Tertiary structure stabilizing domain for chimeric receptor binding protein

Abstract

The present invention relates to a protein comprising a knot-like domain, and stabilization of a tertiary structure of a chimeric receptor binding protein (chimeric RBP) using same. The protein comprising a knot-like domain, according to the present invention, has an effect of providing structural stability in generating a chimeric RBP by fusing two different domains derived from different species, and, in particular, is excellent at stabilizing a triple-stranded helical bundle structure in a shaft domain so as to enable binding of receptor binding domains (RBDs) derived from various different species, and thus can be effectively used in virus or bacteriophage engineering for changing or expanding host selectivity.

Description

tertiary structure stabilization domain of chimeric receptor binding protein

[0001] The present invention relates to a knot-like domain that can be usefully used for engineering viruses, particularly bacteriophages, and to the stabilization of the tertiary structure of helical bundles using the same. Specifically, the present invention relates to a protein containing a knot-like domain, a chimeric receptor binding protein (chimeric RBP) containing the same, and an engineered virus, etc.

[0002] This application claims priority based on Korean Patent Application No. 10-2025-0012273 filed on January 31, 2025, and all contents disclosed in the specification and drawings of said application are incorporated into this application.

[0003]

[0004] In modern medicine, antibiotics have been utilized as a key tool for treating infectious diseases. However, due to the widespread use and overuse of antibiotics, antibiotic-resistant pathogens, such as multidrug-resistant bacteria, are rapidly increasing. These resistant bacteria cause infections that are difficult to control with existing treatments, and this has emerged as a major health issue causing over 700,000 deaths worldwide annually. Due to the rise of such drug-resistant bacteria and the rapid decline in the discovery of new antibiotics, therapeutic approaches using bacteriophages are once again garnering attention.

[0005] Bacteriophages are viruses that selectively infect only bacteria; they are attracting attention as next-generation antimicrobial agents because they can effectively eliminate not only antibiotic-susceptible bacteria but also antibiotic-resistant ones. Bacteriophages have the advantage of high host specificity, allowing them to selectively eliminate pathogenic bacteria without affecting the gut commensal flora. However, naturally occurring bacteriophages must meet strict conditions to be used as therapeutic agents, and finding ideal therapeutic bacteriophages is a complex process that requires significant time and capital. Natural phages act only on specific pathogens, and due to frequent mutations within pathogens, they cannot target all strains. Some natural phages can deliver toxin genes or antibiotic resistance genes into host cells, making direct medical use difficult. Furthermore, since viruses and bacteriophages generally have a very narrow host range, it is often necessary to develop "cocktail" combinations of viruses or phages to target specific pathogenic populations. In this case, the development process is complex and requires a longer development period because stability, efficacy, storage conditions, etc., must be verified separately for each virus or phage.

[0006] To address these issues, active research is underway to redesign bacteriophages using genetic engineering techniques to confer new host ranges or enhance the infectivity of existing phages. Advancements in these technologies have primarily focused on redesigning receptor-binding proteins through genetic modification of viruses to enable them to recognize and infect new host cells. For example, in the case of adenoviruses, techniques are used to expand the infectivity range by fusing specific peptides to fiber proteins, or to alter the host range by replacing fiber proteins or fiber domains with those derived from other adenovirus serotypes. Similarly, for bacteriophages, research is being conducted to target new bacterial species by modifying tail fiber proteins or receptor-binding proteins (RBPs). In other words, host selectivity can be altered or regulated by changing the receptor-targeting function of RBPs through the replacement of a bacteriophage's spike or fiber protein with that of another bacteriophage.

[0007] However, when the entire fibrous or spike protein is replaced for RBP modification, if the structure of the base domain of the proximal end—which self-assembles into the bacteriophage tail or viral capsid—is not identical to that of the original protein, there is a high likelihood that normal self-assembly will be disrupted due to incompatibility of the protein-protein binding interface. This is particularly pronounced when the inter-partition interactions required for multimer structure formation, polypeptide folding pathways, and structural motifs mediating binding signals with the bacteriophage or virus capsid are incompatible with the existing base domain. To overcome this problem, it is necessary to preserve the natural self-assembly process intact by maintaining the structural integrity of the base domain that binds to the capsid or tail. Therefore, a method is utilized to effectively design chimeric receptor-binding proteins (chimeric RBPs) by leaving the basal domain intact while replacing only the shaft domain or the subsequent receptor binding domain (RBD) with a sequence possessing the characteristics of a different host cell target molecule. However, this strategy also carries the risk that the tertiary structure of the shaft domain (particularly the helical bundle structure) will collapse.

[0008] The shaft domain serves as the central axis providing mechanical stability throughout the entire RBP. In the spike and fiber proteins of many bacteriophages and viruses, the shaft forms a triple-helix bundle; maintaining this helical bundle structure requires a close interplay of various factors, including repeating pattern complementarity, domain boundary suitability, and interface compatibility. In particular, if a different sequence is inserted or replaced in the shaft region during heterogeneous fusion, the existing repeating pattern may be disrupted, potentially causing the hydrophobic core to collapse. Furthermore, if the physicochemical properties of interfacial amino acids change significantly, the interstrand packing angle may shift, making it difficult to maintain the shape of the original helical bundle.

[0009] To address the above problems, methods such as selecting the replacement point between the shaft and the RBD to align as much as possible with a 'domain boundary' where secondary structural elements or repeating patterns are clearly distinguishable, or inserting a short linker at the junction between the shaft domain and the RBD, may be considered. However, to date, there is no method or linker that can be universally used for the fusion of heterogeneous shafts and RBDs when designing various heterogeneous RBPs to modify the host selectivity of viruses or bacteriophages.

[0010]

[0011] The present invention aims to solve all the problems of the aforementioned prior art.

[0012] One objective of the present invention is to provide a knot-like domain that can be used more universally than existing methods or linkers and guarantees structural integrity when generating a new RBP by fusing parts of receptor binding proteins (RBPs) derived from heterogeneous viruses (or bacteriophages) together.

[0013] Specifically, the present invention has one objective of providing a knot-like domain capable of stabilizing the tertiary structure of a shaft domain and a receptor binding domain (RBD) derived from a heterogeneous virus (or bacteriophage) when fused.

[0014] In addition, the present invention has another objective of providing a chimeric receptor binding protein (chimeric RBP) comprising the above-mentioned knot-like domain.

[0015] In addition, the present invention has another objective of providing an engineered virus comprising the chimeric RBP, or a vector comprising a polynucleotide encoding the same.

[0016] In addition, another objective of the present invention is to provide a pharmaceutical composition comprising the engineered virus or the vector as an active ingredient.

[0017] In addition, another objective of the present invention is to provide a method for stabilizing the tertiary structure of a chimeric RBP using the above-mentioned knot-like domain.

[0018] The objectives of the present invention are not limited to those mentioned above. The objectives of the present invention will become more apparent from the following description and will be realized by the means and combinations thereof described in the claims.

[0019]

[0020] A representative configuration of the present invention for achieving the above objective is as follows.

[0021] According to one aspect of the present invention, a protein is provided comprising a knot-like domain composed of a plurality of subunits, wherein each of the subunits comprises (i) a β-sheet structure in which at least four β-strands are arranged anti-parallel; and (ii) at least one loop or short α-helix connecting the β-strands, wherein the plurality of subunits are assembled into a multimer by protein folding and simultaneously form a knot-shaped topology, and wherein the knot-like domain is connected between (a) a shaft domain comprising a helix bundle structure derived from viruses of different species; and (b) a receptor binding domain (RBD), thereby stabilizing the tertiary structure of the helix bundle.

[0022] According to another aspect of the present invention, a chimeric receptor binding protein (chimeric RBP) is provided, wherein (a) a shaft domain derived from a first virus and comprising a helix bundle structure; (b) a protein comprising a knot-like domain according to the present invention; and (c) a receptor binding domain (RBD) derived from a second virus heterogeneous to the first virus are sequentially linked.

[0023] According to another aspect of the present invention, a method for stabilizing the tertiary structure of a chimeric RBP is provided, comprising the step of introducing a protein containing the knot-like domain between a shaft domain containing a helix bundle structure and a heterologous receptor binding domain (RBD).

[0024] Generally, bacteriophage tail fiber proteins and tail spike proteins, as well as viral capsid spike proteins and viral capsid fiber proteins, function to bind to receptors present on the surface of host cells in order to recognize and bind to specific host cells; these are referred to as receptor binding proteins (RBPs). These RBPs have a multiplexed structure, and in many cases, a homo-trimer structure.

[0025] The structure of the aforementioned receptor-binding protein (RBP) can generally be divided into a proximal end, a distal end, and a shaft or stem, constituting a functional module. The proximal end includes a base domain that binds to the tail portion of a bacteriophage or the capsid of a virus, while the distal end includes a knob-shaped host binding domain (HBD) or receptor binding domain (RBD) suitable for binding to receptors on the surface of host cells. The region referred to as the shaft or stem refers to a rod-shaped connection domain between the anchorage site and the host binding domain.

[0026] Host selectivity can be altered or regulated by changing the receptor targeting function of receptor-binding proteins (RBPs) through the redesign of spike or fibrous proteins of bacteriophages or viruses. Host selectivity can refer, in a narrow sense, to the targeting of different strains within the same species as the target host cell, or in a broad sense, to the selectivity between different species that are less phylogenetically related to the target host cell.

[0027] In this case, if the entire fibrous protein or spike protein of a different type of phage or virus with low structural similarity is replaced, there is a high probability that it will not self-assemble normally into the bacteriophage tail or viral capsid if the structure of the base domain of the proximal end, which self-assembles into the bacteriophage tail or viral capsid, is not identical to that of the base domain to which the original fibrous protein or spike protein was bound. Therefore, a chimera RBP can be designed by not replacing the entire fibrous protein or spike protein, but by maintaining their existing base domains to ensure there are no issues with self-assembly, and by replacing the shaft domain or the host binding domain (RBD) following the upper domain with the host binding domain (RBD) of the spike or fibrous protein of a bacteriophage or virus that targets a different host cell.

[0028] However, even with the above design strategy, there is a possibility that structural deformation or instability may occur due to chimeric fusion. That is, if a heterogeneous host binding domain (RBD) is fused to the shaft domain, the heterogeneous fusion binding protein may not be able to fold normally.

[0029] Accordingly, the inventors completed the present invention by devising a strategy of introducing a knot-like domain to stabilize the helical bundle structure of a heterogeneous fusion binding protein, particularly the shaft domain, in order to produce a bacteriophage or virus having a new host range by assembling a chimeric receptor binding protein in which the host receptor binding domain has been replaced.

[0030] [Correction pursuant to Rule 91 13.02.2026] A knot-like domain according to the present invention is connected between a shaft domain containing a helix bundle structure and a receptor binding domain (RBD) derived from a virus of a different species from said domain, thereby stabilizing the tertiary structure of said helix bundle. According to Example 2 and FIG. 2, when a heterogeneous shaft domain and a receptor binding domain are fused, a phenomenon in which the helix bundle structure within the shaft domain collapses occurs in many cases. This structural collapse phenomenon can be resolved by introducing a knot-like domain according to the present invention between the two heterogeneous domains.

[0031] In addition to these internal structural stabilization effects, knot-like domains provide additional mechanical stability. That is, long, delicate tail fibers are subjected to significant physical stress as they approach host cells; knot-like structures, such as knot-like domains, can act like molecular springs to disperse excessive forces and maintain the structure.

[0032] Furthermore, knot-like domains have the effect of regulating flexibility upon receptor binding. That is, when the shaft needs to move flexibly while the distal end binding to the receptor contacts the host surface, the knot structure can act as a buffer, preventing excessive deformation while allowing a certain degree of rotation and bending.

[0033] The structural stability enhancement effect of the knot-like domain described above also helps increase infection efficiency and ensure specificity. In other words, by stabilizing the three-dimensional arrangement of the protein through the tail fibers via the knot structure, host-specific receptor recognition can be made more sophisticated.

[0034] In one embodiment, the knot-like domain is a multimer comprising 2 to 6 subunits. Preferably, the knot-like domain may be a homo-trimer comprising 3 identical subunits.

[0035] In another embodiment, the knot-like domain may form a tertiary structure through non-covalent interactions between each subunit.

[0036] In another embodiment, the non-covalent interaction may be formed between a beta strand within one subunit and a beta strand within another subunit. Specifically, the non-covalent interaction may be formed between (i) any one of at least four beta strands arranged continuously from the N-terminus to the C-terminus within one subunit; and (ii) beta strands existing at the same relative position and / or adjacent relative position within another subunit. For example, a non-covalent interaction may be formed between an amino acid residue in the first beta strand of the first subunit and an amino acid residue in the second beta strand of the second subunit.

[0037] In another embodiment, the non-covalent interaction may be selected from the group consisting of ionic bonds, hydrogen bonds, and hydrophobic interactions. For example, a plurality of hydrophobic interactions and a plurality of ionic bonds may be formed between the first subunit and the second subunit. Alternatively, a plurality of hydrophobic interactions and a plurality of hydrogen bonds may be formed between the second subunit and the third subunit. These non-covalent interactions may vary depending on the properties of the amino acid residues constituting the beta strand within each subunit, but they commonly function to provide structural stability to the knot-like domain.

[0038] In another embodiment, the distance between the two amino acids where the non-covalent interaction is formed may be about 3 Å to 5 Å. For example, hydrophobic interactions or van der Waals interactions may be formed in the range of about 3.4 Å to 4.0 Å. When the side chains of the corresponding amino acids are placed within the distance range, the likelihood of them attracting each other or forming spatial interlocking through non-covalent interactions increases, and as a result, contributes to the tertiary structural stability of the entire knot-like domain.

[0039] The term “about” as used in the present invention means an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that varies by about 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% with respect to a reference amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length. For example, when the term “about” is used in relation to a value x expressed as a number or numerical value, it may mean x ± 10%.

[0040] In another embodiment, each beta strand within the subunit may consist of 3 to 12 amino acids.

[0041] In another embodiment, the beta strands within the subunit may be 4 or 5, preferably 4.

[0042] In another embodiment, the subunit may include two or more short alpha helix structures.

[0043] In another embodiment, the short alpha helix within the subunit may consist of 3 to 15 amino acids.

[0044] In another embodiment, the amino acid sequence of the subunit may include a sequence derived from the wild-type sequence of the subunit constituting the knot-like domain present in the tail fiber protein of a bacteriophage, or an amino acid sequence having a sequence similarity of 70% or more with said sequence.

[0045] In another embodiment, the bacteriophage may be selected from the group consisting of Enterococcus phage, Staphylococcus phage, Pseudomonas phage, Acinetobacter phage, and T4 phage.

[0046] In another embodiment, the wild-type sequence of the subunit constituting the knot-like domain may include any one of the amino acid sequences of SEQ ID NOs 6 to 9 and SEQ ID NOs 31 to 37. Here, the amino acid sequences of SEQ ID NOs 6 to 9 are each derived from a part of the amino acid sequences indicated by SEQ ID NOs 1 to 5, which are knot-like domains found in the upper basal plate protein of Enterococcus phage. For example, the amino acid sequence of SEQ ID NO 7 is a sequence constituting one subunit of the knot-like domain and is derived from the upper basal plate protein sequence of Enterococcus phage indicated by the amino acid sequence of SEQ ID NO 2 or SEQ ID NO 3.

[0047] In another embodiment, the number of subunits may be equal to the number of helical strands within the helical bundle structure. For example, if the knot-like domain is homotripty, the shaft domain forms a triple-stranded helical structure. In this case, the fused receptor binding domain (RBD) may also be tritripty.

[0048] In another embodiment, the virus may be a virus belonging to a class or family selected from the group consisting of Caudoviricetes, Adenoviridae, Reoviridae, and Orthomyxoviridae, but the type of virus or bacteriophage from which each domain is derived is not limited to the said family. Here, the virus belonging to the class Codoviricetes may preferably belong to a family selected from the group consisting of Straboviridae, Herelleviridae, Autographiviridae, Demerecviridae, Drexlerviridae, Ackermannviridae, Chaseviridae, Schitoviridae, and Peduviridae.

[0049] In another embodiment, the virus may be selected from the group consisting of Enterococcus phage, Lactococcus phage, Staphylococcus phage, Salmonella phage, Listeria phage, Pseudomonas phage, Acinetobacter phage, Escherichia phage, adenovirus, reovirus, and influenza virus, but the type of virus or bacteriophage from which each domain is derived is not limited thereto.

[0050] In another embodiment, the helical bundle structure may be composed of 2 to 6 helical strands. The helical strands may have a structure selected from the group consisting of, for example, alpha-helix, beta-helix, and beta-spiral as a unit structure of the helical bundle structure.

[0051] In another embodiment, the helical strand may include part or all of the helical strand sequence contained within any one of the amino acid sequences of SEQ ID NOs 10 to 30.

[0052] In another embodiment, the helix bundle structure may be a triple-stranded helix bundle (TSHB) in which three helix strands form a coiled coil structure.

[0053] In another embodiment, the RBD of the second virus may have a different host range compared to the RBD of the first virus. For example, the RBD of the second virus may have an extended host range compared to the RBD of the first virus.

[0054] In another embodiment, the shaft domain or RBD may be a shaft domain or RBD contained in any one protein selected from the group consisting of a phage-derived tail fiber protein, a phage-derived tail spike protein, a virus-derived capsid spike protein, and a virus-derived capsid fiber protein, or a shaft domain or RBD having a sequence similarity of 70% or more to such a protein. For example, the RBD may be an RBD present inside the tail spike protein of Salmonella Enteritidis phage P22. Alternatively, the shaft domain may be a shaft domain of a tail fiber protein derived from Staphylococcus aureus phage P68. Variants of the sequences of these domains having a sequence similarity of 70% or more are also included within the scope of the present invention.

[0055] In another embodiment, the shaft domain may be connected to the N-terminal direction of the knot-like domain; and the RBD may be connected to the C-terminal direction of the knot-like domain.

[0056] According to another aspect of the present invention, an engineered virus comprising the chimeric RBP is provided.

[0057] In one embodiment, the engineered virus may be a bacteriophage capable of infecting a specific group of bacteria.

[0058] In another embodiment, the engineered virus may have its ability to reinfect a host removed.

[0059] In another embodiment, the engineered virus may further include a payload. For example, the payload may include a therapeutic protein or a functional variant thereof; an antibody or antibody fragment; an enzyme; a component of a gene editing system; an RNAi preparation; or a combination thereof.

[0060] In another embodiment, the payload may be a substance exhibiting an anticancer effect, a gene expressing the same, or a factor that promotes the secretion or expression of the substance or gene.

[0061] According to another aspect of the present invention, a pharmaceutical composition comprising the engineered virus as an active ingredient is provided.

[0062] In one embodiment, the pharmaceutical composition may be a pharmaceutical composition for the prevention or treatment of cancer.

[0063] According to another aspect of the present invention, a vector comprising a polynucleotide encoding the engineered virus operably connected to a promoter is provided.

[0064] In one embodiment, the vector may further include a polynucleotide encoding a payload. For example, the polynucleotide encoding the payload may be a polynucleotide encoding a gene that expresses a substance exhibiting an anticancer effect or a factor that promotes the expression thereof.

[0065] According to another aspect of the present invention, a pharmaceutical composition comprising the vector as an active ingredient is provided.

[0066] In one embodiment, the pharmaceutical composition may be a pharmaceutical composition for the prevention or treatment of cancer.

[0067] As used in the present invention, the term "treatment" generally means obtaining desired pharmacological and / or physiological effects. These effects are therapeutic in that they partially or completely cure the disease and / or side effects caused by such disease. Desired therapeutic effects include, but are not limited to, prevention of the onset or recurrence of the disease, improvement of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, improvement or alleviation of the disease state, and remission or improved prognosis. Preferably, "treatment" may mean medical intervention for an already manifested disease or disorder.

[0068] The term "prevention" as used in this invention refers to any act of suppressing symptoms of a target disease or delaying its progression through the administration of the composition of this invention.

[0069] In other embodiments, the pharmaceutical composition may be formulated according to the mode of administration used. For example, if the pharmaceutical composition is an injectable pharmaceutical composition, it may be preferable to use an isotonic formulation. Additives for isotonicity may generally include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In one embodiment, an isotonic solution such as phosphate-buffered saline is preferred. Examples of stabilizers include gelatin and albumin.

[0070] In another embodiment, the composition may further include a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be a functional molecule as a vehicle, adjuvant, carrier, or diluent.

[0071] (Pharmaceutical) The actual dosage of a composition may vary significantly depending on various factors, such as the concentration of the active ingredient (e.g., an engineered virus or a vector encoding it), the site of application, the condition of the target to be treated, the route of administration, and the method of administration. The composition may be manufactured and supplied in various formulations, such as injectables, oral preparations, and topical preparations. In the case of engineered viruses or vectors for the treatment or prevention of cancer, injectable forms are generally commonly used. In particular, viruses or vectors intended for gene delivery may be administered via intravenous (IV), intramuscular (IM), subcutaneous (SC), or intratumoral injection (IT) into the lesion (cancer tissue).

[0072] The above pharmaceutical composition may include pharmaceutically acceptable excipients (e.g., physiological saline, buffer solution, sugar solution, etc.), and stabilizers, preservatives, viscosity enhancers, etc. may be used as formulation aids. Additionally, surfactants or emulsifiers may be added to ensure effective solubility.

[0073]

[0074] The protein containing a knot-like domain according to the present invention has the effect of providing structural stability when generating a chimeric RBP by fusing two different domains derived from different species. Specifically, the protein containing the knot-like domain binds between two different domains of heterogeneous origin to provide tertiary structural stability, and is particularly excellent at stabilizing the triple-stranded helix bundle structure within the shaft domain, thereby enabling the fusion of various heterogeneous receptor binding domains (RBDs), and thus can be usefully utilized in the engineering of viruses or bacteriophages to modify or expand host selectivity.

[0075]

[0076] Figure 1 illustrates examples of various forms of triple-stranded helix bundle (TSHB) structures found in naturally occurring proteins or artificially synthesized:

[0077] A, Avian reovirus fusion domain; B, N-terminus of Escherichia coli triautotransporter adesin EibD; C, Heteromer region of laminin-111; D, Phage 22 tail-needle gp26; E, Synthetic triple-stranded alpha-helical bundle; F, Fibritin deletion mutant E bacteriophage T4; G, Influenza hemagglutinin pH4.0; H, Influenza hemagglutinin; I, Type C mannose-binding protein; J, Phage structural protein [Clostridioides difficil]; K, Baseplate upper protein [Lactococcus phage TP901-1]; L, BppU family phage baseplate upper protein [Enterococcus sp. BWR-S5]; M, Phage baseplate upper protein [Enterococcus faecalis]; N, Baseplate upper protein [Lactococcus phage Tuc2009]; O, Phage baseplate upper protein [Enterococcus faecium]; P, Virtual protein [Enterococcus]; Q, Virtual protein OIC_03754 [Enterococcus faecium EnGen0007]; R, Baseplate / receptor binding protein [Lactococcus phage 50902]; S, Phage baseplate upper protein [Listeria]; T, Phage baseplate upper protein [Listeria monocytogenes]; U, KE1 synthetic triple-stranded alpha helical bundle.

[0078] [Correction pursuant to Rule 91 13.02.2026] Figure 2 shows an example of a chimera RBP in which the structure of TSHB is maintained or collapsed by fusion of heterologous RBDs, as a result of generating a fusion receptor-binding protein (chimera RBP) by combining the receptor-binding domain (RBD) of Staphylococcus aureus phage P68 tail fiber protein with TSHB and performing structure prediction through alphafold.

[0079] Figure 3 illustrates the three-dimensional structure of a knot-like domain, a knot-shaped structure found in the tail fiber proteins of some bacteriophages and existing between the shaft domain and the distal end (RBD).

[0080] Figure 4 illustrates the results of evaluating the structural stability of the fused protein through a methodology for introducing a knot-like domain between TSHB and RBD and AlphaFold-based structure prediction as a strategy to overcome the structural instability of TSHB caused by the fusion of heterogeneous RBDs.

[0081] [Correction pursuant to Rule 91 13.02.2026] Figures 5a and 5b illustrate examples of structural collapse of TSHB by the fusion of heterogeneous RBDs and results in the normal alignment of the TSHB helix by introducing a knot-like domain into the fusion protein in which such structural collapse of TSHB occurs.

[0082] Figures 6a and 6b illustrate the results of the knot-like domain maintaining the structural stability of TSHB when the RBD of a heterologous tail fiber protein or tail spike protein is fused to the shaft domain: Figure 6a shows the stability of the three-dimensional structure with and without the knot-like domain when the P68 tail fiber protein of Staphylococcus aureus phage is fused to the heterologous shaft domain (top) and when the P22 tail spike protein of Salmonella Enteritidis phage is fused (bottom); Figure 6b shows the three-dimensional structure when the S. aureus phage P68 tail fiber protein is fused to the shaft domain by the knot-like domain (left) and when it is directly fused (right).

[0083] FIGS. 7a to 7d illustrate the detailed structures of knot-like domains: FIGS. 7a, 7b, and 7d illustrate the three-dimensional structures of knot-like domains derived from the upper basal plate protein of Enterococcus faecalis phage having different sequences; FIG. 7c illustrates the three-dimensional structure of knot-like domains derived from the upper basal plate protein of Enterococcus lactis phage.

[0084] FIGS. 8a through 8d illustrate non-covalent interactions between each subunit of a knot-like domain: FIG. 8a shows non-covalent interactions of knot-like domains within the BppU (WP_010708439.1) of Enterococcus faecalis phage; FIG. 8b shows non-covalent interactions of knot-like domains within the BppU (WP_010826292.1) of Enterococcus faecalis phage; FIG. 8c shows non-covalent interactions of knot-like domains within the BppU (WP_343436617.1) of Enterococcus lactis phage; FIG. 8d shows non-covalent interactions of knot-like domains within the BppU (WP_125203782.1) of Enterococcus faecalis phage.

[0085] FIGS. 9a to 9c illustrate inter-stranded binding existing between beta strands (β-strands) constituting knot-like domains.

[0086] FIGS. 10a to 10g illustrate the three-dimensional structures of knot-like domains derived from different species and having very low sequence similarity: FIG. 10a shows the three-dimensional structure of a specific segment (residues 124–208) of the Pseudomonas aeruginosa PA0620 R2 piosin fiber (6CL6); FIG. 10b shows the three-dimensional structure of a specific segment (residues 421–533) of the Staphylococcus aureus phage Phi11 host recognition mechanism (5EFV); FIG. 10c shows the three-dimensional structure of a specific segment (residues 298–405) of the Staphylococcus aureus phage P68 tail fiber (6IAB); Fig. 10d shows the three-dimensional structure of a specific segment (residues 455-553) of the tail spike (6EU4) of Acinetobacter phage vb_AbaP_AS12; Fig. 10e shows the three-dimensional structure of a specific segment (residues 38-113) of the Pseudomonas aeruginosa LESB58 R1 piosin fiber (6CL5); Fig. 10f shows the three-dimensional structure of a specific segment (residues 3-92) of the N-truncated R2 piosin tail fiber (6CU2) of Pseudomonas aeruginosa PAO1; Fig. 10g shows the three-dimensional structure of a specific segment (residues 221-344) of the C-terminal region (4UXF) of the bacteriophage T4 proximal long tail fiber protein gp34.

[0087]

[0088] The following detailed description of the invention will be described with reference to specific drawings regarding specific embodiments in which the invention may be practiced, but the invention is not limited thereto and is limited only by the appended claims, including all equivalents thereof as appropriately described. It should be understood that various embodiments of the invention are different but need not be mutually exclusive. For example, specific materials, forms, structures, and properties described herein may be modified from one embodiment to another or combined without departing from the technical spirit and scope of the invention. Technical and academic terms used herein have the same meaning as commonly used in the field to which the invention belongs, unless otherwise defined. For the purpose of interpreting this specification, the following definitions shall apply, and terms used in the singular shall include the plural where appropriate and vice versa.

[0089] Numerical ranges include the values ​​defined in the above ranges. All maximum numerical limits given throughout this specification include all lower numerical limits as clearly stated. All minimum numerical limits given throughout this specification include all higher numerical limits as clearly stated. All numerical limits given throughout this specification will include all better numerical ranges within a wider numerical range, as clearly stated.

[0090] All technical terms used in this invention, unless otherwise defined, include all meanings recognizable by a person skilled in the art and are used in the sense generally understood, and may be interpreted appropriately according to the context. Furthermore, while preferred methods or samples are described in this specification, similar or equivalents are also included within the scope of this invention.

[0091] The invention provided by this specification will be described in more detail below through examples. These examples are intended solely to illustrate the contents disclosed by this specification, and it will be obvious to those skilled in the art that the scope of the contents disclosed by this specification is not to be interpreted as being limited by these examples.

[0092]

[0093] Examples

[0094] Example 1. Strategy for Predicting the Structure of Heterogeneous Fusion Binding Proteins

[0095] The redesign of chimeric receptor binding proteins (chimeric RBPs) is primarily based on the protein's three-dimensional structure. This three-dimensional structure information is essential for understanding the protein's functional regions and interaction mechanisms, and for designing to confer new functions or enhance existing functions.

[0096] According to existing traditional methodologies, the three-dimensional structure of RBPs (based on data such as cryo-EM and X-ray crystallography) is analyzed to identify specific sites (domains) that bind to host cells, and the functions of each domain (binding site, structural stabilization, cell wall degradation after binding, etc.) are segmented and defined to serve as "modules" for constructing a chimera. The design of chimeric RBPs with new target recognition capabilities (host range, binding affinity, etc.) is achieved by recombining structural and functional domains within RBPs of heterologous origin through domain swapping methods, such as cross-overing binding site domains of RBPs derived from different viruses or inserting protein motifs specific to specific host receptors.

[0097] For chimeric fusion of spike or fiber proteins whose tertiary structures have not been fully elucidated, it is common practice to use a protein with a known tertiary structure as a standard and utilize amino acid sequence homology. However, in cases where a standard protein with a determined tertiary structure does not exist or where structure prediction based on comparison with a standard protein is difficult due to low amino acid sequence homology, algorithms for predicting the protein's secondary and tertiary structures can be used. Recently, with the advancement of deep learning-based protein structure prediction technologies such as AlphaFold, the possibility of accurately designing RBP domain swapping or mutation sites is increasing. In particular, the AlphaFold2 multimer function has been verified to be capable of predicting the structure of multimodal proteins (e.g., monotrimers), such as spike or fiber proteins of bacteriophages or viruses, with a high significance rate.

[0098] Experimental analysis can confirm whether heterologous receptor binding proteins containing redesigned spike or fibrous proteins can form a normal tertiary structure without structural deformation or instability caused by heterologous fusion. Experimental analyses include, for example, binding assays or competition assays that verify whether heterologous fusion binding proteins are biosynthesized and purified to bind to target host cells, or acquisition of function tests using bacteriophages or viruses in which the heterologous fusion binding proteins have self-assembled.

[0099] However, biochemical functional analysis of heterogeneous fusion binding proteins as described above requires enormous costs and time. Accordingly, the inventors adopted a strategy to verify the normal function of the redesigned RBP by analyzing whether there are any problems with the structure formation of the heterogeneous fusion binding protein through protein tertiary structure prediction using AlphaFold2, etc.

[0100]

[0101] Example 2. Confirmation of tertiary structural collapse of heterogeneous fusion binding protein

[0102] 2.1. Structure and Role of the Triple-Stranded Helix Bundle (TSHB)

[0103] FIG. 1 illustrates the structures of various forms of triple-stranded helix bundles (TSHBs) found in naturally occurring proteins or artificially synthesized. Information regarding each protein in FIG. 1 is disclosed in Table 1 below, and exemplary sequence information regarding monomers is disclosed in SEQ ID NOs. 10 to 30, respectively.

[0104]

[0105] 구분참조 서열(PDB / GenBank)단백질 이름 / 유형생물 정보서열번호APDB 2JJLAvian Reovirus fusion domainAvian orthoreovirus10BPDB 2XQHN-term of the trimeric autotransporter adhesin EibDEscherichia coli11CPDB 5MC9Heterotrimeric region of laminin-111Mus musculus12DPDB 3C9IPhage 22 tail-needle gp26Salmonellaphage P2213EPDB 1COSSynthetic triple-stranded alpha-helical bundle-14FPDB 1AA0Fibritin deletion mutant EBacteriophage T415GPDB 1HTMInfluenza haemagglutinin pH4.0Influenza A virus16HPDB 2HMGInfluenza haemagglutininInfluenza A virus17IPDB 1RTMC-type mannose binding proteinRattus norvegicus18JVIB22946.1Phage structural proteinClostridioides difficile19KNP_112711.1Baseplate upper proteinLactococcusphage TP901-120LWP_202061325.1BppU family phage baseplate upper proteinEnterococcussp. BWR-S521MEHU8862223.1BppU family phage baseplate upper proteinEnterococcus faecalis22NNP_108728.1Baseplate upper proteinLactococcusphage Tuc200923OWP_002332773.1Phage baseplate upper proteinEnterococcus faecium24PWP_002286491.1Hypothetical proteinEnterococcus25QELB00360.1Hypothetical protein OIC_03754Enterococcus faeciumEnGen000726RANT43434.1Baseplate / receptor binding proteinLactococcusphage 5090227SWP_003743966.1Phage baseplate upper proteinListeria28TWP_003722526.1Phage baseplate upper proteinListeriamonocytogenes29U3TQ2KE1 synthetic triple-stranded alpha-helical bundle-30.

[0106]

[0107] Each protein is primarily derived from bacteriophages, viruses, or specific microorganisms, and these structures are known to be involved in conferring mechanical stability within the protein, ligand binding, protein-protein interactions, or performing specific biological functions. In particular, TSHBs are commonly found in the baseplates and tail structures of bacteriophages, or in protein domains responsible for interactions with host cells. Therefore, the redesign of receptor-binding proteins (RBPs) to alter host selectivity frequently involves fusing the host binding domain (RBD) of the spike or fiber protein of a bacteriophage or virus targeting a specific host cell with the shaft or stem portion of a heterologous bacteriophage or virus.

[0108] 2.2. Redesign and Structural Stability Evaluation of Chimera RBP

[0109] The inventors created a fusion receptor binding protein (chimera RBP) sequence by combining an RBD derived from the P68 tail fiber protein of Staphylococcus aureus phage with the C-terminus or N-terminus of TSHB having the various structures above, and predicted the three-dimensional structure of these chimera RBPs using AlphaFold.

[0110] [Correction pursuant to Rule 91, Feb. 2026, 2013] As a result, as shown in FIG. 2, it was found that while the structure of the fused TSHB was maintained in some chimeric RBPs, in most cases, structural instability of the TSHB was caused by the fusion of heterogeneous RBDs. Specifically, in some combinations shown at the top of FIG. 2 (e.g., TSHB corresponding to the reference sequences of 2JJL, 2XQH, or ELB00360.1), it was confirmed that the TSHB backbone (gray portion) was maintained relatively stably and combined with the RBDs indicated in green without significant conflict. On the other hand, in the combinations shown at the bottom of FIG. 2 (e.g., TSHB having the reference sequences of WP_202061325.1 or NP_108728.1), as indicated by the red circle, the TSHB structure was not properly maintained and collapsed, showing a form in which the triple helix structure had collapsed.

[0111] [Correction pursuant to Rule 91 13.02.2026] As shown in Fig. 5a, such breakdown of the TSHB structure also occurred in the fusion of various other TSHBs (e.g., KE1 synthetic triple-stranded alpha-helical bundle, helical bundle derived from the upper protein of the basal plate of Lactococcus phage, etc.). TSHB is an important part that provides skeletal stability to the entire protein in tail fibers or spike structures, and when heterogeneous RBDs are fused, the interaction between TSHB regions is broken or the triple-stranded bundle structure is frequently not maintained due to twisting of the α-helical, etc.

[0112] The experimental results described above suggest the possibility that the structural stability of chimeric RBPs may be compromised, leading to defects or damage to their actual functions (such as host binding ability). Furthermore, these results demonstrate that even with the same RBD (phage P68), the structure of the chimeric protein can vary significantly depending on which TSHB it is fused with.

[0113]

[0114] Example 3. Confirmation of the structural stabilization effect of the knot-like domain on the fusion protein

[0115] 3.1. Elucidation of the Structure of Note-like Domains

[0116] In some bacteriophage tail fiber proteins, a knot-like domain exhibiting a knot-like structure is found between the shaft of the TSHB and the distal RBD.

[0117] FIG. 3 illustrates the three-dimensional structure of the above-mentioned knot-like domain and includes a shaft (green spiral structure), an RBD proximity region at the distal end (indicated in red and yellow tones), and a knot-like domain (highlighted in light green in the upper figure, with the three parts of knot 1 to knot 3 at the bottom distinguished in blue, orange, and green).

[0118] Specifically, the shaft domain is an elongated axial portion that helps the bacteriophage maintain flexibility and constant strength when in contact with the surface of a host cell. The RBD is the site where the virus recognizes and binds to the host cell, possessing a contact site capable of interacting with bacterial outer membrane proteins, polysaccharides, or specific receptors on the cell surface. Between these two functionally and structurally characteristic portions, a knot-shaped knot-like domain exists. The domain consists of three parts (knot 1, knot 2, and knot 3), each protein chain cross-linking to form a knot-shaped three-dimensional structure. Unlike protein polypeptides arranged simply as α-helices or β-sheets, this knot structure forms a unique topology in which "loops" are intertwined. This knot-shaped topology is formed simultaneously with the assembly of multiple subunits into a multimer form by protein folding, and the formed knot structure is expected to provide mechanical stability to the entire structure of the knot-like domain and other domains bound thereto.

[0119] 3.2. Triple Helix Structure Stabilization Effect of Knot-like Domains

[0120] The inventors evaluated the stability of the tertiary structure by introducing the knot-like domain between two domains during heterogeneous coupling of the shaft and RBD, under the assumption that the knot-like domain has the function of stabilizing the shaft structure (multi-helical structure).

[0121] As shown in Figure 4, a fusion receptor binding protein (chimera RBP) with a knot-like domain introduced between the shaft's TSHB and heterologous-derived RBD was created, and its structure was predicted using AlphaFold. As a result, it was found that Chimera 1, created by directly combining TSHB and RBD without a knot-like domain, failed to maintain the structure of TSHB and collapsed, whereas Chimera 2, created by introducing a knot-like domain between TSHB and RBD in which the knot-like domain connects TSHB and RBD, was found to maintain the tertiary structure of TSHB stably.

[0122] [Correction pursuant to Rule 91, 13.02.2026] It was confirmed that the structural stabilization effect of such knot-like domains on TSHB can function identically in other TSHBs having different sequences and tertiary structures. Figures 5a and 5b show the phenomenon where structural stability is compromised when TSHB (gray) and heterologous RBD (light green) are directly bound, and the effect of maintaining the tertiary structure of TSHB by inserting a knot-like domain (pink) between them. More specifically, TSHB structures derived from upper basal plate proteins of Enterococcus phages, such as Enterococcus faecalis, Enterococcus faecium, and Enterococcus sp. BWR-S5, undergo structural collapse when heterologous RBD (light green) is directly bound, whereas the intrinsic helical arrangement of TSHB is stabilized when a knot-like domain (pink) is inserted between them. In the case of TSHB derived from E. faecalis phage, it is observed that the original tertiary structure collapses when the long coiled coil shape comes into direct contact with the RBD. However, it was found that when a knot-like domain is inserted in the middle, the RBD can be folded normally while maintaining the TSHB shaft shape characteristic of E. faecalis. Similarly, when fusing a heterologous RBD to the TSHB of Lactococcus phage, the shaft becomes compressed, distorted, or unstable upon direct binding, but when bound via a knot-like domain, the TSHB helix is ​​aligned normally, and the receptor binding site of the RBD is fully exposed.

[0123] In addition, referring to Figures 6a and 6b, it can be seen that knot-like domains can play a key role in maintaining structural stability in the fusion of tail fiber proteins as well as tail spike proteins. Specifically, when the P68 tail fiber protein of Staphylococcus aureus phage was directly fused to a heterologous shaft domain, the TSHB structure collapsed, but when a knot-like domain was introduced, a stabilized chimeric RBP could be obtained. Similarly, when the P22 tail spike protein of Salmonella Enteritidis phage was directly fused to a heterologous shaft domain, the tertiary structure of TSHB collapsed, and it was confirmed that this structural instability was completely resolved by introducing a knot-like domain between the shaft domain and the RBD of the spike protein.

[0124] The above results suggest that knot-like domains serve as structural buffer zones for each domain within the chimeric RBP. In other words, through the aforementioned series of in silico experiments, it was proven that in generating chimeric receptor-binding proteins through domain swapping, the knot-like domain stabilizes the long, sensitive helical bundle structure of TSHB to prevent collapse, while simultaneously enabling the RBD to properly perform its recognition function for external receptors. In conclusion, when attempting to adjust host selectivity by fusing a modular structure composed of receptor-binding domains at long fiber / spike ends with heterologous shaft domains, using the knot-like domain according to the present invention ensures the stability of the tertiary structure of the chimeric RBP, thereby enabling more versatile domain swapping.

[0125]

[0126] Example 4. Confirmation of the structural stabilization mechanism of the fusion protein of the note-like domain

[0127] 4.1. Characterization of Subunits Constituting Note-like Domains

[0128] Next, we sought to identify the mechanism by which knot-like domains stabilize the structure of chimeric receptor binding proteins through an analysis of the structural characteristics of each subunit constituting the knot-like domain and the interactions between them. Accordingly, the inventors analyzed knot-like domains of various sequences found in Enterococcus phages and derived four knot-like domain sequences from five types of BppU (baseplate upper protein) sequences. These were found to maintain a generally similar 'knot-shaped' folding, while differing in detailed structures such as loop length, arrangement, and β-strand arrangement.

[0129] FIGS. 7a through 7d show four types of knot-like domains having different amino acid sequences. All four types of knot-like domains are homotrimers formed by the combination of three identical protein subunits. That is, while each subunit consists of a single polypeptide chain, multiple subunits interlock and are intricately assembled to form an overall symmetric and stable trimer structure. Table 2 below discloses exemplary sequences for each of the aforementioned subunits.

[0130]

[0131] 참조 서열(GenBank)단백질 이름 / 유형생물(숙주) 정보서열번호WP_010708439.1BppU family phage baseplate upper proteinEnterococcus faecalis1WP_010826292.1BppU family phage baseplate upper proteinEnterococcus faecalis2WP_010826651.1BppU family phage baseplate upper proteinEnterococcus faecalis3WP_343436617.1BppU family phage baseplate upper protein, partialEnterococcus lactis4WP_125203782.1BppU family phage baseplate upper proteinEnterococcus faecalis5ACZ63892.1Hypothetical proteinEnterococcusphage phiFL1C6MDN6212561.1DUF2479 domain-containing proteinLactococcussp.7MDT6469736.1BppU family phage baseplate upper proteinEnterococcus faecium8WP_125203782.1BppU family phage baseplate upper proteinEnterococcus faecalis9

[0132]

[0133] The subunit (indicated as 1207 / 14-10 RBP) constituting the knot-like domain disclosed in FIG. 7a is derived from the upper basal plate protein of Enterococcus faecalis phage (SEQN 1), and more specifically has the amino acid sequence of SEQN 6. The subunit (indicated as 2365-1 RBP) constituting the knot-like domain disclosed in FIG. 7b is derived from the upper basal plate protein of E. faecalis phage of a different sequence (SEQN 2 or SEQN 3), and more specifically has the amino acid sequence of SEQN 7. The subunit (indicated as PR0572 RBP) constituting the knot-like domain disclosed in FIG. 7c is derived from the upper basal plate protein of Enterococcus lactis phage (SEQN 4), and more specifically has the amino acid sequence of SEQN 8. The subunit (indicated as UK0456 RBP) constituting the note-like domain disclosed in FIG. 7d is derived from the basal plate upper protein of another sequence of E. faecalis phage (sequence number 5), and more specifically has the amino acid sequence of sequence number 9.

[0134] The knot-like domains described above were found to have the following structural commonalities. First, each subunit had a fundamentally similar framework, with a core formed by a β-sheet structure in which at least four β-strands are arranged in reverse, and three to six short alpha-helical structures positioned at the N-terminus, C-terminus, or between each β-strand of the β-sheet structure. Additionally, the β-strands were found to be linearly connected by loop structures and alpha-helical structures, forming a knot-like topology in the multimeric structure. Subunits with this unique structure impart rigidity to the overall structure of the knot-like domain through additional non-covalent interactions (see Example 4.2).

[0135] 4.2. Analysis of Non-Covalent Interactions within Note-like Domains

[0136] Since it was discovered that each subunit constituting the knot-like domain is not connected by covalent bonds but can function to provide stability to the helical bundle structure of the shaft, the inventors hypothesized that other non-covalent interactions exist through which the knot-like domains can be firmly bound and investigated them.

[0137] As a result, as shown in FIGS. 8a to 8d and FIGS. 9a to 9c, it was confirmed that when the knot-like domain as a whole consists of three subunits (indicated as A, B, and C in FIG. 8, respectively) coming together to form a trimer, the hydrophobic residues of each subunit are densely clustered in the center to form a strong hydrophobic interaction core. For example, the spherical notations (gray) in FIGS. 8a to 8d are mainly amino acids with hydrophobic side chains such as isoleucine (Ile), valine (Val), tyrosine (Tyr), phenylalanine (Phe), and alanine (Ala), and residues such as Tyr / Phe / Ile of subunit A and Ile / Val / Ala of subunit B are sterically cross-arranged, providing mechanical and thermodynamic stability to the overall structure of the domain through hydrophobic interactions that minimize water access.

[0138] More specifically, FIG. 8a shows the major non-covalent interactions formed between subunits A and B in a knot-like domain having the amino acid sequence of SEQ ID NO. 6. Each amino acid residue was numbered based on the amino acid sequence of SEQ ID NO. 6. For example, the distance between the benzene ring (CD1 et al.) of tyrosine (Tyr) at number 67 and the allyl branch carbon (CD1) of isoleucine (Ile) at number 43 was measured to be approximately 3.6 to 3.7 Å, and the side chains present on the beta strands within different subunits (the second beta strand of subunit A and the first beta strand of subunit B) form an internal core and exhibit hydrophobic contact. In addition, it was confirmed that CE2 of tyrosine 67 (Tyr) and CG2 of valine 66 (Val) are closely connected by a distance of about 3.7 Å, and that hydrophobic interactions occur between the gamma carbon of the 2nd side chain of valine and a portion of the tyrosine ring. Furthermore, hydrophobic interactions were confirmed between tyrosine 67 (Tyr) and isoleucine 78 (Ile), phenylalanine 72 (Phe) and alanine 63 (Ala), and isoleucine 85 (Ile) and isoleucine 78 (Ile). These dense hydrophobic interactions forming inter-stranded bindings maintain the interface between subunits robustly, thereby giving the knot-like domain high structural stability.

[0139] Figure 8b shows the major non-covalent interactions formed between subunits A and C in a knot-like domain having the amino acid sequence of SEQ ID NO. 7. Each amino acid residue was numbered based on the amino acid sequence of SEQ ID NO. 7. For example, the beta carbon (CB) of the side chain of histidine (His) at number 83, located on the second beta strand of subunit A, forms a hydrophobic contact with the ring end (CZ, CE1) of phenylalanine (Phe) located on the first beta strand of subunit C at a distance of about 3.0 to 3.5 Å. Hydrophobic interactions were also identified between histidine (His) at number 83 and tyrosine (Tyr) at number 81, tyrosine (Tyr) at number 93 and tyrosine (Tyr) at number 79 or arginine (Arg) at number 97, and between phenylalanine (Phe) at number 108 and isoleucine (Ile) at number 106. Characteristically, the negative charge (-COO) of the glutamate (Glu) side chain at position 95 - ) and the positive charge of the 97th arginine (Arg) side chain (-NH2 + It was found that ) forms ionic bonds, which, unlike other surrounding hydrophobic interactions, form strong electrostatic attraction between polar regions to reinforce the bonding between subunits.

[0140] FIG. 8c shows the major non-covalent interactions formed between subunits A and B in a knot-like domain having the amino acid sequence of SEQ ID NO. 8. Each amino acid residue was numbered based on the amino acid sequence of SEQ ID NO. 8. For example, the side chain of glutamate (Glu) at position 68 in the second beta strand of subunit A has a negative charge at its end, but the carbons preceding it (CB, CG, CD) have a hydrophobic methylene portion, and this region forms hydrophobic interactions by being in close proximity (or CB) to the aromatic ring edge (or 3.8 Å) of tyrosine (Tyr) at position 44 in the first beta strand of subunit B or phenylalanine (Phe) at position 62 in the second beta strand. In addition, it was confirmed that valine (Val) at number 71 forms hydrophobic interactions with tyrosine (Tyr) at number 44, phenylalanine (Phe) at number 62, or phenylalanine (Phe) at number 78, respectively, and isoleucine (Ile) at number 73 forms hydrophobic interactions with phenylalanine (Phe) at number 62 or phenylalanine (Phe) at number 78, respectively. Characteristically, the terminal (NZ) of the lysine (Lys) side chain at number 75 within the third beta strand of subunit A carries a positive charge, and the terminals (OE1, OE2) of the glutamate (Glu) side chain at number 77 within the third beta strand of subunit B carry a negative charge, and these two form a strong electrostatic attraction (ionic bond).

[0141] FIG. 8d shows the major non-covalent interactions formed between subunits A and B in a knot-like domain having the amino acid sequence of SEQ ID NO. 9. Each amino acid residue was numbered based on the amino acid sequence of SEQ ID NO. 9. For example, asparagine (Asn) at position 63, located on the third beta strand of subunit A, has a polarity due to having an amide group (-CONH2) at its end, but the beta and gamma carbon regions have a methylene structure and possess some hydrophobic properties. As a result, it forms a van der Waals hydrophobic contact with the branched carbon (CG2) of isoleucine (Ile) at position 41 in the second beta strand of subunit B at a distance of about 4.0 to 4.7 Å. In addition, a weak hydrophobic bond was observed between asparagine (Asn) at number 63 and serine (Ser) at number 59, and it was confirmed that hydrophobic contact also exists between aspartic acid (Asp) at number 65 and serine (Ser) at number 43. Furthermore, the negatively charged sites (OD1, OD2, etc.) at the end of the side chain of aspartic acid (Asp) at number 65 formed hydrogen bonds (Asp65@OD1-Ser43@OG and Asp65@CD2-Ser59@OG) with the -OH groups of serine (Ser) at number 43 or serine (Ser) at number 59, exhibiting a double interaction pattern along with hydrophobic contact.

[0142] In short, the knot-like domain allows the trimer structure of the knot-like domain to have high structural stability by having various residues present in the beta strand form complex non-covalent networks in different ways. That is, a hydrophobic central core forms the backbone, hydrogen bonds and ionic bonds at some positions further enhance structural stability, and aromatic residues provide additional ð-ð contacts to firmly connect the subunits.

[0143]

[0144] Example 5. Discovery of additional knot-like domains through structure-based search

[0145] In tail fiber / spike family proteins, certain regions may preserve their three-dimensional topology and folding patterns despite having low amino acid sequence similarity. Therefore, it is difficult to reliably identify structural buffer regions suitable for heterogeneous domain fusion using sequence similarity-based search or simple linker insertion alone, and there is a need for a module that is defined from a structural perspective and can be used universally.

[0146] As analyzed in Example 4 above, knot-like domain sequences derived from BppU (baseplate upper protein) family proteins found in Enterococcus phages, etc., generally maintain a knot-like folding shape while varying in detailed amino acid sequences; therefore, in Example 5, we aimed to (i) discover additional domains that are structurally similar to the knot-like domains but sequence-dissimilar from different phages (or phage-derived fiber / spike family proteins) through a structure-based search, and (ii) confirm that the domains are reproduced in a stable folding structure using a protein structure prediction tool (e.g., AlphaFold2 multimer), thereby (iii) confirming that the discovered domains are general-purpose modules useful for designing chimeric RBPs.

[0147] As a result of the Foldseek search, numerous regions with structural similarities to BppU knot-like domains were identified in different biological types, and information regarding this is shown in Table 3. The respective three-dimensional structures are shown in Figures 10a to 10g, respectively, in the order of Table 3. The following seven exemplary knot-like domains originate from different species and have very low sequence similarity, but they form a knot-like topology and are positioned between the shaft domain and the RBD to function as structural stabilization modules.

[0148]

[0149] PDB ID Protein Name / Type Biological Information Residue Sequence Number 6CL6R2 pyocin fiber PA0620(C-term 323-691)Pseudomonas aeruginosa PAO1124-208315EFVPhi11 host-recognition deviceDubowvirus dv11421-533326IAB tail fiberStaphylococcus phage P68298-405336EU4gp42 tailspikeAcinetobacter phage vB_AbaP_AS12455-553346CL5R1 pyocin fiber PALES_06171 (C-term 323-701)Pseudomonas aeruginosa LESB5838-113356CU2N-truncated R2-type pyocin tail fiberPseudomonas aeruginosa PAO13-92364UXFgp34 C-term regionTequatrovirus T4221-34437

[0150]

[0151] Referring to Table 3 and Figures 10a to 10g above, residues 124-208 (sequence number 31) of Pseudomonas aeruginosa R2 piosin PA0620 fibrous protein (6CL6) and knot structures (residues 24-109) of BppU (WP_010708439.1; sequence number 1) were found to have very low sequence similarity but very similar three-dimensional structures (Figure 10a).

[0152] In addition, regions corresponding to residues 421-533 (SEQ No. 32) of the host recognition site (5EFV) of Staphylococcus aureus phage Phi11 (Dubowvirus) and residues 298-405 (SEQ No. 33) of the tail fiber (6IAB) of phage P68 also showed high structural similarity to the knot structure of BppU (WP_010826292.1; SEQ No. 2) (residues 10-126 and 21-124, respectively) (Figs. 10b and 10c).

[0153] In addition, residues 455-553 of the Acinetobacter phage vb_AbaP_AS12 tail spike (6EU4) (sequence number 34) and residues 38-113 of the Pseudomonas aeruginosa piosin fiber R1 (6CL5) (sequence number 35) were found to have low sequence similarity but high structural similarity with the knot structure of BppU (WP_010826292.1; sequence number 2) (residues 21-126 and 41-126, respectively).

[0154] Meanwhile, residues 3-92 of the R2 piosin tail fiber (6CU2) derived from P. aeruginosa PAO1 (SEQ No. 36) and residues 221-344 of the long tail fiber gp34 (4UXF) of T4 phage (SEQ No. 37) were observed to have high structural similarity to the knot structure of BppU (WP_343436617.1; SEQ No. 4) (residues 24-126 and 17-100, respectively).

[0155] Although there was almost no sequence homology among the seven proteins mentioned above, it was confirmed that the folding patterns of the discovered regions, such as beta sheet configuration, loop structure, and knot-shaped phase, matched the structural characteristics of the BppU knot-like domain.

[0156] Therefore, it was confirmed that the structural characteristics of the knot-like domains defined in the present invention are not limited to specific amino acid sequences or specific sequence groups, but are also found in proteins derived from various bacteriophages, and that these can also be used as structural stabilization modules. The exemplary knot-like domains additionally discovered in Example 5 were confirmed to be valid as structurally stabilized domains in the design and selection stages of knot-like domain proteins, as the candidate domains were predicted to have actual foldable structures through protein structure prediction AI (e.g., AlphaFold2), in addition to confirming similar folding in structure-based search (Foldseek), and in particular, the knot-type folding was predicted in a direction where it does not collapse even when assuming multimer (preferably trimer) assembly.

[0157] As described above, the structure-based search results of Example 5 demonstrate that knot-like structural elements observed in different phages (and phage-like fibrous structures) can converge and be preserved in tertiary structure despite being sequenceally different. Therefore, not only the knot-like domains additionally discovered in Example 5 but also various proteins having structures three-dimensionally similar to them can be very usefully utilized as structural buffer zones and stabilization modules inserted between (a) heterogeneous shaft domains and (b) heterogeneous RBDs in chimeric RBP designs. In particular, since domains that provide structurally compatible folding while being highly sequencely dissimilar can be selectively utilized, they contribute to securing the tertiary structural stability of chimeric RBPs during domain swapping and module recombination processes, and can ultimately be applied to engineered virus / bacteriophage designs to modify or expand host selectivity as intended in the present invention.

[0158]

[0159] Representative embodiments or combinations of embodiments based on the above description are as follows:

[0160] [Example of Implementation 1]

[0161] A protein comprising a knot-like domain composed of a plurality of subunits, wherein each of the subunits comprises (i) a β-sheet structure in which at least four β-strands are arranged anti-parallel; and (ii) at least one loop or short α-helix connecting the β-strands, wherein the plurality of subunits are assembled into a multimer by protein folding and simultaneously form a knot-shaped topology, and wherein the knot-like domain is connected between (a) a shaft domain comprising a helix bundle structure derived from viruses of different species; and (b) a receptor binding domain (RBD), thereby stabilizing the tertiary structure of the helix bundle.

[0162] [Example of Implementation 2]

[0163] In the above-described embodiment, the note-like domain is a protein that is a multimer comprising 2 to 6 subunits.

[0164] [Example of Implementation 3]

[0165] In any one of the embodiments described above, the note-like domain is a protein that is a homo-trimer containing three identical subunits.

[0166] [Example of Implementation 4]

[0167] In any one of the embodiments described above, the knot-like domain is a protein that forms a tertiary structure through non-covalent interactions between each subunit.

[0168] [Example 5 of Implementation]

[0169] In any one of the embodiments described above, the non-covalent interaction is a protein formed between a beta strand in one subunit and a beta strand in another subunit.

[0170] [Example 6 of Implementation]

[0171] In any one embodiment of the above-described embodiments, the non-covalent interaction comprises (i) any one of at least four beta strands arranged continuously in the direction from the N-terminus to the C-terminus within one subunit; and (ii) proteins formed between beta strands existing at the same relative position and / or adjacent relative positions within another subunit.

[0172] [Example 7]

[0173] In any one of the embodiments described above, the non-covalent interaction is a protein selected from the group consisting of ionic bonds, hydrogen bonds, and hydrophobic interactions.

[0174] [Example of Implementation 8]

[0175] A protein characterized in that, in any one of the aforementioned embodiments, the distance between the two amino acids where the non-covalent interaction is formed is 3 Å to 5 Å.

[0176] [Example of Implementation 9]

[0177] In any one of the embodiments described above, each beta strand within the subunit is a protein composed of 3 to 12 amino acids.

[0178] [Example of Implementation 10]

[0179] In any one of the embodiments described above, the subunit is a protein comprising two or more short alpha helix structures.

[0180] [Example of Implementation 11]

[0181] In any one of the embodiments described above, the short alpha helix within the subunit is a protein composed of 3 to 15 amino acids.

[0182] [Example 12]

[0183] In any one of the embodiments described above, the amino acid sequence of the subunit is a sequence derived from the wild-type sequence of a subunit constituting a knot-like domain present in a tail fiber protein of a bacteriophage, or a protein comprising an amino acid sequence having a sequence similarity of 70% or more with said sequence.

[0184] [Example 13]

[0185] In any one of the embodiments described above, the bacteriophage is a protein selected from the group consisting of Enterococcus phage, Staphylococcus phage, Pseudomonas phage, Acinetobacter phage, and T4 phage.

[0186] [Example 14]

[0187] In any one of the embodiments described above, the wild-type sequence of the subunit constituting the note-like domain comprises any one sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs 6 to 9 and SEQ ID NOs 31 to 37.

[0188] [Example 15]

[0189] In any one of the embodiments described above, the number of subunits is equal to the number of helical strands within the helical bundle structure.

[0190] [Example 16]

[0191] A chimeric receptor binding protein (chimeric RBP) having the following components sequentially connected: (a) a shaft domain derived from a first virus and comprising a helix bundle structure; (b) a protein comprising a knot-like domain according to any one of claims 1 to 15; and (c) a receptor binding domain (RBD) derived from a second virus that is heterogeneous to the first virus.

[0192] [Example 17]

[0193] In the above-described embodiment, the virus is a chimeric RBP that is a virus belonging to a class or family selected from the group consisting of Caudoviricetes, Adenoviridae, Reoviridae, and Orthomyxoviridae.

[0194] [Example 18]

[0195] In any one of the embodiments described above, the virus is a chimeric RBP selected from the group consisting of Enterococcus phage, Lactococcus phage, Staphylococcus phage, Salmonella phage, Listeria phage, Pseudomonas phage, Acinetobacter phage, Escherichia phage, adenovirus, reovirus, and influenza virus.

[0196] [Example of Implementation 19]

[0197] In any one of the embodiments described above, the helical bundle structure is a chimera RBP composed of 2 to 6 helical strands.

[0198] [Gu Hyeon-ye 20]

[0199] In any one of the embodiments described above, the helical strand is a chimeric RBP selected from the group consisting of an alpha helix, a beta helix, and a beta spiral.

[0200] [Example of Implementation 21]

[0201] In any one of the embodiments described above, the helical strand comprises a part or all of the helical strand sequence included in any one sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs 10 to 30.

[0202] [Example of Implementation 22]

[0203] In any one of the embodiments described above, the helix bundle structure is a chimera RBP in which three helix strands form a coiled coil structure, a triple-stranded helix bundle (TSHB).

[0204] [Example of Implementation 23]

[0205] In any one of the embodiments described above, the RBD of the second virus is a chimeric RBP having a different host range compared to the RBD of the first virus.

[0206] [Example of Implementation 24]

[0207] In any one of the embodiments described above, the RBD of the second virus is a chimeric RBP having an extended host range compared to the RBD of the first virus.

[0208] [Example 25]

[0209] In any one of the embodiments described above, the shaft domain or RBD is a shaft domain or RBD included in any one protein selected from the group consisting of a phage-derived tail fiber protein, a phage-derived tail spike protein, a virus-derived capsid spike protein, and a virus-derived capsid fiber protein, or a chimeric RBP that is a shaft domain or RBD having a sequence similarity of 70% or more thereto.

[0210] [Example 26]

[0211] In any one of the embodiments described above, the shaft domain is connected to the N-terminal direction of the knot-like domain; and the RBD is a chimera RBP connected to the C-terminal direction of the knot-like domain.

[0212] [Example of Implementation 27]

[0213] An engineered virus comprising a chimeric receptor binding protein (chimeric RBP) according to any one of the embodiments described above.

[0214] [Example of Implementation 28]

[0215] In any one of the aforementioned embodiments, the virus is an engineered virus that is a bacteriophage capable of infecting a specific group of bacteria.

[0216] [Example of Implementation 29]

[0217] In any one of the embodiments described above, the engineered virus is an engineered virus in which the ability to reinfect a host is removed.

[0218] [Example 30]

[0219] In any one of the embodiments described above, the engineered virus is an engineered virus comprising a payload.

[0220] [Example 31 of implementation]

[0221] In any one of the embodiments described above, the payload comprises an engineered virus comprising a therapeutic protein or a functional variant thereof; an antibody or antibody fragment; an enzyme; a component of a gene editing system; an RNAi preparation; or a combination thereof.

[0222] [Example of Implementation 32]

[0223] In any one of the embodiments described above, the payload is an engineered virus that is a substance exhibiting an anticancer effect, a gene expressing the same, or a factor that promotes the secretion or expression of said substance or gene.

[0224] [Example 33]

[0225] A pharmaceutical composition for the prevention or treatment of cancer comprising an engineered virus according to any one of the embodiments described above as an active ingredient.

[0226] [Example of Implementation 34]

[0227] Use of a composition comprising an engineered virus as an active ingredient according to any one of the aforementioned embodiments for the prevention or treatment of cancer.

[0228] [Example 35]

[0229] Use for producing a drug used for cancer prevention or treatment of an engineered virus according to any one of the embodiments described above.

[0230] [Example 36]

[0231] A method for preventing or treating cancer, comprising the step of administering a composition containing an engineered virus as an active ingredient according to any one of the embodiments described above to a subject in need thereof.

[0232] [Example 37]

[0233] A vector comprising a polynucleotide encoding an engineered virus according to any one of the aforementioned embodiments, operably connected to a promoter.

[0234] [Example 38]

[0235] In any one of the embodiments described above, the vector is a vector further comprising a polynucleotide that encodes a payload.

[0236] [Example of Implementation 39]

[0237] In any one of the embodiments described above, the polynucleotide encoding the payload is a vector in which the polynucleotide encoding the payload is a polynucleotide encoding a gene expressing a substance exhibiting an anticancer effect or a factor promoting the expression thereof.

[0238] [Example of Implementation 40]

[0239] A pharmaceutical composition for the prevention or treatment of cancer comprising a vector according to any one of the embodiments described above as an active ingredient.

[0240] [Example 41]

[0241] A composition comprising a vector according to any one of the aforementioned embodiments as an active ingredient for the prevention or treatment of cancer.

[0242] [Example of Implementation 42]

[0243] Use for producing a drug used for cancer prevention or treatment of a vector according to any one of the embodiments described above.

[0244] [Example 43]

[0245] A method for preventing or treating cancer, comprising the step of administering a composition containing a vector according to any one of the embodiments described above as an active ingredient to a subject in need thereof.

[0246] [Example of Implementation 44]

[0247] A method for stabilizing the tertiary structure of a chimeric receptor binding protein (chimeric RBP), comprising the step of introducing a protein comprising a knot-like domain according to any one of claims 1 to 15 between a shaft domain comprising a helix bundle structure and a heterologous receptor binding domain (RBD).

[0248]

[0249] The description of the invention set forth above is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A protein comprising a knot-like domain composed of multiple subunits, Each of the above subunits comprises (i) a β-sheet structure in which at least four β-strands are arranged anti-parallel; and (ii) at least one loop or short α-helix connecting the β-strands, wherein a plurality of subunits are assembled into a multimer by protein folding and simultaneously form a knot-shaped topology. The above note-like domain is characterized by being connected between (a) a shaft domain containing a helix bundle structure derived from a virus of a different species; and (b) a receptor binding domain (RBD), thereby stabilizing the tertiary structure of the helix bundle. protein.

2. In Paragraph 1, The above note-like domain is a multimer comprising 2 to 6 subunits. protein.

3. In Paragraph 2, The above note-like domain is a homo-trimer containing three identical subunits. protein.

4. In Paragraph 1, The above-mentioned note-like domain forms a tertiary structure through non-covalent interactions between each subunit. protein.

5. In Paragraph 4, The above non-covalent interaction is formed between a beta strand within one subunit and a beta strand within another subunit. protein.

6. In Paragraph 4, The above non-covalent interaction is formed between (i) any one of at least four beta strands arranged continuously in the direction from the N-terminus to the C-terminus within one subunit; and (ii) beta strands existing at the same relative position and / or adjacent relative position within another subunit. protein.

7. In Paragraph 4, The above non-covalent interaction is selected from the group consisting of ionic bonds, hydrogen bonds, and hydrophobic interactions. protein.

8. In Paragraph 4, Characterized by the distance between the two amino acids where the above non-covalent interaction is formed being 3 Å to 5 Å. protein.

9. In Paragraph 1, Each beta strand within the above subunit consists of 3 to 12 amino acids. protein.

10. In Paragraph 1, The above subunit includes two or more short alpha helix structures. protein.

11. In Paragraph 1, The short alpha helix within the above subunit consists of 3 to 15 amino acids. protein.

12. In Paragraph 1, The amino acid sequence of the above subunit is a sequence derived from the wild-type sequence of a subunit constituting a knot-like domain present in the tail fiber protein of a bacteriophage, or an amino acid sequence having a sequence similarity of 70% or more to the said sequence. protein.

13. In Paragraph 12, The above bacteriophage is selected from the group consisting of Enterococcus phage, Staphylococcus phage, Pseudomonas phage, Acinetobacter phage, and T4 phage. protein.

14. In Paragraph 12, The wild-type sequence of the subunit constituting the above note-like domain comprises any one sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs. 6 to 9 and SEQ ID NOs. 31 to 37. protein.

15. In Paragraph 1, The number of the above subunits is equal to the number of helical strands within the helical bundle structure. protein.

16. Chimeric receptor binding protein (chimeric RBP) with the following components sequentially linked: (a) a shaft domain derived from the first virus and containing a helix bundle structure; (b) a protein comprising a knot-like domain according to any one of claims 1 to 15; and (c) Receptor binding domain (RBD) derived from the first virus and the second virus, which is heterologous.

17. In Paragraph 16, The above virus is a virus belonging to a class or family selected from the group consisting of Caudoviricetes, Adenoviridae, Reoviridae, and Orthomyxoviridae. Chimera RBP.

18. In Paragraph 16, The above virus is selected from the group consisting of Enterococcus phage, Lactococcus phage, Staphylococcus phage, Salmonella phage, Listeria phage, Pseudomonas phage, Acinetobacter phage, Escherichia phage, adenovirus, reovirus, and influenza virus. Chimera RBP.

19. In Paragraph 16, The above helical bundle structure consists of 2 to 6 helical strands. Chimera RBP.

20. In Paragraph 19, The helical strand is selected from the group consisting of alpha helix, beta helix, and beta spiral. Chimera RBP.

21. In Paragraph 19, The helical strand comprises a part or all of the helical strand sequence contained within any one sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs. 10 to 30. Chimera RBP.

22. In Paragraph 19, The above helical bundle structure is a triple-stranded helix bundle (TSHB) in which three helical strands form a coiled coil structure. Chimera RBP.

23. In Paragraph 16, The RBD of the second virus above has a different host range compared to the RBD of the first virus. Chimera RBP.

24. In Paragraph 16, The RBD of the second virus above has an extended host range compared to the RBD of the first virus. Chimera RBP.

25. In Paragraph 16, The above shaft domain or RBD is a shaft domain or RBD included in any one protein selected from the group consisting of phage-derived tail fiber proteins, phage-derived tail spike proteins, virus-derived capsid spike proteins, and virus-derived capsid fiber proteins, or a shaft domain or RBD having a sequence similarity of 70% or more thereto. Chimera RBP.

26. In Paragraph 16, The above shaft domain is connected to the N-terminal direction of the knot-like domain; The above RBD is connected in the C-terminal direction of the knot-like domain. Chimera RBP.

27. A chimeric receptor binding protein (chimeric RBP) according to paragraph 16 Engineered virus.

28. In Paragraph 27, The above virus is a bacteriophage capable of infecting specific bacterial groups. Engineered virus.

29. In Paragraph 27, The above-mentioned engineered virus has had its ability to reinfect the host removed. Engineered virus.

30. In Paragraph 27, The above-mentioned engineered virus includes a payload Engineered virus.

31. In Paragraph 30, The above payload comprises a therapeutic protein or a functional variant thereof; an antibody or antibody fragment; an enzyme; a component of a gene editing system; an RNAi agent; or a combination thereof. Engineered virus.

32. In Paragraph 30, The above payload is a substance exhibiting an anticancer effect, a gene expressing the same, or a factor that promotes the secretion or expression of said substance or gene. Engineered virus.

33. Comprising an engineered virus pursuant to Paragraph 32 as an active ingredient Pharmaceutical composition for the prevention or treatment of cancer.

34. A polynucleotide encoding an engineered virus according to claim 27, operably connected to a promoter vector.

35. In Paragraph 34, The above vector further includes a polynucleotide encoding a payload. vector.

36. In Paragraph 35, The polynucleotide encoding the above payload is a polynucleotide encoding a gene that expresses a substance exhibiting an anticancer effect or a factor that promotes the expression thereof. vector.

37. A vector according to paragraph 34 as an effective component Pharmaceutical composition for the prevention or treatment of cancer.

38. A method for stabilizing the tertiary structure of a chimeric receptor binding protein (chimeric RBP), comprising the step of introducing a protein comprising a knot-like domain according to any one of claims 1 to 15 between a shaft domain comprising a helix bundle structure and a heterologous receptor binding domain (RBD).

Images