risk ratio.

[0062] As used throughout this application, the terms "a" and "an" are used in their intended meaning to mean "at least one," "at least a first," "one or more," or "a plurality" of the referenced components or steps, unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0063] As used herein, the term "and / or" is inclusive of the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0064] As used herein, the term "comprising" is intended to mean that the products, compositions, and methods include the components or steps mentioned, but do not exclude others. When used to define products, compositions, and methods, "consisting essentially of" means excluding any other components or steps of substantial importance. Thus, a composition consisting essentially of the recited components does not exclude trace amounts of contaminants and pharma- ceutically acceptable carriers. "Consisting of" means excluding more than trace amounts of other components or steps.

[0065] As used herein, the term "invasive" when referring to a microorganism, e.g., a bacterium or bacterial therapeutic particle (BTP), refers to a microorganism that is capable of delivering at least one molecule, e.g., an RNA or an RNA-encoded DNA molecule, to a target cell. An invasive microorganism may be a microorganism that is capable of crossing a cell membrane, thereby entering the cytoplasm of said cell, and delivering at least a portion of its contents, e.g., an RNA or an RNA-encoded DNA, into the target cell. The process of delivery of at least one molecule to the target cell preferably does not significantly alter the invasion apparatus.

[0066] As used herein, the term "inter-kingdom" refers to a delivery system that uses bacteria (or another invasive microorganism) to generate nucleic acids, proteins, antibodies, antibody derivatives, polypeptides, gene editing systems (CRISPR and other gene editing nucleases), eukaryotic translatable mRNA, or combinations thereof, and deliver the nucleic acids, proteins, antibodies, antibody derivatives, polypeptides, gene editing systems (CRISPR and other gene editing nucleases), eukaryotic translatable mRNA, or combinations thereof intracellularly (i.e., across kingdoms: from prokaryotes to eukaryotes, or across phyla: from invertebrates to vertebrates) in a target tissue for processing without integration into the host genome.

[0067] Invasive microorganisms include microorganisms that can naturally deliver at least one molecule to a target cell, such as by crossing a cell membrane, e.g., a eukaryotic cell membrane, and entering the cytoplasm, as well as microorganisms that are not naturally invasive and have been modified, e.g., genetically modified, to be invasive. In another preferred embodiment, a microorganism that is not naturally invasive can be modified to be invasive by linking the bacterium or BTP to an "invasion factor," also called an entry factor or "cytoplasmic targeting factor." As used herein, an "invasion factor" is a factor, e.g., a protein or group of proteins, that, when expressed by a non-invasive bacterium or BTP, renders the bacterium or BTP invasive. As used herein, an "invasion factor" is encoded by a "cytoplasmic targeting gene." Invasive microorganisms are generally described in the art, for example, in U.S. Patent Application Publication Nos. 20100189691 A1 and 20100092438 A1, and Xiang, S. et al., Nature Biotechnology 24, 697-702 (2006), each of which is incorporated by reference in its entirety for all purposes.

[0068] In a preferred embodiment, the invasive microorganism is Escherichia coli, as taught in the examples of this application. However, it is contemplated that additional microorganisms can potentially be adapted to function as interkingdom delivery vehicles for the delivery of NA. These non-toxic, invasive bacteria and BTPs exhibit or are modified to exhibit invasive properties and can invade host cells via various mechanisms. In contrast to the uptake of bacteria or BTPs by specialized phagocytes, which usually results in the destruction of bacteria or BTPs in specialized lysosomes, invasive bacteria or BTP strains have the ability to invade non-phagocytic host cells. Naturally occurring examples of such intracellular bacteria include Yersinia, Rickettsia, Legionella, Brucella, Mycobacterium, Helicobacter, Coxiella, Chlamydia, Neisseria, Brucella, Bordetella, Borrelia, Listeria, Shigella, Salmonella, Staphylococcus, Streptococcus, Porphyromonas, Treponema, and Vibrio, but this property can also be transferred to other bacteria or BTPs such as E. coli, Lactobacillus, Lactococcus, or Bifidobacterium, including probiotics, via the introduction of invasion-related genes (P. Courvalin, S. Goussard, C. Grillot-Courvalin, CRAcad. Sci. Paris 318, 1207 (1995)). Factors to consider or address when evaluating additional bacterial species as candidates for use as inter-kingdom NA delivery vehicles include the candidate's pathogenicity, or lack thereof, the tropism of the candidate bacterium for target cells or the extent to which the bacterium can be engineered to deliver NAs inside target cells, and any synergistic value the candidate bacterium may provide by triggering the host's innate immunity.

[0069] Nucleic acids are defined as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or any closely related compound. They may be coding or non-coding, synthetic or naturally occurring, single or double stranded segments, and often consist of many (two or more) molecules of nucleotides linked together. Examples include, but are not limited to, small interfering RNA / small hairpin RNA (siRNA / shRNA), microRNA (miRNA), antagomiR, RNA or DNA aptamers, messenger RNA (mRNA), splice switching oligonucleotides, antisense oligonucleotides, antigene oligonucleotides, DNAzymes, RNA decoys, ribozymes, peptide nucleic acids, oligomers, and defective interfering particles.

[0070] Therapeutic nucleic acids are NAs as described herein or closely related compounds used to treat or study diseases, or to achieve desired genetic modifications, or for gene transfer purposes. They are used in cases where specific inhibition or interruption or alteration of the function of a particular gene or other molecule involved in a disease is considered therapeutically desirable.

[0071] A synthetic binding protein is an artificial protein tailored to bind to a target molecule of interest. A synthetic binding domain is a binding domain of a synthetic binding protein. Synthetic binding proteins (SBPs) are smaller, more stable, less immunogenic, and have better tissue penetration than typical non-synthetic alternatives. SBPs include affibodies, anticalins, DARPins, i-bodies, monobodies / adnectins, nanobodies, repebodies, scFabs, scFvs, and vNARs. It is contemplated that SBPs and / or their binding domains, including those mentioned above, can be utilized in chimeric basin polypeptides. SBPs are reported in Sha F, Salzman G, Gupta A, Koide S. "Monobodies and other synthetic binding proteins for expanding protein science." Protein Sci. 2017 May;26(5):910-924. doi:10.1002 / pro.3148. Epub 2017 Mar 24. PMID:28249355; PMCID:PMC5405424 and Xiaona Wang, Fengcheng Li, Wenqi Qiu, Binbin Xu, Yanlin Li, Xichen Lian, Hongyan Yu, Zhao Zhang, Jianxin Wang, Zhaorong Li, Weiwei Xue, Feng Zhu, SYNBIP: "synthetic binding proteins for research, diagnosis and therapy", Nucleic Acids Discussed in Research, Volume 50, Issue d1, 7 January 2022, Pages D560-d570, https: / / doi.org / 10.1093 / nar / gkab926.

[0072] Nanobodies, also known as single domain antibodies (sdAbs), are antibody fragments that consist of a single monomeric variable antibody domain.

[0073] Affibody molecules are small, robust proteins engineered to bind with high affinity to multiple target proteins or peptides, mimicking monoclonal antibodies and therefore being members of the family of antibody mimetics. These molecules can be used for molecular recognition in diagnostic and therapeutic applications.

[0074] DARPins (designed ankyrin repeat proteins) are engineered antibody-mimetic proteins that typically exhibit highly specific and high affinity target protein binding. They are essentially derived from natural ankyrin repeat proteins, one of the most common classes of binding proteins, which are responsible for diverse functions such as cell signaling, regulation and structural integrity. DARPins consist of at least three repeat motifs or modules, of which the most N-terminal and most C-terminal modules are called "caps" because they shield the hydrophobic core of the protein.

[0075] Anticalin proteins are artificial proteins that can bind to antigens, either proteins or small molecules. They are not structurally related to antibodies, which makes them a kind of antibody mimic. Instead, they are derived from human lipocalins, a family of natural binding proteins. Anticalin proteins are used instead of monoclonal antibodies, but are about eight times smaller, with a size of about 180 amino acids and a mass of about 20 kDa.

[0076] As used herein, a disease is prevented when (1) the pharmaceutical composition is administered internally (by ingestion, inhalation, injection, etc.), topically (on the skin for absorption by the body), or otherwise to a subject, and (2) the pharmaceutical composition prevents the subject from acquiring and experiencing symptoms / clinical disease normally associated with the disease, or when the subject acquires and experiences, with varying degrees of severity, some or all of the symptoms / clinical disease normally associated with the disease, prior to or after exposure to the disease, and the subject is restored from the disease to a normal state of health.

[0077] Kits for carrying out the methods of the invention are further provided. A "kit" is intended to be any article of manufacture (e.g., package or container) that contains at least one reagent, such as a pH buffer of the invention. The kit may be advertised, distributed, or sold as a unit for carrying out the methods of the invention. Additionally, the kit may include a package insert that describes the kit and methods for its use. Any or all of the kit reagents may be provided in a container that protects them from the external environment, such as a sealed container or pouch.

[0078] In an advantageous embodiment, the kit container may further include a medicamentously acceptable carrier. The kit may further include a sterile diluent, which is preferably stored in a separate additional container. In another embodiment, the kit further includes a package insert including printed instructions directing the use of a combination treatment of a pH buffer and an anti-pathogen agent as a method for treating and / or preventing disease in a subject. The kit may also include additional containers containing additional anti-pathogen agents (e.g., amantadine, rimantadine, and oseltamivir), agents that enhance the effects of such agents, or other compounds that improve the efficacy or tolerability of the treatment. The kit may also include at least one reagent used to perform certain conventional techniques (i.e., nucleic acid extraction) that are within the skill of the art.

[0079] Sequence identity / similarity: The identity / similarity between two or more nucleic acid sequences, or between two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are.

[0080] Methods for alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, Comput. Appl. Biosci. 5:151-3, 1989; Corpet et al., Nucl. Acids. Res. 16:10881-90, 1988; Huang et al. Comput. Appl. Biosci. 8, 155-65, 1992, and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990 presents a detailed discussion of sequence alignment methods and homology calculations.

[0081] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894), and on the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. blastn is used to compare nucleic acid sequences, while blastp is used to compare amino acid sequences. Additional information can be found at the NCBI website.

[0082] Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. Percent sequence identity is determined by dividing the number of matches by either the length of the sequence set forth in the identified sequence, or by the combined length (such as 100 contiguous nucleotides or amino acid residues from the sequence set forth in the identified sequence), and then multiplying the resulting value by 100.

[0083] The practice of the present invention can employ conventional techniques and explanations of organic chemistry, polymer technology, molecular biology (including recombinant technology), cell biology, biochemistry, and immunology that are within the scope of the art, unless otherwise indicated. Such conventional techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using labels. Specific illustrations of suitable techniques can be obtained by referring to the examples herein above. However, of course, other equivalent conventional procedures can also be used. Such prior techniques and descriptions can be found in Genome Analysis: A Laboratory Manual Series (Volumes I-IV), Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primers: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press), Stryer, L. (1995) Biochemistry (4th ed.) Freeman, NY, Gait, Oligonucleotide Synthesis: A Practical Approach 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rd ed., WH Freeman Pub., New York, NY, and Berg et al. (2002) Biochemistry, 5th ed., WH Freeman Pub., New York, NY. Methods for the preparation of such compositions can be found in standard laboratory manuals, such as those in US Pat. No. 6,393,311, published by Johns Hopkins, New York, NY, all of which are incorporated herein by reference in their entireties for all purposes.

[0084] All references cited in this application are incorporated herein by reference in their entirety to the extent not inconsistent herewith.

[0085] Since the advantages set forth above, and those made apparent from the foregoing description, are efficiently attained, and certain changes may be made in the above construction without departing from the scope of the invention, it will be understood that it is intended that all matter contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

[0086] It is also to be understood that the following claims are intended to encompass all of the generic and specific features of the invention described herein, as well as all statements of the scope of the invention that may, as a matter of language, be said to fall within these scopes.

[0087] [Table 1]

[0088] [Table 2] TIFF2025514169000004.tif56159

[0089] [Table 3]

[0090] [Table 4] TIFF2025514169000007.tif225159

[0091] Invasin amino acid (1-986) [SEQ ID NO: 1] D1 = AA 1-595 D2 = AA 596-694 D3 = AA 695-794 D4 / D5 = 795-986 10 20 30 40 50 MMVFQPISEF LLIRNAGMSM YFNKIISFNI ISRIVICIFL ICGMFAMAGAS 60 70 80 90 100 EKYDANAPQQ VQPYSVSSSA FENLHPNNEM ESSINPFSAS DTERNAAIID 110 120 130 140 150 RANKEQETEA VNKMISTGAR LAASGRASDV AHSMVGDAVN QEIKQWLNRF 160 170 180 190 200 GTAQVNLNFD KNFSLKESSL DWLAPWYDSA SFLFFSQLGI RNKDSRNTLN 210 220 230 240 250 LGVGIRTLEN GWLYGLNTFY DNDLTGHNHR IGLGAEAWTD YLQLAANGYF 260 270 280 290 300 RLNGWHSSRD FSDYKERPAT GGDLRANAYL PALPQLGGKL MYEQYTGERV 310 320 330 340 350 ALFGKDNLQR NPYAVTAGIN YTPVPLLTVG VDQRMGKSSK HETQWNLQMN 360 370 380 390 400 YRLGESFQSQ LSPSAVAGTR LLAESRYNLV DRNNNIVLEY QKQQVVKLTL 410 420 430 440 450 SPATISGLPG QVYQVNAQVQ GASAVREIVW SDAELIAAGG TLTPLSTTQF 460 470 480 490 500 NLVLPPYKRT AQVSRVTDDL TANFYSLSAL AVDHQGNRSN SFTLSVTVQQ 510 520 530 540 550 PQLTLTAAVI GDGAPANGKT AITVEFTVAD FEGKPLAGQE VVITTNNGAL 560 570 580 590 600 PNKITEKTDA NGVARIALTN TTDGVTVVTA EVEGQRQSVD THFVKGTIAA 610 620 630 640 650 DKSTLAAVPT SIIADGLMAS TITLELKDTY GDPQAGANVA FDTTLGNMGV 660 670 680 690 700 ITDHNDGTYS APLTSTTLGV ATVTVKVDGA AFSVPSVTVN FTADPIPDAG 710 720 730 740 750 RSSFTVSTPD ILADGTMSST LSFVPVDKNG HFISGMQGLS FTQNGVPVSI 760 770 780 790 800 SPITEQPDSY TATVVGNSVG DVTITPQVDT LILSTLQKKI SLFPVPTLTG 810 820 830 840 850 ILVNGQNFAT DKGFPKTIFK NATFQLQMDN DVANNTQYEW SSSFTPNVSV 860 870 880 890 900 NDQGQVTITY QTYSEVAVTA KSKKFPSYSV SYRFYPNRWI YDGGRSLVSS 910 920 930 940 950 LEASRQCQGS DMSAVLESSR ATNGTRAPDG TLWGEWGSLT AYSSDWQSGE 960 970 980 986 YWVKKTSTDF ETMNMDTGAL QPGPAYLAFP LCALSI

[0092] Amino acid sequence of Inv from Yersinia pseudotuberculosis [SEQ ID NO:39] D1 = AA 1-595 D2 = AA 596-694 D3 = AA 695-794 D4 / D5 = 795-985 10 20 30 40 50 MVFQPISEFL LIRNAGMSMY FNKIISFNII SRIVICIFLI CGMFMAGASE 60 70 80 90 100 KYDANAPQQV QPYSVSSSAF ENLHPNNEME SSINPFSASD TERNAAIIDR 110 120 130 140 150 ANKEQETEAV NKMISTGARL AASGRASDVA HSMVGDAVNQ EIKQWLNRFG 160 170 180 190 200 TAQVNLNFDK NFSLKESSLD WLAPWYDSAS FLFFSQLGIR NKDSRNTLNL 210 220 230 240 250 GVGIRTLENG WLYGLNTFYD NDLTGHNHRI GLGAEAWTDY LQLAANGYFR 260 270 280 290 300 LNGWHSSRDF SDYKERPATG GDLRANAYLP ALPQLGGKLM YEQYTGERVA 310 320 330 340 350 LFGKDNLQRN PYAVTAGINY TPVPLLTVGV DQRMGKSSKH ETQWNLQMNY 360 370 380 390 400 RLGESFQSQL SPSAVAGTRL LAESRYNLVD RNNIVLEYQ KQQVVKLTLS 410 420 430 440 450 PATISGLPGQ VYQVNAQVQG ASAVREIVWS DAELIAAGGT LTPLSTTQFN 460 470 480 490 500 LVLPPYKRTA QVSRVTDDLT ANFYSLSALA VDHQGNRSNS FTLSVTVQQP 510 520 530 540 550 QLTLTAAVIG DGAPANGKTA ITVEFTVADF EGKPLAGQEV VITTNNGALP 560 570 580 590 600 NKITEKTDAN GVARIALTNT TDGVTVVTAE VEGQRQSVDT HFVKGTIAAD 610 620 630 640 650 KSTLAAVPTS IIADGLMAST ITLELKDTYG DPQAGANVAF DTTLGNMGVI 660 670 680 690 700 TDHNDGTYSA PLTSTTLGVA TVTVKVDGAA FSVPSVTVNF TADPIPDAGR 710 720 730 740 750 SSFTVSTPDI LADGTMSSTL SFVPVDKNGH FISGMQGLSF TQNGVPVSIS 760 770 780 790 800 PITEQPDSYT ATVVGNTAGD VTITPQVDTL ILSTLQKKIS LFPVPTLTGI 810 820 830 840 850 LVNGQNFATD KGFPKTIFKN ATFQLQMDND VANNTQYEWS SSFTPNVSVN 860 870 880 890 900 DQGQVTITYQ TYSEVAVTAK SKKFPSYSVS YRFYPNRWIY DGGTSLVSSL 910 920 930 940 950 EASRQCQGSD MSAVLESSRA TNGTRAPDGT LWGEWGSLTA YSSDWQSGEY 960 970 980 985 WVKKTSTDFE TMNMDTGALV QGPAYLAFPL CALAI

[0093]

Table 5

[0094] Invasion D1 - D3 nucleic acid sequence [SEQ ID NO: 38] ATGATGGTTT TCCAGCCAAT CAGTGAGTTT CTCTTGATAA GGAATGCGGG 50 AATGTCTATG TATTTTAATA AAATAATTTC ATTTAATATT ATTTCACGAA 100 TAGTTATTTG TATCTTTTTG ATATGTGGAA TGTTCATGGC TGGGGCTTCA 150 GAAAAATATG ATGCTAACGC ACCGCAACAG GTCCAGCCTT ATTCTGTCTC 200 TTCATCTGCA TTTGAAAATC TCCATCCTAA TAATGAAATG GAGAGTTCAA 250 TCAATCCCTT TTCCGCATCG GATACAGAAA GAAATGCTGC AATAATAGAT 300 CGCGCCAATA AGGAGCAGGA GACTGAAGCG GTGAATAAGA TGATAAGCAC 350 CGGGGCCAGG TTAGCTGCAT CAGGCAGGGC ATCTGATGTT GCTCACTCAA 400 TGGTGGGCGA TGCGGTTAAT CAAGAAATCA AACAGTGGTT AAATCGATTC 450 GGTACGGCTC AAGTTAATCT GAATTTTGAC AAAAATTTTT CGCTAAAAGA 500 AAGCTCTCTT GATTGGCTGG CTCCTTGGTA TGACTCTGCT TCATTCCTCT 550 TTTTTAGTCA GTTAGGTATT CGCAATAAAG ACAGCCGCAA CACACTTAAC 600 CTTGGCGTCG GGATACGTAC ATTGGAGAAC GGTTGGCTGT ACGGACTTAA 650 TACTTTTTAT GATAATGATT TGACCGGCCA CAACCACCGT ATCGGTCTTG 700 GTGCCGAGGC CTGGACCGAT TATTTACAGT TGGCTGCCAA TGGGTATTTT 750 CGCCTCAATG GATGGCACTC GTCGCGTGAT TTCTCCGACT ATAAAGAGCG 800 CCCAGCCACT GGGGGGGATT TGCGCGCGAA TGCTTATTTA CCTGCACTCC 850 CACAACTGGG GGGGAAGTTG ATGTATGAGC AATACACCGG TGAGCGTGTT 900 GCTTTATTTG GTAAAGATAA TCTGCAACGC AACCCTTATG CCGTGACTGC 950 CGGGATCAAT TACACCCCCG TGCCTCTACT CACTGTCGGG GTAGATCAGC 1000 GTATGGGGAA AAGCAGTAAG CATGAAACAC AGTGGAACCT CCAAATGAAC 1050 TATCGCCTGG GCGAGAGTTT TCAGTCGCAA CTTAGCCCTT CAGCGGTGGC 1100 AGGAACACGT CTACTGGCGG AGAGCCGCTA TAACCTTGTC GATCGTAACA 1150 ATAATATCGT GTTGGAGTAT CAGAAACAGC AGGTGGTTAA ACTGACATTA 1200 TCGCCAGCAA CTATCTCCGG CCTGCCGGGT CAGGTTTATC AGGTGAACGC 1250 ACAAGTACAA GGGGCATCTG CTGTAAGGGA AATTGTCTGG AGTGATGCCG 1300 AACTGATTGC CGCTGGCGGC ACATTAACAC CACTGAGTAC CACACAATTC 1350 AACTTGGTTT TACCGCCTTA TAAACGCACA GCACAAGTGA GTCGGGTAAC 1400 GGACGACCTG ACAGCCAACT TTTATTCGCT TAGTGCGCTC GCGGTTGATC 1450 ACCAAGGAAA CCGATCTAAC TCATTCACAT TGAGCGTCAC CGTTCAGCAG 1500 CCTCAGTTGA CATTAACGGC GGCCGTCATT GGTGATGGCG CACCGGCTAA 1550 TGGGAAAACT GCAATCACCG TTGAGTTCAC CGTTGCTGAT TTTGAGGGGA 1600 AACCCTTAGC CGGGCAGGAG GTGGTGATAA CCACCAATAA TGGTGCGCTA 1650 CCGAATAAAA TCACGGAAAA GACAGATGCA AATGGCGTCG CGCGCATTGC 1700 ATTAACCAAT ACGACAGATG GCGTGACGGT AGTCACAGCA GAAGTGGAGG 1750 GGCAACGGCA AAGTGTTGAT ACCCACTTTG TTAAGGGTAC TATCGCGGCG 1800 GATAAATCCA CTCTGGCTGC GGTACCGACA TCTATCATCG CTGATGGTCT 1850 AATGGCTTCA ACCATCACGT TGGAGTTGAA GGATACCTAT GGGGACCCGC 1900 AGGCTGGCGC GAATGTGGCT TTTGACACAA CCTTAGGCAA TATGGGCGTT 1950 ATCACGGATC ACAATGACGG CACTTATAGC GCACCATTGA CCAGTACCAC 2000 GTTGGGGGTA GCAACAGTAA CGGTGAAAGT GGATGGGGCT GCGTTCAGTG 2050 TGCCGAGTGT GACGGTTAAT TTCACGGCAG ATCCTATTCC AGATGCTGGC 2100 CGCTCCAGTT TCACCGTCTC CACACCGGAT ATCTTGGCTG ATGGCACGAT 2150 GAGTTCCACA TTATCCTTTG TCCCTGTCGA TAAGAATGGC CATTTTATCA 2200 GTGGGATGCA GGGCTTGAGT TTTACTCAAA ACGGTGTGCC GGTGAGTATT 2250 AGCCCCATTA CCGAGCAGCC AGATAGCTAT ACCGCGACGG TGGTTGGGAA 2300 TAGTGTCGGT GATGTCACAA TCACGCCGCA GGTTGATACC CTGATACTGA 2350 GTACATTGCA GAAAAAAATA TCCCTATTCC CG 2382

[0095]

Table 6

[0096] Chimeric invasin-furin-her2nb fusion protein (nucleic acid sequence) [SEQ ID NO: 41] atgatggttt tccagccaat cagtgagttt ctcttgataa ggaatgcggg 50 aatgtctatg tattttaata aaataatttc atttaatatt atttcacgaa 100 tagttatttg tatctttttg atatgtggaa tgttcatggc tggggcttca 150 gaaaaatatg atgctaacgc accgcaacag gtccagcctt attctgtctc 200 ttcatctgca tttgaaaatc tccatcctaa taatgaaatg gagagttcaa 250 tcaatccctt ttccgcatcg gatacagaaa gaaatgctgc aataatagat 300 cgcgccaata aggagcagga gactgaagcg gtgaataaga tgataagcac 350 cggggccagg ttagctgcat caggcagggc atctgatgtt gctcactcaa 400 tggtgggcga tgcggttaat caagaaatca aacagtggtt aaatcgattc 450 ggtacggctc aagttaatct gaattttgac aaaaattttt cgctaaaaga 500 aagctctctt gattggctgg ctccttggta tgactctgct tcattcctct 550 tttttagtca gttaggtatt cgcaataaag acagccgcaa cacacttaac 600 cttggcgtcg ggatacgtac attggagaac ggttggctgt acggacttaa 650 tacttttat gataatgatt tgaccggcca caaccaccgt atcggtcttg 700 gtgccgaggc ctggaccgat tatttacagt tggctgccaa tgggtatttt 750 cgcctcaatg gatggcactc gtcgcgtgat ttctccgact ataaagagcg 800 cccagccact gggggggatt tgcgcgcgaa tgcttattta cctgcactcc 850 cacaactggg ggggaagttg atgtatgagc aatacaccgg tgagcgtgtt 900 gctttattg gtaaagataa tctgcaacgc aacccttatg ccgtgactgc 950 cgggatcaat tacaccccg tgcctctact cactgtcggg gtagatcagc gtatggggaa aagcagtaag catgaaacac agtggaacct ccaaatgaac 1050 tatcgcctgg gcgagagttt tcagcgcaa cttagccctt cagcggtggc 1100 aggaacacgt ctactggcgg agagccgcta taaccttgtc gatcgtaaca 1150 atatatcgt gttggagtat cagaaacagc aggtggttaa actgacatta 1200 tcgccagcaa ctatctccgg cctgccggt caggtttatc aggtgaacgc 1250 acaagtacaa ggggcatctg ctgtaaggga aattgtctgg agtgatgccg 1300 aactgattgc cgctggcggc acattaacac cactgagtac cacacaattc 1350 aacttggttt taccgcctta taaacgcaca gcacaagtga gtcgggtaac 1400 ggacgacctg acagccaact tttattcgct tagtgcgctc gcggttgatc 1450 accaaggaaa ccgatctaac tcattcacat tgagcgtcac cgttcagcag 1500 cctcagttga cattaacggc ggccgtcatt ggtgatggcg caccggctaa 1550 tgggaaaact gcaatcaccg ttgagttcac cgttgctgat tttgagggga 1600 aacccttagc cgggcaggag gtggtgataa ccaccaataa tggtgcgcta 1650 ccgaataaaa tcacggaaaa gacagatgca aatggcgtcg cgcgcattgc 1700 attaaccaat acgacagatg gcgtgacggt agtcacagca gaagtggagg 1750 ggcaacggca aagtgttgat acccactttg ttaagggtac tatcgcggcg 1800 gataaatcca ctctggctgc ggtaccgaca tctatcatcg ctgatggtct 1850 aatggcttca accatcacgt tggagttgaa ggatacctat ggggacccgc 1900 aggctggcgc gaatgtggct tttgacacaa ccttaggcaa tatgggcgtt 1950 atcacggatc acaatgacgg cacttatagc gcaccattga ccagtaccac 2000 gttgggggta gcaacagtaa cggtgaaagt ggatggggct gcgttcagtg 2050 tgccgagtgt gacggttaat ttcacggcag atcctattcc agatgctggc 2100 cgctccagtt tcaccgtctc cacaccggat atcttggctg atggcacgat 2150 gagttccaca ttatcctttg tccctgtcga taagaatggc catttatca 2200 gtgggatgca gggcttgagt tttactcaaa acggtgtgcc ggtgagtatt 2250 agccccatta ccgagcagcc agatagctat accgcgacgg tggttgggaa 2300 tagtgcggt gatgtcacaa tcacgccgca ggttgatacc ctgatactga 2350 gtacattgca gaaaaaaata tccctattcc cggtacctac gctgaccggt 2400 attctggtta acgggcaaaa tttcgctacg gataaagggt tcccgaaaac 2450 gatctttaaa aacgccacat tccagttaca gatggataac gatgttgcta 2500 ataatactca gtatgagtgg tcgtcgtcat tcacacccaa tgtatcggtt 2550 aacgatcagg gtcaggtgac gattacctac caaacctata gcgaagtggc 2600 tgtgacggcg aaaagtaaaa aattcccaag ttattcggtg agttatcggt 2650 tctacccaaa tcggtggata tacgatggcg gcagatcgct ggtatccagt 2700 ctcgaggcca gcagacaatg ccaaggttca gatatgtctg cggttcttga 2750 atcctcacgt gcaaccaacg gaacgcgtgc gcctgacggg acattgtggg 2800 gcgagtgggg gagcttgacc gcgtatagtt ctgattggca atctggtgaa 2850 tattgggtca aaaagaccag cacggatttt gaaaccatga atatggacac 2900 aggcgcactg caaccagggc ctgcatactt ggcgttcccg ctctgtgcgc 2950 tgtcaatacg ccgcgcgcgc agcgtggcga gcatggaagt tcagctggtt 3000 gaatctggtg gtggtctggt tcaggcgggt ggttctctgc gtctgtcttg 3050 cgcggcgtct ggtatcacct tctctatcaa caccatgggt tggtaccgtc 3100 aggcgccggg taaacagcgt gaactggttg cgctgatctc ttctatcggt 3150 gacacctact acgcggactc tgttaaaggt cgtttcacca tctctcgtga 3200 caacgcgaaa aacaccgttt acctgcagat gaactctctg aaaccggaag 3250 acaccgcggt ttactactgc aaacgtttcc gtaccgcggc gcagggtacc 3300 gactactggg gtcagggtac ccaggttacc gtttcttctc accaccacca 3350 ccaccactaa tag

[0097] HER2 synthetic nanobody (nucleic acid sequence) [SEQ ID NO: 42] atggaagtt cagctggtt gaatctggt ggtggtctg gttcaggcg ggtggttct 50 ctgcgtctg tcttgcgcg gcgtctggt atcaccttc tctatcaac accatgggt 100 tggtaccgt caggcgccg ggtaaacag cgtgaactg gttgcgctg atctcttct 150 atcggtgac acctactac gcggactct gttaaaggt cgtttcacc atctctcgt 200 gacaacgcg aaaaacacc gtttacctg cagatgaac tctctgaaa ccggaagac 250 accgcggtt tactactgc aaacgtttc cgtaccgcg gcgcagggt accgactac 300 tggggtcag ggtacccag gttaccgtt tcttctcac caccaccac caccac 346 References Isberg et al. 1987: Isberg RR, Voorhis DL, Falkow S. Identification of invasin: a protein that allows enteric bacteria to penetrate cultured mammalian cells. Cell. 1987 Aug 28;50(5):769-78. doi: 10.1016 / 0092-8674(87)90335-7. PMID: 3304658. Chain et al. 2004: Chain PS, Carniel E, Larimer FW, Lamerdin J, Stoutland PO, Regala WM, Georgescu AM, Vergez LM, Land ML, Motin VL, Brubaker RR, Fowler J, Hinnebusch J, Marceau M, Medigue C, Simonet M, Chenal-Francisque V, Souza B, Dacheux D, Elliott JM, Derbise A, Hauser LJ, Garcia E. Insights into the evolution of Yersinia pestis through whole-genome comparison with Yersinia pseudotuberculosis. Proc Natl Acad Sci U S A. 2004 Sep 21;101(38):13826-31. doi: 10.1073 / pnas.0404012101. Epub 2004 Sep 9. PMID: 15358858; PMCID: PMC518763. Leong et al. 1990: Leong JM, Fournier RS, Isberg RR. Identification of the integrin binding domain of the Yersinia pseudotuberculosis invasin protein. EMBO J. 1990 Jun;9(6):1979-89. PMID: 1693333; PMCID: PMC551907.

Claims

1. An expression cassette for generating a chimeric invasin (Inv) polypeptide, comprising a prokaryotic promoter and a nucleic acid sequence encoding an Inv polypeptide fused to a linker polypeptide at the carboxyl terminus of the Inv polypeptide, wherein the expression of the nucleic acid encoding the chimeric invasin polypeptide is controlled by the prokaryotic promoter.

2. An expression cassette for generating an invasin polypeptide of a chimeric species according to claim 1, further comprising a sequence encoding a binding domain, wherein the binding domain is fused to the amine terminus of the linker polypeptide.

3. The aforementioned binding domains are shown in Tables 1-3: Table 1 Table 2 Table 3 An expression cassette for generating an invasin polypeptide of a chimeric species according to claim 2, wherein the binding domain is from a protein listed in or the binding domain is a synthetic binding domain.

4. An expression cassette for generating a chimeric invasin polypeptide according to claim 1, wherein the nucleic acid sequence of the invasin region of the chimeric polypeptide encodes an Inv polypeptide having an amino acid sequence having at least 90% sequence identity with amino acids 1 to 986 of SEQ ID NO:

1.

5. An expression cassette for generating a chimeric invasin polypeptide according to claim 1, wherein the nucleic acid sequence of the invasin region of the chimeric polypeptide encodes an Inv polypeptide having an amino acid sequence that has 100% sequence identity with amino acids 1 to 986 of SEQ ID NO:

1.

6. An expression cassette for generating a chimeric invasin polypeptide according to claim 1, wherein the nucleic acid sequence of the invasin region of the chimeric polypeptide is at least 90%, 95%, or 99% identical to nucleic acids 1 to 2958 of SEQ ID NO:

37.

7. An expression cassette for generating a chimeric invasin polypeptide according to claim 1, wherein the nucleic acid sequence of the invasin region of the chimeric polypeptide is 100% identical to nucleic acids 1 to 2958 of SEQ ID NO:

37.

8. An expression cassette for generating an invasin polypeptide of a chimeric species according to claim 1, wherein the nucleic acid sequence encodes a linker polypeptide selected from the group consisting of [SEQ ID NO: 2] to [SEQ ID NO: 20] and / or a protease cleavage site selected from the group consisting of [SEQ ID NO: 22] to [SEQ ID NO: 36].

9. The nucleic acid sequence is optionally converted by peptidase or protease into aminopeptidase N (APN / CD13), aminopeptidase A (APA), aminopeptidase P (APP), dipeptidylpeptidase 9 (DPP9), pyroglutamylpeptidase II (TRHDE), dipeptidylpeptidase IV (DPP) IV / CD26), angiotensin-converting enzyme (ACE / CD143), angiotensin-converting enzyme-2 (ACE2 / ACEH), carboxypeptidase M (CPM), carboxypeptidase P (CPP), γ-glutamyl transpeptidase 8 (γ-GT / CD224), membrane dipeptidase (MBD), neprilysin (NEP / CD10), endothelin-converting enzyme (ECE-1), prostasin (PRSS8), matryptase (ST14) Matryptase-2 (TMPRSS6), Matryptase-3 (TMPRSS7), Polycellase-1 (TMPRSS9), Transmembrane Protease, Serine 2 (TMPRSS2), Transmembrane Protease, Serine 3 (TMPRSS3), Transmembrane Protease, Serine 4 (TMPRSS4), Transmembrane Protease, Serine 5 / Spinesin (TMPRSS5), Choline, Furin, DPPIV (DPP4), MT1-MMP (MMP14), MT4-M MP (MMP17), gelatinase (MMP2; MMP9), disintegrin and metalloproteinase domain-containing protein 8 (ADAM8), disintegrin and metalloproteinase domain-containing protein 9 (ADAM9), disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), disintegrin and metalloproteinase domain-containing protein 12 (ADAM12), disintegrin and metalloproteinase domain-containing protein 15 (ADAM15), disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), disintegrin and metalloproteinase domain-containing protein 33 (ADAM33), hepsin (HPN), fibroblast-activating protein (FAP), neutral endopeptidase / neprilysin (MMEL1), mepurin A (MEP1A), mepurin B (MEP1B), testisin (PENFFG;An expression cassette for generating an invasin polypeptide of a chimera according to claim 1, encoding a protease cleavage site selected from the group consisting of TSEDFLVV), HAT, transmembrane protease serine 11E (DESC1), transmembrane protease serine 11A (TMPRSS11A), transmembrane protease serine 11F (TMPRSS11F), transmembrane protease serine 11B (TMPRSS11B), MSPL (TMPRSS13), enteropeptidase (TMPRSS15), and insulin-modulating aminopeptidase (IRAP).

10. The aforementioned prokaryotic promoter a) A promoter selected from the group consisting of T7, lacUV5, gapA, T5, recA, Ptac, Patac, pAl, lac, Sp6, araBad, and trp, or b) An expression cassette for generating the invasin polypeptide of the chimeric polypeptide according to claim 1, which is a hybrid or synthetic prokaryotic promoter.

11. A bacterium expressing a chimeric invasin polypeptide, wherein the bacterium comprises the expression cassette described in any one of claims 1 to 10. Optionally, the bacteria include Clostridium difficile, Escherichia coli, Neisseria tetanus, Helicobacter pylori, Fusobacterium nucleatum, Gardnerella vaginalis, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Listeria monocytogenes, Staphylococcus aureus, Campylobacter jejuni, Vibrio vulnificus, Salmonella typhi, Clostridium botulinum, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium repromatosis, Corynebacterium diphtheriae, Klebsiella pneumoniae, Acinetobacter baumannii, and Streptococcus mutans. The bacteria are selected from the group consisting of Group B Streptococcus, Staphylococcus aureus, Streptococcus agalactia, Streptococcus pneumoniae, Enterococcus, Enterococcus faecalis, Listeria, Yersinia, Rickettsia, Sigella, Salmonella, Legionella, Chlamydia, Brucella, Neisseria, Brucorderia, Bordetella, Borrelia, Coxiella, Mycobacterium, Helicobacter, Staphylococcus, Streptococcus, Porphyromonas, Vibrio, Treponema, Lactobacillus, and Bifidobacterium. Preferably, the bacterium is Escherichia coli.

12. A bacterium according to claim 11 for use in a method for treating or preventing a disease in a subject, wherein the bacterium is engineered to express therapeutic nucleic acids, proteins, antibodies, antibody derivatives, polypeptides, gene editing systems (CRISPR and other gene editing nucleases), eukaryotically translatable mRNA, or combinations thereof from a sequence on the bacterial chromosome or plasmid.

13. A chimeric invasin polypeptide comprising an inv polypeptide and a linker polypeptide, wherein the linker polypeptide has a first end (N-terminus) and a second end (C-terminus), and the first end (N-terminus) of the linker polypeptide is attached to the C-terminus of the inv polypeptide, optionally a) The invasin polypeptide further comprises a peptidase or protease cleavage site, b) The chimerine basin polypeptide further comprises a heterologous protein binding domain or a synthetic binding domain attached to the second terminus (C terminus) of the linker polypeptide, and / or c) A chimeric invasin polypeptide wherein the amino acid sequence of the Inv polypeptide of the chimeric invasin is 90%, 95%, 99%, or 100% identical to amino acids 1 to 986 of SEQ ID NO:

1.

14. The binding domains in b) above are shown in Tables 1-3: Table 4 Table 5 Table 6 The invasin polypeptide of the chimeric species according to claim 13, wherein the binding domain is from the proteins listed above.

15. A composition for selective conjugation of a substrate to a target molecule comprising a chimeric invasin polypeptide according to claim 13 or 14, conjugated at the amino terminus of the Inv polypeptide on the surface of a biological or synthetic substrate, wherein the substrate is selected from the group consisting of beads, viruses, exosomes, rigid substrates (e.g., for the manufacture of lateral flow strips), paper-based biosensors, plastic substrates (e.g., for the manufacture of plastic-based biosensors), graphene-based substrates, or nanomaterials (e.g., lipid nanoparticles, metal nanoparticles, mesoporous silica nanoparticles, nanowires, ITO, organic polymers).

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