Bacterial delivery of antibodies, antibody derivatives, and polypeptides to eukaryotic cells

Abstract

The present invention relates to the treatment, prevention and diagnosis of disease.The present invention describes a bacteria-mediated platform for producing antibodies, antibody derivatives, proteins / polypeptides and delivering them intracellularly to targeted eukaryotic cells and tissues using invasive non-pathogenic bacteria.The bacteria can contain prokaryotic expression cassettes encoding protein cargo.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 195,982, filed June 2, 2021.

[0002] Technical Field The present invention relates to the treatment, prevention and diagnosis of disease. More specifically, the present invention relates to a bacteria-mediated platform that uses invasive, non-pathogenic bacteria to produce and intracellularly deliver polypeptides, proteins, antibodies and antibody derivatives to target eukaryotic cells and tissues. [Background technology]

[0003] The use of agonistic and antagonistic antibodies and antibody derivatives is a rapidly growing area of ​​interest in the development of novel therapeutic agents, mainly due to their target specificity and low immunogenicity. The use of antibodies allows the development of therapeutic agents for diseases for which no druggable targets existed before. To date, the market has been dominated by antibody-based drugs that target extracellular targets (i.e., on the cell surface). Intracellular targets remain largely inaccessible. Despite the enormous therapeutic potential of modulating intracellular targets with agonistic or antagonistic antibodies, their use in this capacity is limited due to the inability of antibodies to cross the target cell membrane and enter the cytoplasm.

[0004] To date, efficient in vivo delivery of proteins, antibodies, and antibody derivatives to target cells remains challenging. Most of the current antibody delivery strategies are primarily based on mechanical approaches (e.g., electroporation, hydrodynamic injection, microinjection) and viral vector delivery (e.g., lentivirus, adenovirus, adeno-associated virus). Many of these methods are useful in vitro but cannot be easily clinically translated to animal or human patients. Non-viral delivery methods, such as liposomes and nanoparticles, are also used, but the size and number of antibodies or other proteins they can carry are greatly limited. Antibodies and antibody derivatives must be delivered more efficiently to achieve both extracellular and especially intracellular therapeutic effects. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention provides a system for the production and / or intracellular delivery of polypeptides, proteins, antibodies, and antibody derivatives into eukaryotic cells using a non-pathogenic bacterial delivery platform. The delivered proteins can be agonistic or antagonistic to intracellular pathways by interacting with specific target molecules in the target cells. For example, the target molecule can be active when not complexed with the antibody derivative, but inactive when complexed. For example, the delivery of single domain antibodies (nanobodies) that complex with the survivin molecule to inhibit the proliferation of cancer cells. Antibodies, antibody derivatives, and polypeptides can include: IgG, IgG fragments (Fab, Fab', F(ab') 2 ), V HH Moieties, single chain variable fragments (scFv), di-scFv, single domain antibodies (sdAb), nanobodies, camelid IgG antibodies, llama IgG antibodies, peptibodies, any other immunological polypeptides, and any polypeptides composed of amino acid residues that form therapeutic protein molecules, it is contemplated that the present invention can provide for the effective delivery of these molecules to target eukaryotic cells.

[0006] In a first aspect, the present invention provides a bacterial-mediated platform that uses invasive, non-pathogenic bacteria to produce polypeptides, proteins, antibodies, and antibody derivatives ("protein cargo") and deliver them intracellularly to target eukaryotic cells and tissues. The bacteria may contain a prokaryotic expression cassette encoding the protein cargo under the control of a prokaryotic promoter. The novel bacterial delivery platform for therapeutic antibodies, antibody derivatives, or proteins can provide tissue- and cell-specific delivery of proteins and / or antibodies, as well as intracellularization, in eukaryotic cells at any cell cycle stage (dividing, non-dividing, quiescent). Targeting to the desired eukaryotic cells can be controlled by the selection of entry factors that interact with specific target cell surface moieties.

[0007] In a second aspect, the present invention provides a method for replacing proteins (e.g., enzyme replacement therapy) or modulating specific activities and / or pathways in eukaryotic cells. In the case of protein replacement, the method may include delivering a eukaryotic protein encoded and produced by a prokaryotic bacterial cell to replace a specific eukaryotic protein that is non-functional (e.g., due to mutation) or missing (e.g., due to haploinsufficiency). In the case of modulating specific activities and / or pathways in eukaryotic cells, the method includes transmitting one or more cargo molecules, such as antibodies, antibody derivatives, or IgG, IgG fragments [Fab, Fab', F(ab'), IgG fragments, collectively known as proteins / polypeptides, under the control of one or more prokaryotic promoters. 2 ], V HHThe method may include contacting a bacterium containing an expression cassette encoding a portion, a single chain variable fragment (scFv), a di-scFv, a single domain antibody (sdAb), a nanobody, a camelid IgG antibody, a llama IgG antibody, a peptibody, any other immune polypeptide, or any polypeptide composed of amino acid residues that form a therapeutic protein molecule, wherein said encoded cargo molecule is produced by a bacterium that has been engineered to enter a eukaryotic cell, and wherein a region on one or more antibodies or other antibody derivatives binds a target molecule in the target cell and modulates the activity of the target molecule in the eukaryotic cell.

[0008] In a third aspect, the present invention provides a bacteria-mediated platform for the production and intracellular delivery of therapeutic nanobodies. Producing structurally intact and functional antibodies in bacteria is challenging, especially due to their large size and disulfide bonds, which can only be formed in the periplasmic space of E. coli cells. For this reason (and many others), there has been an increasing interest in single domain antibodies (sdAbs), also known as nanobodies. These proteins range in size from 12-15 kDa (versus 150-160 kDa for typical antibodies) and contain only one monomeric variable antibody domain. Importantly, as relatively large macromolecules, nanobodies may be less sensitive to the fine structure of the target protein, for example by binding to the entire surface of the protein (rather than to small pockets) to exert their biochemical effects. For this reason, nanobodies are a valuable mutation-refractory therapeutic modality. Due to the inhibitory mechanism of nanobodies, nanobody-based therapeutics are expected to be less affected by acquired resistance, especially by concomitant mutations in the target protein.

[0009] In a fourth aspect, the present invention provides a system for producing proteins and delivering them to eukaryotic cells. The system uses a bacterium engineered to be invasive and to have at least one expression cassette encoding a protein that is exogenous to the bacterium. Transcription of the nucleic acid encoding the exogenous protein is under the control of a prokaryotic promoter and terminator.

[0010] In an advantageous embodiment, the prokaryotic promoter and terminator are synthetic. The synthetic promoter or terminator may have transcription-enhancing or transcription-terminating activity in E. coli.

[0011] In a further advantageous embodiment, the encoded protein is an antibody or an antibody derivative. The antibody or antibody derivative may essentially consist of one protein domain extracted from a multi-domain antibody. An antibody derivative may consist of one V HH Consists essentially of an antibody domain or nanobody. The antibody or antibody derivative may have a biologically active peptide (having an effect on a living organism, tissue, cell, or biochemical process) grafted to the Fc domain or other antibody domain (e.g., peptibody) of the antibody or antibody derivative. The biologically active peptide may be a peptide having antioxidant, antimicrobial, immunomodulatory, cytomodulatory, and / or metabolic altering properties or effects.

[0012] The structural domain of an antibody or antibody derivative may have an amino acid sequence that binds an epitope that targets an intracellular protein, where the intracellular protein is a therapeutically relevant protein or is therapeutically relevant as a binding target for the antibody or antibody derivative.

[0013] Antibodies or antibody derivatives can form complexes with target proteins, thereby modulating specific activities or cellular pathways in eukaryotic cells by rendering the target protein biologically inactive with antibodies that mask, occupy, or otherwise interfere with binding sites or epitopes that are important for the interaction of the target protein with other molecules, whereas the uncomplexed target is a biologically active protein.

[0014] In certain embodiments, the antibody or antibody derivative binds to an intracellular factor in cancer cells and regulates specific activities or cellular pathways, including those related to cell survival, proliferation, and sensitivity to chemotherapeutic agents, so that the antibody or antibody derivative has an anti-tumorigenic effect.In an advantageous embodiment, the intracellular factor that is bound by the antibody or antibody derivative in cancer cells can be mutated HRAS, NRAS, or KRAS protein.In other words, the antibody or antibody derivative binds mutated HRAS, NRAS, or KRAS protein.

[0015] In further advantageous embodiments, binding by the antibody or antibody derivative modulates a specific activity or cellular pathway, thereby enhancing the therapeutic effectiveness of a chemotherapeutic agent or other therapy administered to or performed on the subject. The antibody or antibody derivative may be administered prior to, consecutively with, or after the chemotherapeutic agent or other therapy.

[0016] In certain embodiments, the antibody or antibody derivative comprises a region that binds an epitope on an apoptosis-regulating protein or apoptosis-related protein.The antibody or antibody derivative may comprise a region that binds survivin (BIRC5), BCL-2, MCL-1, XIAP, BRUCE, or any other inhibitor of apoptosis (IAP) family protein, or a protein that comprises one or more characteristic BIR domains.The antibody or antibody derivative may comprise a region that binds an epitope on a virus, bacteria, protozoan, or fungal protein, whereby binding the epitope inhibits the replication of the virus, bacteria, protozoan, or fungus.

[0017] The bacterium according to the fourth aspect may be a non-pathogenic bacterium engineered to have at least one invasion factor that promotes the invasion of the non-pathogenic bacterium into a eukaryotic cell or causes the release of the non-pathogenic bacterium from the phagosome of the eukaryotic cell. The invasion factor may be encoded by the inv, hlyA, or hlyE genes, or any fragments, or chimeric or recombinant forms thereof. The invasion factor may be a chimeric recombinant invasion protein comprising a non-binding domain of an invasin protein fused to a binding domain from a heterologous protein. The binding domain from the heterologous protein may be a binding domain of GalNAc binding protein, lectin, group of cell adhesion molecules (CAM), group of sulfated glycosaminoglycan (GAG) binding proteins, selectin, integrin, laminin, cadherin, fibronectin, collagen, thrombospondin, vitronectin, tenascin, apolipoprotein B, E, and AV, lipoprotein lipase, hepatic lipase, Siglec, galectin, immunoglobulin, and annexin, FimH, papG, PrsG, Afa-IE, DraA, MrpH, RodA, Mp1, hydrophobin, heat shock protein, CspA, hemagglutinin, neuraminidase, capsid protein, glycoprotein, and envelope protein. In certain embodiments, the invasion factor is engineered to be present on the bacterial chromosome.

[0018] The expression cassette encoding the protein is engineered to be carried by a prokaryotic plasmid or on a bacterial chromosome. The expression cassette can be on a plasmid having a length of about 7,000 base pairs or less, about 6,000 base pairs or less, about 5,000 base pairs or less, about 4,000 base pairs or less, or about 3,000 base pairs or less. The reduction in size of the plasmid compared to larger plasmids reduces the plasmid-induced load on the host bacterial cell, thereby increasing the growth rate of the bacteria.

[0019] A protein encoded by the invading bacterium is delivered to the cytoplasm of a target eukaryotic cell, in certain embodiments, the protein is functional within the eukaryotic cell and increases the levels of the protein within the target eukaryotic cell to compensate for a clinically significant deficiency in endogenous levels of the protein, thereby conferring a therapeutic benefit to the target cell and the subject.

[0020] In a fifth aspect, the present invention provides a method for modulating a specific activity and / or pathway in a eukaryotic cell, the method comprising expressing one or more antibodies, one or more VRNAs under the control of one or more prokaryotic promoters. HH The method includes contacting a eukaryotic cell with a bacterium comprising an expression cassette encoding an antibody domain, a nanobody, and / or other antibody derivative, wherein the bacterium is engineered to enter the eukaryotic cell and to express one or more antibodies, one or more V HH Regions on antibody domains, nanobodies, and / or other antibody derivatives bind target molecules, whereby binding modulates the activity of the target molecule.

[0021] In a sixth aspect, the present invention provides a composition comprising an engineered bacterium carrying a plasmid, the plasmid carrying an origin of replication, a selectable marker and one or more antibodies, a V gene under the control of one or more prokaryotic promoters and terminators. HH The plasmid essentially consists of an expression cassette encoding an antibody domain, a nanobody, an antibody derivative, or a combination thereof, and the bacterium is engineered to be invasive to eukaryotic cells. HH The antibody may encode an antibody domain, a nanobody, an antibody derivative, or a combination thereof, and may include an antibody, a V HH At least two of the antibody domains, nanobodies, antibody derivatives or combinations thereof are under the control of different promoters, thereby producing two or more antibodies, one V HH It allows for differential expression of antibody domains, nanobodies, antibody derivatives, or combinations thereof. In an advantageous embodiment, an antibody, one V HHOne of the antibody domains, nanobodies, antibody derivatives, or combinations thereof is an anti-survivin nanobody.

[0022] In a seventh aspect, the present invention provides a method for replacing or supplementing an endogenous eukaryotic protein in a eukaryotic target cell, the method comprising the step of contacting the eukaryotic target cell with a bacterium comprising an expression cassette encoding and producing a eukaryotic protein requiring replacement or supplementation in the target cell, wherein transcription of the protein is under the control of a prokaryotic promoter, the bacterium is a non-pathogenic bacterium engineered to be invasive for eukaryotic cells, and the eukaryotic protein expressed in the exogenously delivered bacterium has the same biological or biochemical activity as the endogenous eukaryotic protein, which activity is present in the eukaryotic cell (i.e. the protein performs its normal function).

[0023] For a more complete understanding of the present invention, please see the following detailed description taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 shows the pSiVEC2_survivin_nb plasmid. The cloned nanobody ("nb") sequence also encodes a 6XHis affinity tag translationally fused to the C-terminus of nb. In pSiVEC2_survivin_nb, the prokaryotic expression of the anti-survivin nb protein is controlled by a prokaryotic promoter (i.e., a promoter active only in bacterial cells). Thus, bacteria produce (transcribe and translate) and deliver anti-survivin nb. [Diagram 2] Figure 1 shows an image of a Western blot of four independent clones (labeled #9, #10, #12, and #16) of FEC21 / pSiVEC2_survivin_nb. A strong band representing a protein of approximately 15 kDa, which was absent in the negative control lane, confirms strong bacterial expression of anti-survivin nb. [Diagram 3]1 is a graph showing that A549 epithelial cancer cells receiving anti-survivin nb via the bacterial delivery system described herein have a sustained reduction in proliferation compared to cells receiving a scrambled sequence (no nb control). [Figure 4] Graph showing that A549 epithelial cancer cells receiving anti-survivin nb via the bacterial delivery system described herein have 1) a sustained decrease in proliferation (compare "control" to "anti-survivin") and 2) increased sensitivity to anti-survivin as shown by a stronger effect on proliferation in the presence of cisplatin (compare the effect of the addition of cisplatin between "control" to "control + cisplatin (25 mM)" and "anti-survivin" to "anti-survivin + cisplatin"). [Diagram 5]

[0023] Figure 1 is a panel of brightfield images showing that the addition of cisplatin to anti-survivin nb-treated A549 epithelial cancer cells increased the number of apoptotic cells that persisted for at least 98 hours after cisplatin addition. Triangles indicate exemplary apoptotic cells. [Figure 6] FIG. 1 shows the E. coli optimized anti-survivin nanobody ("nb") sequence (boxed asterisk(s) at the C-terminus indicate the stop codon). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The use of agonistic and antagonistic antibodies and antibody derivatives is a rapidly growing area of ​​interest in the development of novel therapeutic agents, mainly due to their target specificity and low immunogenicity. The use of such molecules has enabled the development of therapeutic agents for diseases for which no druggable targets existed before. To date, the market has been dominated by antibody-based drugs directed at extracellular targets (i.e. targets on the cell surface). Intracellular targets remain largely inaccessible. Despite the enormous therapeutic potential of modulating intracellular targets with agonistic or antagonistic antibodies, their use in this capacity is limited due to the inability of antibodies to cross the target cell membrane and enter the cytoplasm. Free antibodies in circulation are non-specifically taken up by immune cells by their Fc domain, a protein typically found on the cell surface, thereby limiting the ability of antibodies to reach the desired intracellular target through systemic depletion. For these reasons, intracellular targets are often referred to as "undruggable" in the context of therapeutic antibodies, antibody derivatives, and proteins / polypeptides. This challenge has been partially addressed by the discovery and development of smaller antibody derivatives that do not contain the Fc region, e.g. single domain antibodies or nanobodies. Nevertheless, such antibody derivatives are still rapidly cleared by the body; therefore, a delivery approach is needed that (1) targets the antibodies to the desired cells, (2) protects the antibodies during their trafficking and entry into the target cells, and (3) allows the antibodies to cross the target cell membrane and enter the cytoplasm. These problems can be solved by mechanically introducing the antibodies, antibody derivatives, and proteins / polypeptides into the target cells (e.g., by microinjection, electroporation), by directly modifying the antibodies, antibody derivatives, and proteins / polypeptides to cross the cell membrane and enter their target cells due to their inherent but heterogeneous properties (e.g., via conjugation to cell-penetrating peptides), and by associating the antibodies, antibody derivatives, and proteins / polypeptides with various types of delivery vehicles (e.g., nanoparticles, liposomes, etc.).However, each of these potential solutions has significant limitations that limit their usefulness, including: (1) microinjection and electroporation are typically used under in vitro conditions, and both exhibit high cytotoxicity and are very labor intensive; (2) cell-penetrating peptides facilitate more efficient delivery, but do not protect the antibody during delivery or allow specific cellular targeting; (3) nanoparticles (including liposomes, LNPs, etc.) protect the antibody during delivery and allow some degree of targeting, but they can suffer from large production requirements, low encapsulation efficiency, limited loading capacity, and cytotoxicity. Finally, a common problematic feature of these delivery systems is endosomal trapping and subsequent degradation, which prevents the antibody from reaching its cytoplasmic target intact. An ideal delivery approach would have several key features, including: (1) efficient, stable, and robust prokaryotic encoding and production of the antibody, (2) high delivery efficiency and intracellularization in vivo, (3) delivery of the antibody in an active form, (4) targeting to specific cells / tissues, (5) protection of the antibody from degradation / clearance, and (6) lack of toxicity and immunogenicity. To date, no delivery platform meets all of these functional requirements.

[0026] The efficiency and effectiveness of encoding and producing proteins in prokaryotes prior to delivery can be hindered by reliance on inefficient or suboptimal naturally occurring prokaryotic regulatory sequences (e.g., promoters and terminators) from other species (i.e., heterologous) or from the host species (i.e., homologous). The activity of heterologous prokaryotic regulatory sequences (either in promoting or terminating transcription) can be suboptimal, thus diminishing the therapeutic potential of the delivery system due to limited production of the therapeutic moiety. The use of homologous prokaryotic regulatory sequences can tolerate homologous recombination events, resulting in unintended mutations to chromosomal and / or plasmid DNA sequences and general bacterial genome instability. The use of highly optimized synthetic regulatory elements, which are regulatory elements rationally and computationally designed (e.g., designed for use in E. coli) with optimal activity in the host species, can overcome these shortcomings. Examples of highly optimized synthetic elements used to regulate the expression of antibodies, antibody derivatives, and proteins / polypeptides inside bacterial cells according to the present invention are shown in Tables 1 and 2 below. Substantial improvements in protein production have been observed by switching to the use of synthetic promoters that are functional in prokaryotic systems but do not naturally occur in prokaryotes. By using synthetic prokaryotic promoters, the bacterial production of these proteins can be dramatically increased, which in turn improves the overall composition of the system and makes more protein available for delivery.

[0027] Collectively, the limitations of current delivery platforms have slowed the transition of intracellular antibody delivery from the laboratory to clinical use and hindered the potential of antibody-based drugs with intracellular targets. Thus, there is an urgent need for more robust production and delivery systems for antibodies, antibody derivatives, antibody fragments, antibody-like molecules, and other proteins or polypeptides that have limited ability to cross the plasma membrane of target cells.

[0028] Bacterial systems according to aspects of the invention include plasmids or bacteria genetically engineered to carry antibody, antibody derivative, or protein / polypeptide coding sequences whose expression is regulated by synthetic prokaryotic expression sequences (e.g., highly optimized for efficient use in E. coli, but not naturally occurring in the host prokaryotic species, i.e., synthetic, prokaryotic promoters and terminators) on the plasmid or on the bacterial chromosome. The bacterial cell thus serves as the site of production of the therapeutic moiety, in addition to then serving as a delivery mode for transporting the bacterially expressed therapeutic moiety to the targeted eukaryotic cell. In one embodiment of the invention, a plasmid using a prokaryotic promoter to drive the expression of the antibody, antibody derivative, or protein / polypeptide is transformed into a non-pathogenic bacterial cell. In another embodiment, the gene(s) encoding the antibody or antibody derivative is inserted into the bacterial chromosome with a prokaryotic promoter (e.g., an optimized synthetic promoter) to drive its expression. Additionally, the bacterial cells are engineered to be invasive, allowing them to enter eukaryotic cells via receptor-mediated phagocytosis. Small structures (e.g., LNPs, protein conjugates, etc.) are taken up by target cells via endocytosis, while larger structures (e.g., bacterial cells) are taken up by phagocytosis. In the context of this bacteria-mediated delivery system, as with other delivery platforms, endosomes and phagosomes are both membrane-bound compartments and are roughly equivalent barriers to cytoplasmic delivery. Furthermore, bacterial cells are genetically modified to allow efficient phagosome escape of delivered antibodies, antibody derivatives, or proteins / polypeptides. Constructs combining bacteria with genes encoding antibodies, antibody derivatives, and proteins / polypeptides (including prokaryotic regulatory / expression sequences), whether plasmid-based or chromosomal-based, constitute a bacteria-mediated antibody delivery platform, by which antibodies, antibody derivatives, and proteins / polypeptides can be produced and delivered intracellularly to targeted eukaryotic cells.Targeting can be directed by the selection of an entry factor, e.g., a moiety displayed on the surface of the bacterial system that preferentially binds a receptor on the surface of the target cell. Entry factors can be factors that promote attachment to and uptake by the target cell (e.g., invasin protein) and / or factors that promote release from the phagosome upon uptake (e.g., listeriolysin O, LLO). Entry factors can be produced from genes expressed from the chromosome or plasmid of the bacterial cell. In certain embodiments, bacteria are engineered to express a specific entry factor that directs the targeting of the delivery platform to the target cell.

[0029] In a preferred embodiment, the present invention provides a bacterial-mediated production and delivery platform composed of invasive non-pathogenic bacteria for intracellular delivery of antibodies, antibody derivatives, and proteins / polypeptides into eukaryotic cells, where the antibodies, antibody derivatives, and proteins / polypeptides produced and delivered are not endogenous to the bacterial vehicle. A "non-pathogenic bacterium" refers to a bacterium that is incapable of causing disease and can either be engineered to be non-pathogenic or naturally non-pathogenic, but may have cytotoxic or deleterious effects in a target cell as a result of the factors (e.g., polypeptides, antibody derivatives) that the bacterium has been engineered to deliver to the target cell.

[0030] This bacteria-mediated delivery system uniquely overcomes the shortcomings of other current technologies as described above, and provides: (1) High delivery efficiency - upon entry into eukaryotic cells, the bacteria are engineered to release antibodies, antibody derivatives, and proteins / polypeptides from the phagosome into the cytoplasm (i.e., although endosomal or phagosomal escape of therapeutic modalities continues to be an issue in some delivery systems, escape from the phagosome is not a limiting step in our engineered system).

[0031] (2) Delivery of antibodies in an active form - the bacteria themselves produce the antibodies, antibody derivatives, or proteins / polypeptides; thus, each cell is pre-loaded with its cargo, thereby enabling the mature antibodies, antibody derivatives, or proteins / polypeptides to rapidly interact with target intracellular molecules after delivery to eukaryotic cells.

[0032] (3) Targeting to specific cells and tissues - bacteria are engineered to be invasive to eukaryotic cells via receptor-mediated phagocytosis. Upon administration to a patient, bacterial cells migrate to distant tissues or remain in local tissues, further ensuring highly efficient and focused delivery to the target tissue. Precise targeting of bacteria to cells expressing specific surface proteins or chemical moieties is also possible by modifying entry factors present on the bacterial cell surface.

[0033] (4) Protection of antibodies, antibody derivatives, or proteins / polypeptides from degradation / clearance - Antibodies, antibody derivatives, or proteins / polypeptides are carried inside bacterial cells, which provides protection from degradation on their way to the eukaryotic cells of their targets.

[0034] (5) Favorable safety profile - the lack of toxicity and immunogenicity demonstrated in vivo indicates that the bacteria is well tolerated and amenable to repeated administration.

[0035] (6) Robust antibody, antibody derivative, and protein / polypeptide expression - The use of highly optimized synthetic regulatory elements designed to be functional in E. coli ensures high expression levels and more reliable termination of therapeutic antibodies, antibody derivatives, and proteins / polypeptides, particularly those polypeptides that are exogenous to or not naturally occurring within the bacterial delivery vehicle.

[0036] The present invention offers many substantial improvements over the current state of the art. The present invention utilizes bacterial cells containing prokaryotic expression cassettes encoded on plasmids or on chromosomes to produce and deliver functional antibodies, antibody derivatives, and proteins / polypeptides as cargo to eukaryotic cells. The advantage of using bacteria encoding prokaryotic expression cassettes is that the antibodies are expressed only by the bacteria and are produced by the bacteria prior to delivery, providing greater safety, the ability to control the dose, and faster time to effect compared to eukaryotic expression systems. Another important advantage of this system is the use of synthetic prokaryotic regulatory elements (promoters and terminators) to drive the expression of the entry factors and antibodies, antibody derivatives, and proteins / polypeptides. This strategy is advantageous because various methodologies have been used to optimize these sequences to ensure high expression levels and more reliable termination of the antibodies, antibody derivatives, and proteins / polypeptides. Once the bacterial cell is incorporated into the cytoplasm of the eukaryotic cell, the antibody sequences encoded on the plasmid cannot be expressed by the eukaryotic cell due to incompatible sequence requirements.

[0037] Another important advantage of the system of the present invention is related to the size of the prokaryotic expression plasmids encoding antibodies, antibody derivatives, and proteins / polypeptides, where the size of the plasmid backbone is reduced to reduce the plasmid-induced burden on the host bacterial cell, thereby increasing the bacterial growth rate.Ideally, the plasmid backbone is less than 10,000 base pairs, and in some cases less than 7,000 base pairs, or less than 5,000 base pairs, or less than 3,000 base pairs.The size of the plasmid can be addressed by introducing specific genes (e.g., invasion factors) into the bacterial chromosome.The reduction in the size of the plasmid can increase the copy number of the plasmid in the cell and increase the expression level of the plasmid genes.

[0038] Based on an in-depth knowledge of E. coli and general bacterial biology and genetics, an additional advantage of the present invention is that the bacterial compositions can be further modified to include additional or new features to increase the expression efficiency of antibodies, antibody derivatives, and protein / polypeptide cargos, to enhance the function of antibodies, antibody derivatives, and protein / polypeptides, to enhance the safety profile of the bacterial cells, to enhance the immune resistance profile of the bacterial cells to repeated administration, and to enhance and / or optimize other aspects of delivery, including the ability to target specific eukaryotic tissues, organs and cells by altering entry factors or modifying the physical size of the bacterial cell.

[0039] For example, the bacterial surface-exposed invasion factor invasin (including fragments or domains of the invasin protein) can be modified via genetic engineering to facilitate binding of the bacteria to different target proteins expressed on the surface of target eukaryotic cells (e.g., cell surface receptors such as integrins) or to various target chemical moieties expressed on the surface of target eukaryotic cells (e.g., surface-accessible N-acetylgalactosamine, GalNAc). As a further example, chimeric or fusion proteins can be made utilizing the transmembrane domain of an invasin polypeptide and the binding domain of a second protein. Examples of binding domains of second proteins include heterologous proteins of animal, bacterial, fungal and / or viral origin having binding domains (e.g., GalNAc binding proteins, lectins, group of cell adhesion molecules (CAMs), group of sulfated glycosaminoglycans (GAG) binding proteins, selectins, integrins, laminins, cadherins, fibronectin, collagens, thrombospondin, vitronectin, tenascin, apolipoproteins B, E and AV, lipoprotein lipase, hepatic lipase, Siglecs, galectins, immunoglobulins and annexins, FimH, papG, PrsG, Afa-IE, DraA, MrpH, RodA, Mp1, hydrophobins, heat shock proteins, CspA, hemagglutinin, neuraminidase, capsid proteins, glycoproteins and envelope proteins, among others). This approach allows the bacteria to be targeted to specific cell types, thereby reducing potential off-target effects or undesired immune stimulation or inducing desired immune stimulation.

[0040] The size of E. coli cells (or other bacteria) can limit their access to target tissues and organs, especially when they pass through the circulatory system and enter small capillaries. Thus, strategies to reduce the size / dimensions of the delivery vehicle can be used to facilitate delivery to specific target areas that may otherwise be difficult to access. Mutations can be introduced into the E. coli genome (e.g., the fabH gene) to reduce the dimensions of the cells.

[0041] A third approach is to address the limitation of bacterial cell components that may prove toxic or otherwise harmful to target cells. Some target cell types may be more sensitive to residual bacterial components (e.g., lipopolysaccharide, LPS) that are deposited after eukaryotic cell invasion and cargo delivery, which can cause cytotoxicity and cell death. In some cases, this cell death may be due to one pathway (e.g., caspase-mediated cell death). Bacteria can be genetically modified to deliver additional heterologous factors (e.g., caspase-inhibiting viral proteins) that improve such effects. This approach offers the added benefit of increasing delivery efficiency by preserving the health of the invaded eukaryotic cell and preventing undesirable cytotoxicity. Additionally, bacteria can be genetically modified to limit virulence and immune stimulation by altering, modifying, mutating, or removing bacterial virulence factors (e.g., mutating the msbB gene, resulting in LPS lacking the myristoyl fatty acid portion of lipid A).

[0042] In some applications, bacteria must pass through an endothelial cell layer to reach a target cell or tissue (e.g., to exit the circulation through a capillary wall). Bacteria have various levels of ability to pass such barriers, and these abilities can be conferred by one or a few bacterially encoded proteins. To improve delivery efficiency, such proteins ("entry proteins") can be borrowed from one bacterial species or strain to introduce this ability into the delivery strain. For example, some strains of E. coli and other bacteria easily pass through the endothelial layer, and genes encoding these proteins can be introduced into the delivery bacterial strain to enhance tissue biodistribution across multiple cell layers, thereby improving delivery efficiency. Examples of such invasion proteins include FimH, OmpA, IbeA, IbeB, IbeC, Opc, PilA, PilB, LOS, Lmb, FbsA, IagA, Vsp1, OspA, 70-kDa PBP, enolase, Isc1, Yps3p, Stx, type III secretion system injection factor, EspF, Map, EspG).

[0043] Approaches to generate antibody conjugates are technically complex, and the conjugated elements may have toxic effects. The bacterial-mediated system described in the present invention overcomes these problems because it does not require the conjugation of the therapeutic moiety to potentially toxic compounds or packaging in particles (e.g., LNPs) that may exhibit toxicity. Furthermore, the present invention eliminates complex manufacturing steps, such as chemical conjugation, production of nanoparticles and packaging into nanoparticles. A notable problem in other systems, especially those involving free antibodies conjugated to cell-penetrating moieties, is the difficulty of protecting the antibody from degradation upon introduction into the body (especially the circulatory system). The bacterial-mediated system taught herein overcomes this challenge because the therapeutic moiety is protected within the bacterial cell until it reaches and is delivered into the target host cell.

[0044] The present invention provides a bacterial-mediated production and delivery platform composed of invasive non-pathogenic bacteria for intracellular delivery of antibodies, antibody derivatives, and proteins / polypeptides into eukaryotic cells. The bacteria may contain a prokaryotic expression cassette encoding the antibody, antibody derivative, or protein / polypeptide cargo. More specifically, the bacterial delivery system is composed of at least one antibody, antibody derivative, or protein / polypeptide nucleotide coding sequence that is integrated into the bacterial chromosome or expressed from at least one plasmid within the bacteria. The bacteria used in the production and delivery system of the present invention can be from various species, including species of Lactobacillus, Yersinia, Escherichia, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Corynebacterium, Citrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Salmonella, Vibrio, Bacillus, Leishmania and Erysipelothrix, Shigella, Listeria, Rickettsia, Acetoanaerobium, Aerococcaceae, Carnobacteriaceae, Enterococcusceae, Leuconostocaceae, Streptococciaceae, Bifidobacterium, etc., bacteria with Generally Regarded as Safe ("GRAS") status. In a preferred embodiment, the bacteria is Escherichia coli. For controlled and enhanced transcription of nucleic acids encoding antibodies, antibody derivatives, and proteins / polypeptides, the nucleic acid coding sequences are controlled by synthetic prokaryotic promoters and synthetic prokaryotic terminators. Additionally, bacteria can deliver antibodies, antibody derivatives, or proteins / polypeptides into the cytoplasm of eukaryotic cells by expressing entry factors (e.g., invasin, complete or fragments thereof, HylA, HlyE, influenza HA-1) that facilitate entry into eukaryotic cells via receptor-mediated phagocytosis, followed by efficient phagosomal escape of the delivered antibodies, antibody derivatives, or proteins / polypeptides.The antibodies, antibody derivatives, or proteins / polypeptides encoded and delivered by bacteria can modify the activity or function of at least one intracellular factor located inside the cytoplasm of eukaryotic cells. The delivered antibodies, antibody derivatives, or proteins / polypeptides can be agonistic or antagonistic to intracellular pathways by interacting with specific target molecules. Examples of intracellular pathways include targeting and / or inactivating the replication and survival mechanisms of infectious pathogens, including intracellular bacterial pathogens, protozoan pathogens, viruses, and fungal pathogens. Other examples of therapeutic applications include producing and delivering antibodies, antibody derivatives, proteins / polypeptides that target and / or inactivate intracellular factors associated with cancer, metabolic diseases, neurodegeneration, protein overexpression / aggregation disorders (e.g., Alzheimer's and Parkinson's diseases, amyotrophic lateral sclerosis, dementia with Lewy bodies, frontotemporal dementia, Huntington's disease, amyloid transthyretin cardiomyopathy, type 2 diabetes, and other forms of amyloidosis), inflammatory disorders, and autoinflammatory diseases. For cancer applications, antibody delivery may offer the advantage of being able to target multiple isoforms of a protein(s) carrying a range of mutations. For example, mutations in the proto-oncogene KRAS are present in many types of human cancer, but the range of mutations is diverse. This diversity, in addition to other structural features of the KRAS protein, makes the discovery of therapeutic KRAS inhibitors extremely challenging. Due to their size and refractoriness to minor structural features that do not affect direct interaction with the epitope, a single antibody or antibody derivative (e.g., nanobody) can be used as a robust inhibitor of a variety of proteins, including multiple RAS isoforms (e.g., KRAS4A, KRAS4B, HRAS, and NRAS) and numerous mutated KRAS proteins. This inhibitory effect, when combined with a robust delivery platform such as the one described herein, opens new opportunities for the development of both RAS-targeted cancer therapeutics and therapeutics that benefit from broad targeting flexibility.Additional therapeutic applications include the production and delivery of polypeptides, such as enzymes or proteins, that can act as antigens to stimulate antibody responses in vaccine applications, and / or to supplement or replace endogenous polypeptides whose production is dysregulated or altered by disease states or genetic disorders. The present invention also has diagnostic applications, including applications related to diagnostic intracellular imaging processes. For example, the delivered protein can be an affibody, or can act as an intracellular probe to detect intracellular targets indicative of disease. This includes the delivery of nanoflares (probe-like molecules) that can be used to detect intracellular targets, such as, for example, mRNAs encoding genes overexpressed in cancer (epithelial-mesenchymal transition, oncogenes, thymidine kinase, telomerase, etc.), intracellular levels of ATP, pH values, and inorganic ions. This also allows for the diagnosis of disease and / or elucidation of intracellular processes in live cells in real time.

[0045] Therapeutic applications (for humans and animals) include, but are not limited to: Virology: Antibodies, antibody derivatives, and proteins / polypeptides can be produced and delivered using the present invention to target and inactivate essential proteins required for intracellular viral replication. This inactivation occurs, for example, through disruption of proper protein folding, blocking of allosteric conformational changes, blocking of active sites, blocking of binding sites of interacting proteins, or blocking of binding sites of cofactors (e.g., ATP, GTP, etc.) by the antibody, antibody derivative, or protein / polypeptide. Potential applications include inhibition of HIV replication by inhibiting HIV integrase activity; inhibition of Norovirus replication; inhibition of Influenza A replication; inhibition of Ebola virus replication; inhibition of Hepatitis virus replication (all types); inhibition of Coronavirus replication (all types).

[0046] Bacteriology: Antibodies, antibody derivatives, and proteins / polypeptides can be produced and delivered using the present invention to target / inactivate essential proteins required for the intracellular lifestyle of intracellular bacterial pathogens, such as proteins required for nutritional uptake from the bacterial host (such as siderophores). Potential applications include the elimination of Ehrlichia chaffeensis, Staphylococcus aureus, Chlamydia, Rickettsia, Coxiella, Mycobacterium species, Brucella, Legionella, Nocardia, Neisseria, Rhodococcus equi, Yersinia, Francisella tularensis species, and Bartonella henselae.

[0047] Protozoa: Antibodies, antibody derivatives, and proteins / polypeptides can be produced and delivered using the present invention to target / inactivate essential proteins required for the intracellular lifestyle of intracellular protozoan pathogens, such as proteins required for nutritional uptake from the protozoan host. Potential applications include elimination of Trypanosoma (e.g., Leishmania spp., Trypanosoma spp.), Apicomplexa (e.g., Plasmodium spp., Toxoplasma gondii, Cryptosporidium parvum).

[0048] Mycology: Antibodies, antibody derivatives, and proteins / polypeptides can be produced and delivered using the present invention to target / inactivate essential proteins required for the intracellular lifestyle of intracellular fungal pathogens, such as proteins required for nutritional uptake from the fungal host. Potential applications include elimination of Pneumocystis jirovecii, Histoplasma capsulatum, Candida albicans, and Cryptococcus neoformans.

[0049] Antibodies, antibody derivatives, and proteins / polypeptides can also be generated and delivered using the present invention to target / inactivate proteins in intracellular signaling pathways with therapeutic potential. Examples of pathways, target proteins, and relevant therapeutic areas include: (1) Apoptosis / cell death / cell proliferation and related disorders, e.g., PI3K, Akt, HIF1A, p53, Blc2, Bcl-XL, Bcl-w, BRUCE, MCL-2, XIAP, cIAP1, C-IAP2, NAIP, livin, survivin (BIRC5), cancer. (2) Warburg effect-related proteins and associated diseases (e.g., GLUT1, GLUT3, PDK1, PDK2, MAGL, HK, PKM2, LDHA, G6PD, MCT1, PKB; cancer, metabolic disorders). (3) Nutrient signaling proteins and associated disorders (e.g., mTor, or any protein found in the mTORC1 or mTORC2 complexes, cancer, reduction of cognitive decline associated with neurodegeneration, metabolic diseases). (4) Wnt pathway and related disorders (e.g., Wnt, Frizzled, LRP5 / 6, etc.; cancer). (5) NFkB pathway and related disorders (e.g., TNF-α, JAK1, etc., cancer, inflammatory disorders). (6) Notch pathway and related disorders (e.g., g-secretase, Notch1, Notch2, Notch3; cancer). (7) Sonic hedgehog pathway and related disorders (e.g., GLI1, GLI2, SMO; cancer). (8) TLR signaling and associated disorders (TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, TLR13; leukemia, colon cancer, myelodysplastic syndromes, autoinflammatory diseases, inflammatory diseases).

[0050] Example: Delivery of single domain antibodies (nanobodies) that inactivate survivin inhibits cancer cell proliferation.

[0051] In eukaryotic cells, survivin / BIRC5, a member of the inhibitor of apoptosis protein family, inhibits apoptosis (i.e., programmed cell death) by blocking caspase activation. Survivin is highly expressed in many proliferating human cancer cell types and is absent in terminally differentiated postmitotic cells, therefore survivin inhibition preferentially affects proliferating cancer cells.

[0052] Cancer cells are characterized by uncontrolled proliferation, often due to defective apoptosis. A powerful therapeutic approach to limit cancer / tumor growth is to increase apoptosis, especially apoptosis caused by excessive DNA damage.

[0053] Disruption of survivin function results in increased apoptosis and decreased cancer cell proliferation. These characteristics highlight survivin as a potential cancer therapeutic target that allows discrimination between cancer and normal cells, and indeed, survivin has been investigated as a cancer therapeutic for over 20 years. Unfortunately, multiple clinical trials of survivin-based therapies have failed. A persistent challenge is the delivery of survivin-inhibiting moieties to cancer cells in the same cellular compartment (cytoplasm) where survivin exerts its function. The bacteria-based delivery system described herein overcomes this challenge by delivering bacterially encoded and expressed single-domain antibodies (nanobodies) that specifically inhibit survivin to the cytoplasm of target cells.

[0054] When the amount of DNA damage accumulated by a cell reaches a threshold, the apoptotic cascade is initiated to remove the cell from the population via apoptosis or programmed cell death. However, survivin inhibits cancer cell death by effectively raising the threshold that must be reached for the initiation of apoptosis. Most chemotherapy drugs used to treat cancer are toxic not only to tumor cells but also to normal tissues; thus, systemic chemotherapy can have significant off-target effects. It is therefore advantageous to use the lowest effective dose to minimize these undesirable effects. Many chemotherapeutic agents activate apoptosis in cells by inducing sufficient DNA damage to exceed the above threshold. Survivin is a barrier to apoptosis activation because of its effect on this threshold. Therefore, the dosage of the chemotherapeutic agent must be high enough to overcome it. Inactivation of survivin, for example by nanobodies, can lower the threshold for apoptosis activation, thereby reducing the effective dose of the chemotherapeutic agent and possibly reducing off-target effects without compromising its antitumor effect. In this approach, the survivin-inactivating nanobodies produced and delivered using the bacterial system of the invention will function as chemotherapy enhancers, or optionally as chemotherapy adjuvant therapies.

[0055] Here, we confirm the successful delivery of functional, bacterially expressed survivin-inhibitory nanobody (nb) proteins (i.e., anti-survivin nb) using the herein described invasive bacterial delivery system, consisting of diaminopimelic acid-requiring Escherichia coli (FEC21), by showing that proliferation of adenocarcinoma human alveolar basal epithelial cells (A549 cells, a common model cancer cell line) is delayed upon treatment of the cells with invasive bacteria that deliver anti-survivin nb to the cytoplasm of cancer cells. These results show the potential of this bacterial delivery system as a new approach to develop cancer therapeutics based on the delivery of protein factors that interfere with the function of cancer cells.

[0056] The E. coli optimized anti-survivin nb (Figure 6, the boxed asterisk indicates the stop codon) sequence was cloned into the empty pSiVEC2 vector to generate the pSiVEC2_survivin_nb plasmid (Figure 1). The cloned sequence also encodes a 6XHis affinity tag translationally fused to the C-terminus of nb. In pSiVEC2_survivin_nb, the prokaryotic expression of the anti-survivin nb protein is controlled by a prokaryotic promoter (i.e., a promoter active only in bacterial cells). Thus, bacteria produce (transcribe and translate) the anti-survivin nb and deliver it to eukaryotic cells.

[0057] pSiVEC2_survivin_nb was transformed into FEC21 E. coli to generate FEC21 / pSiVEC2_survivin_nb strain. FEC21 bacteria were further engineered to be invasive to eukaryotic cells via chromosomal integration of inv and hlyA genes for invasin- and receptor-mediated phagocytosis and HylA-mediated endosomal release, respectively. FEC21 cells transformed with pSiVEC2_survivin_nb were plated on brain heart infusion (BHI) agar containing appropriate antibiotics for selection. FEC21 / pSiVEC2_survivin_nb clones were frozen at -80°C in 20% glycerol.

[0058] Bacterial expression of anti-survivin_nb was confirmed by Western blotting of denatured protein samples of FEC21 / pSiVEC2_survivin_nb cells using an anti-6XHis tag antibody (1:500 dilution) that binds to the C-terminal 6XHis tag fused to anti-survivin_nb cells. Figure 2 shows the results of Western blot of four independent clones of FEC21 / pSiVEC2_survivin_nb (labeled #9, #10, #12, and #16). A strong band representing a protein of approximately 15 kDa, which was absent in the negative control lane, confirms strong bacterial expression of anti-survivin_nb.

[0059] Using a standard invasion assay (i.e., where FEC21 bacteria are incubated with mammalian cells and invade and intracellularize the mammalian cells), we determined whether delivery of bacterially expressed anti-survivin nb by invasive FEC21 (Figure 3, clone #10) bacteria could reduce proliferation of alveolar basal epithelial cells (A549), a common cancer cell line. A549 cells were cultured at 37 °C with 5% CO 2 The cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM GlutaMAX, 100 U / mL penicillin, and 100 μg / mL streptomycin during incubation. Invasive bacteria (by encoding inv and hlyA) enter A549 cells through the RME to deliver cargo (i.e., anti-survivin nb) encoded and expressed by those bacteria. This invasion assay involves the following steps; A549 cells are seeded at a fixed concentration in black 24-well plates. On the day of bacterial invasion, two bacterial stocks are thawed: 1) FEC21 / pSiVEC2_survivin_nb (clone #10) and 2) FEC21 / pSiVEC2_Scramble (an invasive negative control bacterial strain transformed with a plasmid carrying a non-coding scramble sequence). Bacterial cells are centrifuged and incubated at an absorbance of 0.004 (A 600 ) and resuspended in DMEM(-) (serum- and antibiotic-free, high glucose DMEM). A549 cells were washed with DMEM(-) to remove antibiotics, incubated with 0.5 mL of each bacterial suspension for 2 h (37 °C, 5% CO2), and then rinsed with DMEM to remove residual bacteria that did not invade the A549 cells. Cell confluence (an index of cell proliferation) was measured at 0, 18, 28, 52, and 76 h post-invasion using a Nexcelom Celigo instrument (in the brightfield channel).

[0060] Figure 3 shows that A549 cells receiving anti-survivin nb via the bacterial delivery system described herein have a sustained reduction in proliferation compared to cells receiving a scrambled sequence (no nanobody control). Collectively, these results demonstrate that the invasive FEC21 bacterial delivery platform can express and deliver functional anti-survivin nb protein to lung epithelial cells to limit their proliferation. N=6 biological replicates per time point for each condition. Cell confluence at each time point was normalized to the starting confluence for that condition. Data shown is the mean standard deviation.

[0061] Figure 4 shows the use of the same anti-survivin nb as a chemotherapy adjuvant. A549 cells were treated with invasive bacteria expressing anti-survivin nanobody (FEC21 / pSiVEC2_survivin_nb clone #10) as described above and then exposed to the common chemotherapy agent cisplatin (25 mM), which activates apoptosis in cancer cells by inducing DNA damage, for the first 24 hours after bacterial treatment. Cell confluence was measured, analyzed, and plotted as described above. As shown in the plot, delivery of anti-survivin nb via the bacteria-mediated delivery system described herein enhances the effect of cisplatin treatment on cell proliferation, establishing FEC21 / pSiVEC2_survivin_nb as a chemotherapy enhancer, i.e., delivery of anti-survivin nb increases the efficacy of a given dose of cisplatin. Robust statistical significance (p<0.005) was assessed by two-way ANOVA.

[0062] FIG. 5 is a series of images showing increased rates of apoptosis in cells analyzed for production of FIG.

[0063] References [Table 1]

[0064] definition As used in this application, the terms "a" and "an" are used with the understanding that they mean "at least one," "at least a first," "one or more," or "a plurality" of the referenced component or step, unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells (including mixtures thereof).

[0065] Whenever used herein, the term "and / or" includes the meaning of "and", "or" and "any or any other combination of the elements connected by this term".

[0066] 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.

[0067] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, when numerical ranges of various scopes are set forth herein, it is contemplated that any combination of these values ​​inclusive of the recited values ​​may be used.

[0068] As used herein, the term "comprising" is intended to mean that the products, compositions and methods include the recited components or steps, but do not exclude others. When used to define products, compositions and methods, "consisting essentially of" may mean excluding any other essentially significant components or steps. 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 elements of other components or steps.

[0069] The term "administration" and variations thereof (e.g., "administering" a compound) with respect to a compound of the present invention means introducing the compound into the system of a subject in need of treatment. When a compound of the present invention is provided in combination with one or more other active agents, "administration" and variations thereof are understood to include simultaneous and sequential introduction of the compound and the other agent, respectively.

[0070] As used herein, the term "composition" is intended to encompass a product containing the specified ingredients in the specified amounts, as well as any product resulting, directly or indirectly, from a combination of the specified ingredients in the specified amounts.

[0071] The term "therapeutically effective amount" as used herein refers to an amount of an active compound or pharmaceutical agent that induces a biological or medical response in a tissue, system, animal or human as desired by a researcher, veterinarian, physician, or other clinician. In the context of treating cancer, an effective amount includes an amount sufficient to prevent clinical disease or reduce the severity of disease as manifested by clinical disease, clinical symptoms. In some embodiments, an effective amount is an amount sufficient to delay the onset of clinical disease and / or symptoms or to prevent disease. An effective amount may be administered in one or more doses.

[0072] As used herein, "treatment" refers to obtaining beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, reduction in extent of disease, stabilized (i.e., not worsening) disease state or symptoms of disease, prevention or slowing of disease spread, prevention, slowing, slowing of disease progression, and / or maintenance of weight / weight gain. The methods of the present invention contemplate any one or more of these aspects of treatment.

[0073] A "pharmacologically acceptable" component refers to a component that is suitable for use in humans and / or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio.

[0074] A "safe and effective amount" refers to the amount of a component that, when used in the methods of the invention, is sufficient to effect a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response), commensurate with a reasonable benefit / risk ratio.

[0075] As used herein, a "promoter" or "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding or non-coding sequence. For purposes of defining the present invention, a promoter sequence is bounded at its 3' end by a transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a level detectable above background. Within the promoter sequence, a transcription initiation site is found, as well as protein binding domains involved in the binding of RNA polymerase. A variety of promoters, including inducible promoters, may be used to drive vectors as described in the present invention.

[0076] The promoter may be a constitutively active promoter (i.e., a promoter that is in a constitutively active ("ON") state) or it may be an inducible promoter (i.e., a promoter whose state is active ("ON") or inactive ("OFF")) that is controlled by an external stimulus (e.g., temperature, the presence of a particular compound, or protein).

[0077] As used herein, a synthetic prokaryotic promoter is a non-naturally occurring DNA sequence that is rationally designed to have transcription-enhancing activity in bacterial cells (i.e., contains -35, -10, and UP sequences that recruit RNA polymerase). As used herein, a synthetic prokaryotic terminator is a non-naturally occurring DNA sequence that is rationally designed to have transcription-terminating activity in bacterial cells (i.e., contains sequences that terminate transcription via intrinsic or Rho-dependent mechanisms). Furthermore, a "synthetic" DNA sequence is a DNA sequence that does not exist in nature and is rather artificially created (e.g., by bioinformatics or in silico techniques) for a specific purpose.

[0078] Antibodies, or immunoglobulins (Ig), are large Y-shaped proteins with a size of about 150-160 kDa, which consist of two heavy and two light protein chains, and are even smaller than the Fab fragment (about 50 kDa, one light chain and half a heavy chain) and the single-chain variable fragment (about 25 kDa, two variable domains, one from the light chain and one from the heavy chain).

[0079] As used herein, the term "antibody derivative" is a polypeptide whose amino acid chain (i.e., primary sequence) adopts a tertiary folded structure that shares structural homology with any domain or subdomain found in an antibody and / or which structure enables the polypeptide to interact with another cellular component.

[0080] "Affibodies" or affibody molecules are small, robust proteins engineered to bind to target proteins (e.g., antigens) or peptides with high affinity. The binding of affibodies is similar to that of monoclonal antibodies, thus making affibodies antibody mimics. Affibodies can be used for molecular recognition in diagnostic and therapeutic applications.

[0081] "Nanobodies", or single domain antibodies (sdAbs), are antibody fragments consisting of one monomeric variable antibody domain. Like complete antibodies, nanobodies can selectively bind to specific antigens. Nanobodies have a molecular weight of only about 12-15 kDa, making nanobodies / single domain antibodies much smaller than typical antibodies (150-160 kDa) which are composed of two heavy protein chains and two light chains, and even smaller than Fab fragments (about 50 kDa, one light chain and half a heavy chain) and single chain variable fragments (about 25 kDa, two variable domains, one from a light chain and one from a heavy chain).

[0082] V HH An antibody, or nanobody, is an antigen-binding fragment of an antibody that is a heavy chain only.

[0083] As used herein, the term "invasive" refers to a microorganism, such as a bacterium or a bacterial therapeutic particle (BTP), that can deliver at least one molecule, such as an antibody, an antibody derivative, and a protein / polypeptide, to a target cell. An invasive microorganism can be a microorganism that can pass through a cell membrane, thereby entering the cytoplasm of a cell, and deliver at least some of its contents, such as an antibody, an antibody derivative, and a protein / polypeptide, into a target cell. The process of delivering at least one molecule into a target cell preferably does not significantly modify the invasive device.

[0084] As used herein, the term "cellular targeting factor" is a moiety expressed on the surface of a bacterial cell that enables the bacterial cell to specifically interact with a particular type or class of eukaryotic cell (i.e., a target cell).

[0085] As used herein, the term "domain" or "protein domain" refers to a sequence of amino acids within the polypeptide chain of a protein that folds and self-stabilizes independently of the rest of the protein. Some domains fold into a compact three-dimensional structure.

[0086] As used herein, the terms "endogenous" or "endogenously expressed" when referring to antibodies, antibody derivatives, and proteins / polypeptides means that the antibodies, antibody derivatives, and proteins / polypeptides are naturally produced by an organism.

[0087] The term "interkingdom" as used herein refers to a delivery system that uses bacteria (or another invasive microorganism) to produce antibodies, antibody derivatives, and proteins / polypeptides and deliver them intracellularly (i.e., interkingdom: prokaryotes to eukaryotes, or across phyla: invertebrates to vertebrates) in target tissues to modulate the activity of target molecules in eukaryotic cells without integrating into the host genome. Bacteria can deliver the encoded antibodies, one V HH They are "non-pathogenic" with or without a specific expression cassette carrying an antibody domain, nanobody, and / or other antibody derivative (or other therapeutic nucleic acid). In some cases, however, the cargo carried and delivered by the bacteria may have a cytoregulatory or cytotoxic effect on the recipient eukaryotic cell. "Non-pathogenic bacteria" are bacteria that cannot cause disease and can be engineered to be non-pathogenic or are naturally non-pathogenic in the absence of a specific factor (e.g., polypeptide, antibody derivative) that the delivery system has been engineered to deliver to the target cell.

[0088] Invasive microorganisms include microorganisms that can naturally deliver at least one molecule to a target cell, such as by passing through a cell membrane, e.g., a eukaryotic cell membrane, and entering the cytoplasm, as well as microorganisms that are not naturally invasive and that have been modified, e.g., genetically modified, to be invasive. In another preferred embodiment, a naturally non-invasive microorganism 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" provides targeting, uptake, and / or export functions, and can be engineered as a chimeric factor (i.e., a recombinant protein encoded by a heterologous gene sequence fused in frame to another invasion factor or a fragment of its subunit). As used herein, an "invasion factor" is encoded by a "cell targeting gene". Invasive microorganisms are generally described in the art, for example, in U.S. Patent Nos. US20100189691(A1) and US20100092438(A1) and Xiang, S. et al., Nature Biotechnology 24, 697-702 (2006), the contents of each of which are incorporated by reference in their entirety for all purposes.

[0089] 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 delivering gene editing cargo.These non-toxic invasive bacteria and BTPs can exhibit or be modified to exhibit invasive properties and enter host cells via various mechanisms.In contrast to the uptake of bacteria or BTPs by professional 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-phagocyte host cells. Naturally occurring examples of such intracellular bacteria are Yersinia, Rickettsia, Legionella, Brucella, Mycobacterium, Helicobacter, Coxiella, Chlamydia, Neisseria, Burkholderia, 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, Bifidobacterium, including probiotics via the transfer 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 interkingdom delivery vehicles include the candidate's pathogenicity or lack thereof, the tropism of the candidate bacterium for target cells or, alternatively, the extent to which the bacterium can be engineered to deliver gene editing cargo inside target cells, and any synergistic value the candidate bacterium may provide by triggering the host's innate immunity.

[0090] Methods of administration of these improved bacterial delivery vehicles include intraperitoneal and intravenous administration for systemic delivery, intrathecal administration for CNS delivery, intramuscular injection for administration to skeletal muscles, intranasal administration to the nasal cavity for local action, aerosolization to target the upper and lower respiratory tract, ingestion in the oral cavity for absorption into the digestive tract for buccal delivery, application to the delicate genital mucosal epithelium, and topical administration for intraocular delivery. These improved delivery vehicles can be used to prevent and / or treat a wide range of diseases (infectious, allergic, cancerous, genetic, and immunological) in a wide range of species (human, avian, porcine, bovine, canine, equine, feline).

[0091] The term "administration" and variations thereof with respect to the compounds of the present invention (e.g., "administering" a compound) refers to the introduction of the compound into the system 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., cytotoxic agents), "administration" and variations thereof are understood to include simultaneous and sequential introduction of the compound and the other agent, respectively.

[0092] A "subject" is an organism belonging to any multicellular vertebrate phylum, including humans and non-human mammals (e.g., veterinary subjects). In one example, the subject is known to have or is suspected of having an infectious or other condition that is life-threatening or impairs the quality of life.

[0093] As used herein, the terms "treat" and "treatment" refer to the administration of an agent or formulation (e.g., a bacterium) of the invention to a clinically symptomatic subject having an adverse condition, disorder, or disease, thereby affecting a reduction in the severity and / or frequency of the symptoms, eliminating the symptoms and / or their underlying causes, and / or promoting the amelioration or repair of damage.

[0094] The terms "prevent" and "prevention" refer to the administration of an agent or composition to a clinically asymptomatic individual susceptible to a particular deleterious condition, disorder, or disease, and thus relate to the prevention of the occurrence of the symptoms and / or their underlying causes.

[0095] As used herein, a "biologically active peptide" is a peptide that has an effect on a living organism, tissue, cell, or biochemical process. A biologically active peptide can be grafted onto the Fc domain or other antibody domains of an antibody or antibody derivative (e.g., a peptibody) to deliver the function of the biologically active peptide to a target cell along with the antibody or antibody derivative to which it is grafted. The effect of the biologically active peptide can be that of antioxidant properties, antimicrobial properties, immunomodulatory properties, cell modulating properties, and / or metabolic altering properties.

[0096] The antibodies, antibody derivatives, and invasive bacteria containing proteins / polypeptides can be introduced into a subject by intravenous, intramuscular, intradermal, intraperitoneal, oral, intranasal, intraocular, intrarectal, intravaginal, intraosseous, oral, immersion, and intraurethral inoculation routes. The amount of the invasive bacteria of the present invention administered to a subject will vary depending on the species of the subject, as well as the disease or condition being treated. For example, the dosage may be about 10 mg / subject. 3 ~10 11 of viable organisms, preferably about 10 5 ~10 9 The invasive bacteria or BTP of the present invention may be a live organism. The invasive bacteria or BTP of the present invention are generally administered with a pharma- ceutically acceptable carrier and / or diluent. In some cases, the invasive bacteria or BTP of the present invention are formulated as a dry powder, lyophilized, or freeze-dried.

[0097] Those skilled in the art can easily determine the appropriate dose of one of the compositions of the present invention for administering to a subject without undue experimentation.Typically, a doctor will determine the actual dosage that is most suitable for an individual patient, which depends on a variety of factors, including the activity of the specific compound used, the metabolic stability and duration of action of the compound, age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the specific condition, and the individual undergoing treatment.The dosage disclosed herein is an example of the average case.Of course, there may be individual cases where higher or lower dosage ranges are advantageous, and such are within the scope of the present invention.

[0098] For administration by inhalation, the pharmaceutical composition for use according to the present invention is conveniently delivered in the form of an aerosol spray from a pressurized pack or nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount.Capsules and cartridges, for example of gelatin, for use in an inhaler or insufflator can be formulated containing a powder mix of the composition, for example, bacteria, and a suitable powder base, such as lactose or starch.

[0099] The pharmaceutical composition may be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. The preparation for injection may be provided in unit dosage form, for example in ampoules, or in multi-dose containers, with the addition of preservatives. The composition of the present application may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and may contain formulating agents such as suspending agents, stabilizing agents and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water.

[0100] Invasive bacteria containing the introduced antibodies, antibody derivatives, and proteins / polypeptides can be used to infect animal cells cultured in vitro, such as cells obtained from a subject. These in vitro infected cells can then be introduced into the animal, e.g., the subject from whom the cells were originally obtained, intravenously, intramuscularly, intradermally, or intraperitoneally, or by any route of inoculation that allows the cells to enter the host tissue. When delivering antibodies, antibody derivatives, and proteins / polypeptides to individual cells, the dose of live organisms administered is about 0.1-10 per cell. 6 , preferably about 10 2 ~10 4 The multiplicity of infection is the range of bacteria.

[0101] Further provided is a kit for carrying out the method of the present invention. By "kit" is intended any article of manufacture (e.g., package or container) that contains at least one reagent, such as the pH buffer of the present invention. The kit may be advertised, distributed, or sold as a unit for carrying out the method of the present invention. In addition, the kit may include a package insert that describes the kit and the method 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.

[0102] In an advantageous embodiment, the kit container may further comprise a pharma- ceutically acceptable carrier. The kit may further comprise a sterile diluent, which is preferably stored in a separate additional container. In another embodiment, the kit further comprises a package insert comprising printed instructions directing the use of the combined treatment of the pH buffer and the anti-pathogen agent as a method for treating and / or preventing disease in a subject. The kit may also comprise an additional container containing an additional anti-pathogen agent (e.g., amantadine, rimantadine, and oseltamivir), an agent that enhances the effect of such an agent, or another compound that improves the efficacy or tolerability of the treatment.

[0103] The advantages set forth above, and those made apparent from the preceding description, are efficiently attained. Since certain changes may be made in the above configurations without departing from the scope of the invention, it is intended that all matter contained in the above description or shown in the accompanying drawings be interpreted as illustrative and not in a limiting sense.

[0104] All references cited in this application are hereby incorporated by reference in their entirety to the extent not inconsistent herewith.

[0105] Since it is clear that the above-mentioned advantages, and those made apparent from the foregoing description, are efficiently attained, and certain changes may be made in the above-described configurations without departing from the scope of the invention, it is intended that all matter contained in the foregoing description or shown in the accompanying drawings be interpreted as illustrative and not in a limiting sense.

[0106] It is also to be understood that the following claims are intended to cover all of the general and specific features of the invention described herein, and, where language is concerned, all statements of the scope of the invention that may lie therebetween.The invention is now described.

[0107] [Table 2]

[0108] [Table 3]

[0109] [ka]

Claims

**Claim 1** A system for producing a protein and delivering the protein to a eukaryotic cell, the system comprising bacteria engineered to be invasive and to have at least one expression cassette encoding a protein that is exogenous to the bacteria, wherein transcription of the nucleic acid encoding the exogenous protein is under the control of a prokaryotic promoter and terminator. **Claim 2** The system for producing a protein and delivering the protein to a eukaryotic cell according to claim 1, wherein the prokaryotic promoter and terminator are synthetic, whereby the promoter and terminator have the activity of promoting or terminating transcription in Escherichia coli. **Claim 3** The system for producing a protein and delivering the protein to a eukaryotic cell according to claim 1, wherein the encoded protein is an antibody or antibody derivative, and optionally, the antibody or antibody derivative consists essentially of a single protein domain extracted from a multi-domain antibody. **Claim 4** The system for producing a protein and delivering the protein to a eukaryotic cell according to claim 3, wherein the antibody or antibody derivative comprises a biologically active peptide grafted onto the Fc domain or other antibody domain (e.g., peptibody) of the antibody or antibody derivative, and optionally, the biologically active peptide is selected from the group of peptides having antioxidant, antimicrobial, immunomodulatory, cytomodulatory, and / or metabolic-altering properties or effects. **Claim 5** The system for producing a protein and delivering the protein to a eukaryotic cell according to claim 3, wherein the structural domain of the antibody or antibody derivative has an amino acid sequence that binds to an epitope targeting an intracellular protein, and the intracellular protein is a therapeutically relevant protein or is therapeutically relevant as a binding target for the antibody or antibody derivative. **Claim 6** A system for producing the protein according to claim 3 and for delivering the protein to a eukaryotic cell, wherein the antibody or antibody derivative biologically inactivates the target protein by forming a complex with the target protein, thereby hiding, occupying, or otherwise interfering with a binding site or epitope important for the interaction of the target protein with other molecules, and a non-complexed target that is biologically active regulates a specific activity or cellular pathway in the eukaryotic cell.

7. A system for producing the protein according to claim 3 and for delivering the protein to a eukaryotic cell, wherein the antibody or antibody derivative binds to an intracellular factor inside a cancer cell to regulate a specific activity or cellular pathway, including those related to cell survival, proliferation, and sensitivity to chemotherapeutic agents, such that the antibody or antibody derivative has an anti-tumor formation effect, and optionally, the intracellular factor bound by the antibody or antibody derivative inside the cancer cell is a mutated HRAS,NRAS, or KRAS protein.

8. A system for producing the protein according to claim 3 and for delivering the protein to a eukaryotic cell, wherein binding by the antibody or antibody derivative regulates a specific activity or cellular pathway, thereby improving the therapeutic effectiveness of a chemotherapeutic agent or other therapy administered to or performed on a subject.

9. A system for producing the protein according to claim 3 and for delivering the protein to a eukaryotic cell, wherein the antibody or antibody derivative includes a region that binds to an epitope on an apoptosis regulatory protein or apoptosis-related protein, and optionally, the antibody or antibody derivative includes a region that binds to a protein including survivin (BIRC5), BCL-2, MCL-1, XIAP, BRUCE, or any other inhibitor of apoptosis (IAP) family protein, or a protein including one or more characteristic BIR domains.

10. A system for producing the protein according to claim 3 and for delivering the protein to a eukaryotic cell, wherein the antibody or antibody derivative includes a region that binds to an epitope on a viral, bacterial, protozoan, or fungal protein, and binding to the epitope inhibits the replication of the virus, bacterium, protozoan, or fungus.

11. A system for producing the protein according to claim 1 and for delivering the protein to a eukaryotic cell, wherein the bacterium is a non-pathogenic bacterium engineered to have at least one invasion factor to promote the invasion of the non-pathogenic bacterium into the eukaryotic cell or to cause the release of the non-pathogenic bacterium from the phagosome of the eukaryotic cell.

12. The invasion factor is a) encoded by the inv, hlyA, or hlyE gene or any fragment thereof, or by a chimeric or recombinant form thereof, b) a chimeric, recombinant invasion protein comprising the non-binding domain of an invasion protein fused to a binding domain from a heterologous protein, optionally, the binding domain from the heterologous protein being selected from the group consisting of GalNAc-binding proteins, lectins, groups of cell adhesion molecules (CAMs), groups of sulfated glycosaminoglycan (GAG)-binding proteins, selectins, integrins, laminin, cadherin, fibronectin, collagen, thrombospondin, vitronectin, tenascin, apolipoproteins B, E, and A-V, lipoprotein lipase, hepatic lipase, Siglec, galectin, immunoglobulins, and annexin, FimH, papG, PrsG, Afa-IE, DraA, MrpH, RodA, Mpl, hydrophobin, heat shock proteins, CspA, hemagglutinin, neuraminidase, capsid proteins, glycoproteins, and binding domains of envelope proteins, c) engineered to be present on the chromosome of the bacterium, or d) encoded by the invasion proteins FimH, OmpA, IbeA, IbeB, IbeC, Opc, PilA, PilB, LOS, Lmb, FbsA, IagA, Vsp1, OspA, 70-kDa PBP, enolase, Isc1, Yps3p, Stx, type III secretion system injection factors, EspF, Map, EspG), or fragments, chimeric or recombinant forms thereof, of a system for producing the protein according to claim 11 and for delivering the protein to a eukaryotic cell.

13. A system for producing the protein according to claim 1 and for delivering the protein to a eukaryotic cell, wherein the bacterium is a non-pathogenic bacterium engineered to have at least one cell targeting factor (a surface-expressed moiety).

14. The expression cassette is on a plasmid having a length of about 7,000 base pairs or less, whereby a decrease in plasmid size as compared to a larger plasmid reduces the plasmid-induced load on the host bacterial cell, thereby increasing the growth rate of the bacteria. A system for producing the protein according to claim 1 and for delivering the protein to a eukaryotic cell.

15. The protein encoded by the invasive bacterium is delivered to the cytoplasm of the target eukaryotic cell and the protein is functional in the eukaryotic cell and compensates for a clinically significant deficiency in the endogenous level of the protein. A system for producing the protein according to claim 1 and for delivering the protein to a eukaryotic cell to increase the level of the protein in the target eukaryotic cell.