Bacteriophage comprising mutation for improving pharmacokinetic properties
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- ACCURIE BIO INC
- Filing Date
- 2026-02-02
- Publication Date
- 2026-08-06
Smart Images
Figure KR2026001930_06082026_PF_FP_ABST
Abstract
Description
Bacteriophages containing variants for improved pharmacokinetic properties
[0001] The present invention relates to a phage with a mutation induced to increase the duration of action in vivo so that a therapeutic concentration is maintained for a long time, and an engineered phage with said mutation introduced. Specifically, the present invention relates to a phage with improved pharmacokinetic properties comprising at least one missense mutation in a structural protein gene.
[0002] This application claims priority based on Korean Patent Application No. 10-2025-0012279 filed on January 31, 2025, and all contents disclosed in the specification and drawings of said application are incorporated into this application.
[0003]
[0004] Bacteriophages are a type of virus capable of selectively infecting and lysing specific bacteria, and they are attracting attention as an alternative treatment for antibiotic-resistant strains. However, the commercialization of phage therapy has been delayed due to various limitations. Key issues include (i) the need for customized design for specific pathogenic strains because bacteriophages have a very narrow host range, (ii) the potential inclusion of toxins derived from the bacterial lysis process within bacteriophage preparations, and (iii) the difficulty in sustaining therapeutic effects because injected bacteriophages are rapidly eliminated by the host's immune system.
[0005] After being injected into the body, bacteriophages can be eliminated or degraded by the host's immune system. In particular, the reticuloendothelial system (RES) of the immune system plays a role in effectively removing foreign particles from the circulating blood; in this process, bacteriophages are also phagocytosed by macrophages or rapidly excreted by the kidneys. Additionally, they can be neutralized by antibody responses or destroyed by the complement system, which leads to a problem of a shortened duration in the body. Some studies have confirmed that bacteriophages are rapidly eliminated even in experiments using germ-free mice, suggesting that mechanical removal by the reticuloendothelial system, in addition to antibody responses, is a major mechanism of elimination.
[0006] It is known that the retention time of bacteriophages in the blood is determined by the inherent characteristics of the phage. While some phages are rapidly eliminated within a few hours of administration, phage variants with specific mutations (long-circulating phages) can effectively evade the reticuloendothelial system and be maintained for a longer period in vivo. These variants can be selectively obtained through a passaging approach, and mutations in the bacteriophage's capsid protein have been reported to play a major role.
[0007] Therefore, to maximize the efficacy of bacteriophage therapy, it is essential to improve the persistence of the bacteriophage in vivo. In this invention, a mutant strain capable of persisting for a long time in vivo was secured by utilizing a continuous microevolution technique, thereby aiming to improve the pharmacokinetic characteristics of the bacteriophage and maximize therapeutic efficiency.
[0008]
[0009] The present invention aims to solve all the problems of the aforementioned prior art.
[0010] One objective of the present invention is to provide a Lactococcus phage Tuc2009 mutant strain with improved pharmacokinetic properties comprising at least one missense mutation in one or more structural protein genes.
[0011] In addition, the present invention has another objective of providing an engineered phage with improved pharmacokinetic properties by introducing at least one missense mutation into one or more structural protein genes.
[0012] In addition, the present invention has another objective of providing a vector comprising a polynucleotide encoding the said mutant or engineered phage.
[0013] In addition, the present invention has another objective of providing a pharmaceutical composition comprising the above-mentioned mutant, engineered phage, or vector as an active ingredient.
[0014] 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.
[0015]
[0016] A representative configuration of the present invention for achieving the above objective is as follows.
[0017] According to one aspect of the present invention, a Lactococcus phage Tuc2009 mutant strain with improved pharmacokinetic properties is provided, comprising at least one missense mutation in one or more structural protein genes selected from the group consisting of (a) a Tuc2009_32 gene consisting of the nucleotide sequence of SEQ ID NO. 2; (b) a Tuc2009_48 gene consisting of the nucleotide sequence of SEQ ID NO. 4; (c) a Tuc2009_49 gene consisting of the nucleotide sequence of SEQ ID NO. 6; (d) a Tuc2009_51 gene consisting of the nucleotide sequence of SEQ ID NO. 8; and (e) a Tuc2009_53 gene consisting of the nucleotide sequence of SEQ ID NO. 10.
[0018] The technologies disclosed herein aim to provide bacteriophage variants with an extended duration in vivo. Generally, bacteriophages are rapidly eliminated by the reticuloendothelial system (RES) after injection into the body, and the majority (over 90%) are degraded, particularly due to phagocytosis by Kupffer cells in the liver sinusoids. This shortens the time that therapeutic phages maintain an effective concentration in the blood, acting as a major factor limiting therapeutic efficacy.
[0019] The present invention aims to overcome these limitations by improving pharmacokinetic properties through the induction of missense mutations in specific structural protein genes of Tuc2009 bacteriophage. Specifically, it was found that when specific mutations are induced or introduced in the structural protein genes (Tuc2009_32, Tuc2009_48, Tuc2009_49, Tuc2009_51, Tuc2009_53) corresponding to SEQ ID NOs. 2, 4, 6, 8, and 10, the structural stability of the bacteriophage is enhanced and elimination by RES can be avoided.
[0020] These variants survive for a longer period in the body and can maintain an effective concentration in the blood for an extended time.
[0021] In one embodiment, the missense mutation comprises: (i) substitution of the 333rd aspartic acid (D) with alanine (A) based on the amino acid sequence of SEQ ID NO. 3; (ii) substitution of the 458th methionine (M) with threonine (T) based on the amino acid sequence of SEQ ID NO. 5; (iii) substitution of the 636th glutamic acid (E) with alanine (A) based on the amino acid sequence of SEQ ID NO. 5; (iv) substitution of the 280th isoleucine (I) with valine (V) based on the amino acid sequence of SEQ ID NO. 7; (v) substitution of the 145th leucine (L) with glutamine (Q) based on the amino acid sequence of SEQ ID NO. 9; (vi) substitution of the 151st isoleucine (I) with lysine (K) based on the amino acid sequence of SEQ ID NO. 9; (vii) substitution of the 155th asparagine (N) with serine (S) based on the amino acid sequence of SEQ ID NO. 9; and (viii) may result in one or more amino acid substitutions selected from the group in which the 397th leucine (L) is substituted with valine (V) based on the amino acid sequence of SEQ ID NO. 11.
[0022] In another embodiment, the wild type of the Lactococcus phage Tuc2009 may include a whole-length genome sequence consisting of the nucleotide sequence of SEQ ID NO. 1.
[0023] Each sequence referred to by the above sequence number is specifically disclosed in Table 1 below and the sequence list attached to this invention.
[0024]
[0025] Sequence Number Type NCBI Reference Sequence Gene / Protein Description Locus1DNANC_002703.1Lactococcus phage Tuc2009, complete genomeNC_0027032DNANC_002703.1Minor capsid protein 1Tuc2009_323AANP_108711.1Minor capsid protein 1Tuc2009_324DNANC_002703.1Tal2009 (tail-associated lysin)Tuc2009_485AANP_108727Tal2009 (tail-associated lysin)Tuc2009_486DNANC_002703.1BppU (upper baseplate protein)Tuc2009_497AANP_108728.1BppU (upper baseplate protein)Tuc2009_498DNANC_002703.1BppL (lower baseplate protein)Tuc2009_519AANP_108730.1BppL (lower baseplate protein)Tuc2009_5110DNANC_002703.1Neck passage structureTuc2009_5311AANP_108732.1Neck passage structureTuc2009_53
[0026]
[0027] In another embodiment, the pharmacokinetic properties may be one or more selected from the group consisting of resistance to immune response, pH resistance, and temperature resistance.
[0028] In another embodiment, the mutant strain may have an in vivo half-life that is at least twice as long as that of the wild type.
[0029] In another embodiment, the mutant strain may be stable for about 1 hour or more in an environment of pH 3.5 to pH 12.
[0030] In another embodiment, the mutant strain may be stable for about 1 hour or more in an environment of about 4°C to about 50°C.
[0031] 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%.
[0032] According to another aspect of the present invention, an engineered phage with improved pharmacokinetic properties is provided by introducing at least one missense mutation into one or more structural protein genes selected from the group consisting of (a) Minor capsid protein 1 gene; (b) Tail-associated lysin gene; (c) Upper baseplate protein (BppU) gene; (d) Lower baseplate protein (BppL) gene; and (e) Neck passage structure (NPS) gene.
[0033] In one embodiment, the pharmacokinetic characteristic may be any one selected from the group consisting of resistance to immune response, pH resistance, and temperature resistance.
[0034] In another embodiment, the engineered phage may have an increased in vivo duration compared to before the introduction of at least one missense mutation.
[0035] In another embodiment, the engineered phage may be engineered based on Lactococcus phage Tuc2009.
[0036] In another embodiment, the engineered phage comprises a protein encoded by one or more structural protein genes selected from the group consisting of (a) a Tuc2009_32 gene with the nucleotide sequence of SEQ ID NO. 2; (b) a Tuc2009_48 gene with the nucleotide sequence of SEQ ID NO. 4; (c) a Tuc2009_49 gene with the nucleotide sequence of SEQ ID NO. 6; (d) a Tuc2009_51 gene with the nucleotide sequence of SEQ ID NO. 8; and (e) a Tuc2009_53 gene with the nucleotide sequence of SEQ ID NO. 10, wherein at least one missense mutation may be introduced into the structural protein gene.
[0037] In another embodiment, the engineered phage may comprise (i) a structural protein composed of the amino acid sequence of SEQ ID NO. 3 containing a D333A mutation; (ii) a structural protein composed of the amino acid sequence of SEQ ID NO. 5 containing M458T and / or E636A mutations; (iii) a structural protein composed of the amino acid sequence of SEQ ID NO. 7 containing an I280V mutation; (iv) a structural protein composed of the amino acid sequence of SEQ ID NO. 9 containing one or more mutations selected from the group consisting of L145Q, I151K and N155S; and (v) one or more structural proteins selected from the group consisting of the amino acid sequence of SEQ ID NO. 11 containing an L397V mutation.
[0038] In another embodiment, the engineered phage may have a different host range or an extended host range compared to the wild-type Lactococcus phage Tuc2009.
[0039] Bacteriophages can alter or regulate host selectivity by changing the receptor targeting function of receptor-binding proteins (RBPs) through the redesign of spike or fibrillary proteins. 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 broader sense, to selectivity between different species that are less related to the target host cell.
[0040] In another embodiment, the engineered fragment may further include a payload.
[0041] In another embodiment, the payload may comprise 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. For example, 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 said substance or gene.
[0042] According to another aspect of the present invention, a pharmaceutical composition for the prevention or treatment of a disease is provided, comprising a mutant strain or an engineered phage according to the present invention as an active ingredient.
[0043] In one embodiment, the pharmaceutical composition may be a pharmaceutical composition for the prevention or treatment of cancer.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] (Pharmaceutical) The actual dosage of a composition may vary significantly depending on various factors, such as the concentration of the active ingredient (e.g., engineered phage, mutant strain, or 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 bacteriophages 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).
[0049] According to another aspect of the present invention, a vector comprising a polynucleotide encoding a mutant or engineered phage according to the present invention, operably connected to a promoter is provided.
[0050] 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.
[0051] According to another aspect of the present invention, a method for producing a phage with improved pharmacokinetic properties is provided, comprising the step of inducing or introducing at least one missense mutation into one or more structural protein genes selected from the group consisting of (a) a minor capsid protein 1 gene; (b) a tail-associated lysin gene; (c) an upper baseplate protein (BppU) gene; (d) a lower baseplate protein (BppL) gene; and (e) a neck passage structure (NPS) gene.
[0052] In one embodiment, the phage is Lactococcus phage Tuc2009, and the missense mutation comprises: (i) substitution of aspartic acid (D) at the 333rd position based on the amino acid sequence of SEQ ID NO. 3 with alanine (A); (ii) substitution of methionine (M) at the 458th position based on the amino acid sequence of SEQ ID NO. 5 with threonine (T); (iii) substitution of glutamic acid (E) at the 636th position based on the amino acid sequence of SEQ ID NO. 5 with alanine (A); (iv) substitution of isoleucine (I) at the 280th position based on the amino acid sequence of SEQ ID NO. 7 with valine (V); (v) substitution of leucine (L) at the 145th position based on the amino acid sequence of SEQ ID NO. 9 with glutamine (Q); and (vi) substitution of isoleucine (I) at the 151st position based on the amino acid sequence of SEQ ID NO. 9 with lysine (K). (vii) substitution of the 155th asparagine (N) with serine (S) based on the amino acid sequence of SEQ ID NO. 9; and (viii) substitution of the 397th leucine (L) with valine (V) based on the amino acid sequence of SEQ ID NO. 11, resulting in one or more amino acid substitutions selected from the group consisting of these.
[0053]
[0054] The mutation according to the present invention provides the effect of significantly improving the duration and stability of the bacteriophage in vivo. The mutant strain phage produced through a continuous microevolution method has a significantly extended half-life in vivo compared to the wild-type phage and can maintain high stability even in extreme environments such as pH and temperature. Through this, by improving the pharmacokinetic characteristics of the therapeutic bacteriophage, it becomes possible to exert a more effective and sustained therapeutic effect and control infection.
[0055]
[0056] FIG. 1 is a schematic diagram illustrating each step of a microevolution method according to one embodiment.
[0057] Figure 2 is 10 11 The results of measuring blood phage concentrations at 3, 6, 12, 24, and 48 hours after administering Lactococcus lactis phage at a dose of 300 μL of pfu / mL intraperitoneally are shown.
[0058] Figures 3a and 3b are 10 11 The results of measuring the phage concentration in the blood by plaque assay after whole blood was collected from each mouse 24 hours after administering pfu / mL Lactococcus lactis phage intraperitoneally at a dose of 300 μL are shown: Fig. 3a shows the plaque assay results for three lines of round 2 and line 1 of round 3; Fig. 3b shows the plaque assay results for line 1 of rounds 4 through 9.
[0059] Figure 4 shows the results of performing plaque analysis by combining all serum samples from line 1 collected during rounds 1 to 9.
[0060] Figure 5 shows the results of performing plaque analysis on each of the three lines in 10 rounds.
[0061] Figure 6 shows the results of analyzing the survival amount of bacteriophages in the blood over time during rounds 1 to 10.
[0062] Figures 7a and 7b show the mutant phage (indicated by M) and wild-type phage (indicated by W) obtained through 10 rounds of microevolution in 3 x 10 11 Figure 7a shows the results of a comparative analysis of the amount of residual bacteria in the serum 24 hours after administration at a dose of 300 μL / pfu intraperitoneally: Figure 7b shows the results of plaque analysis at 6, 12, 18, 24, 36, and 48 hours after administration; Figure 7b shows the results of plaque analysis performed by integrating serum collected at each time point.
[0063] Figure 8 illustrates the results of a comparative analysis of the survival rates of bacteriophages remaining in the blood after 1 round (wild type) and 10 rounds (mutant strain) over time.
[0064] Figures 9a and 9b illustrate blood concentrations up to 24 hours after phage administration: Figure 9a is a graph comparing the change in survival rate up to 24 hours after administering a phage that has completed 10 rounds with that of a wild-type phage; Figure 9b is a graph comparing the half-life up to 24 hours for a phage that has completed 10 rounds and a wild-type phage.
[0065] Figure 10 shows the results of a comparative analysis of pH stability using a wild-type phage administered in Round 1 and a mutant phage isolated, proliferated, and purified from blood collected 48 hours after administration in Round 10.
[0066] Figure 11 shows the results of a comparative analysis of temperature stability using a wild-type phage administered in Round 1 and a mutant phage isolated, proliferated, and purified from blood collected 48 hours after administration in Round 10.
[0067]
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072]
[0073] Examples
[0074] Example 1. Induction of enhanced in vivo duration of bacteriophages by continuous microevolution
[0075] 1.1. Experimental Method
[0076] Continuous microevolution methods were used to induce and select mutant phages with enhanced persistence in the blood. Microevolution is an experimental method in which microorganisms, such as bacteriophages or bacteria, are continuously cultured under specific environments or selective pressures to evolve them to enhance specific phenotypes or acquire new characteristics. This method is used to accelerate natural evolutionary processes in a laboratory setting and reproduce them in a controlled manner.
[0077] Figure 1 shows the schematic steps of the microevolution method used in Example 1. To improve the duration in vivo, the process of injecting phages into an animal model and recovering and multiplying surviving phages after a certain period of time was defined as one round, and the accumulation of mutations was induced by transferring phage individuals that succeeded in surviving and multiplying in each round to the next round.
[0078] More specifically, the high-titer (10) established through prior toxicity tests 11After administering Lactococcus lactisphage (pfu / mL) intraperitoneally (ip), the decrease in blood phage over time was analyzed, and when the blood phage concentration was 10 8 The time required to decrease to the pfu / mL level was determined. To this end, 6 to 8-week-old BALB / c mice were used to evaluate phage survival rates over 72 hours.
[0079] The experiment is 10 11 After administering 300 μL of pfu / mL Lactococcus lactis phage intraperitoneally, whole blood was collected from each mouse at 3, 6, 12, 24, 48, and 72 hours, and the concentration of phage in the blood was quantitatively analyzed using a plaque assay. The experimental groups were conducted in Round 1 (n=20), Round 2 (n=18), Round 3 (n=15), Rounds 4 to 5 (n=12), and Rounds 6 to 10 (n=9), respectively.
[0080] 1.2. Experimental Results
[0081] A total of 10 rounds of experiments were conducted, and in each round, purified bacteriophages (10 11 pfu / mL) was provided to experimental animals.
[0082] As a result of the first round of experiments, the amount of bacteriophage administered into the abdominal cavity decreased to about half log unit after 24 hours of administration, and after 48 hours, no phage was detected in the blood (see Figure 2; at the 72-hour mark, it was below the detection limit, so up to 48 hours is indicated in the figure). Subsequently, from the second round to the tenth round, the bacteriophage remaining in the blood after 24 hours was recovered, proliferated, and purified, and then used for the next round of experiments.
[0083] As a result of experiments conducted over 2 to 9 rounds, a trend was observed in which the amount of bacteriophages remaining in the blood after 24 hours gradually increased through repeated rounds (see Figs. 3a and 3b). This suggests that the bacteriophages underwent a process of microevolution in which they adapted to selective pressure and increased their survival rate. When the entire mouse population was divided into three lines and quantitative analysis was performed on each line, the trend of microevolution was most pronounced in line 1; therefore, this example focuses on the results of line 1.
[0084] In addition, plaque analysis was performed on the combined total of serum samples collected during rounds 1 to 9. The analysis confirmed that the survival rate of bacteriophages increased with each round (see Fig. 4). This suggests that the bacteriophages continuously adapted to the selective pressure during the experiment, extending their duration in vivo.
[0085] In the results of Round 10, quantitative analysis was performed on three lines, and similar to the results of the previous rounds, the trend of microevolution was most pronounced in Line 1 (see Fig. 5). Accordingly, in the subsequent second experiment, additional experiments were conducted using the bacteriophage recovered from Line 1.
[0086] As a result of analyzing the survival amount of bacteriophages in the blood over time during rounds 1 to 10, the amount of phages surviving in the blood increased as each round progressed (see Fig. 6). This shows that the bacteriophages gradually adapted to the selective pressure through repeated rounds, and their in vivo stability and duration improved.
[0087]
[0088] Example 2. Comparative evaluation of in vivo viability of wild-type and mutant-type phages
[0089] 2.1. Experimental Method
[0090] Experiments were conducted to compare and evaluate the in vivo viability of "long-circulating phage" and "wild type phage," which are mutant types produced by repeating the administration-isolation-proliferation-re-administration process of Lactococcus lactis phage. After completing 10 rounds of microevolution, bacteriophages that survived were isolated from blood serum collected 24 hours after intraperitoneal injection, proliferated, and purified, and were prepared as the phages to be used in the experiment.
[0091] The provided bacteriophage is 3x10 11 It was administered intraperitoneally to 6 to 8-week-old BALB / c mice at a dose of 300 μL / pfu. Whole blood was collected from each mouse at 6, 12, 18, 24, 36, and 48 hours after administration, and the concentration of bacteriophages in the blood was quantitatively analyzed using a plaque assay.
[0092] Experiments were conducted separately (n=3) according to phage type ("long-circulating phage" and "wild-type phage") and time course. This example was designed to confirm the improvement in in vivo stability of bacteriophages generated through microevolution by comparing and analyzing the viability and persistence in the blood between the two phage types.
[0093] 2.2. Experimental Results
[0094] To compare the survival rates of wild-type phages and long-circulating phage mutant strains, plaque analysis was performed using serum collected from the blood after administering each bacteriophage to BALB / c mice. Analysis of serum collected from each mouse at different time points showed that, similar to the first round of the experiment, the survival rate of wild-type phages decreased rapidly over time, and no surviving phages were detected after 36 and 48 hours (see Fig. 7a). In contrast, the mutant phages showed a low rate of titer loss over time, and the survival rate gap with the wild type tended to increase significantly over time.
[0095] In addition, plaque analysis was performed by integrating serum samples collected from each mouse over time, and the results were consistent with those obtained for each individual, confirming that the mutant strain had a significantly higher survival rate compared to the wild type (see Fig. 7b). This further demonstrated the long-term circulation characteristics of the mutant phage.
[0096] Meanwhile, a comparative analysis of the survival rates of bacteriophages remaining in the blood after rounds 1 and 10 revealed that after 48 hours, wild-type phages were not detected in the blood, but mutant phages were still detected. Based on the survival rates at 24 and 48 hours, the data from rounds 1 and 10 were plotted and compared (see Fig. 8). In particular, even when a small amount of wild-type phage was mixed with the mutant phage, the half-lives were calculated to be approximately 1.4 hours for the wild-type phage and approximately 3.6 hours for the mutant phage (see Figs. 9a and 9b).
[0097] The above results demonstrate that mutant phages produced through the microevolution process possess the characteristic of maintaining a high survival rate while circulating for a long time in vivo, and that their pharmacokinetic properties are significantly improved compared to wild-type phages.
[0098]
[0099] Example 3. Comparative evaluation of the in vivo stability of phages
[0100] Stability with respect to pH and temperature was compared and analyzed using wild-type phage administered in Round 1 and mutant phage isolated, proliferated, and purified from blood collected 48 hours after administration in Round 10.
[0101] 3.1. Evaluation of pH stability
[0102] As a result of the pH stability test, the wild-type phage was stable for more than 1 hour at pH 3.5 to 11, but at pH 12, a loss of 1 log unit of potency occurred every 10 minutes, and it was completely inactivated after 30 minutes (see the top graph in Fig. 10).
[0103] On the other hand, the mutant strain phage maintained stability in the pH range of 3.5 to 11, and a loss of 1 log unit of potency was observed only after 1 hour at pH 12 or higher (see bottom graph of Fig. 10).
[0104] 3.2. Evaluation of Temperature Stability
[0105] As a result of the temperature stability test, the wild-type phage was stable without loss of potency for 1 hour at 4°C, 30°C, 37°C, and 42°C, and maintained activity after 24 and 48 hours. However, at 50°C and 60°C, a loss of potency of 1 log unit occurred every 10 minutes up to 30 minutes, and it became inactive after 1 hour (see top graph in Fig. 11).
[0106] In comparison, the mutant phage maintained activity without loss of potency for 1 hour at 4°C, 30°C, 37°C, 42°C, and 50°C, and was inactivated only at 60°C (see bottom graph in Fig. 11).
[0107] 3.3. Sintering
[0108] The results of Example 3 show that the mutant phage produced through the administration-isolation-proliferation-re-administration process not only had improved survival rates in the blood but also significantly increased stability in extreme pH and high-temperature environments. This suggests that the mutant phage acquired resistance to physical and chemical stress through the process of microevolution. These characteristics can be utilized as important design elements in the development of therapeutic bacteriophages and can further expand the potential for application in vivo and under various environmental conditions.
[0109]
[0110] Example 4. Confirmation of mutation location and content through genetic analysis of the variant strain
[0111] 4.1. Analysis Method
[0112] DNA from a bacteriophage population obtained from serum collected 48 hours after administration in Round 10 was analyzed using next-generation sequencing (NGS). During the analysis, the location and type of mutations occurring within the Tuc2009 structural protein gene were specifically evaluated, and data were obtained to infer the effects of these mutations on the in vivo characteristics and stability of the bacteriophages. This example was performed for the purpose of identifying genetic changes induced during the repeated administration-separation-proliferation-re-administration process and evaluating whether the mutations contributed to the functional improvement of the phages.
[0113] 4.2. Analysis Results
[0114] Next-generation sequencing (NGS) analysis of the DNA of the mutant phage population revealed that multiple missense mutations were induced in the Tuc2009 structural protein gene. These mutations were found at eight locations across a total of five genes.
[0115] Table 2 below summarizes variations at a total of eight positions expected to be significant among various missense mutations. The description for each item in Table 2 is as follows:
[0116] - Ref: Represents the Tuc2009 genome sequence information registered in the database. This is used as a reference sequence, and the registered GenBank access number is NC_002703.1. The above sequence served as a reference point for comparing the variants observed in the examples.
[0117] - Alt: A genomic sequence obtained from a phage sample that survived for 48 hours in Round 10, representing an altered sequence that has undergone mutation when compared to a reference sequence (Ref). This information allows for the identification of genetic changes that occurred during the process of microevolution.
[0118] - Pos: Represents the location of the mutated gene as a number on the Tuc2009 genome, used to identify the precise location of the mutation.
[0119] - p.(protein change): Indicates which amino acid substitutions the mutation caused at the protein level.
[0120] - Gene name: Indicates the name of the gene in which the mutation occurred or the gene number assigned on the reference genome (annotation). This was used to clarify that the mutation occurred in a specific gene.
[0121] - Description: Explains the name and function of the mutated gene.
[0122] - Transcript: Describes detailed information about the mutation.
[0123] - codon_change: Indicates how the gene's codons have been changed by mutation.
[0124] - protein_change: Indicates which amino acid substitutions the mutation caused at the protein level.
[0125] - cDNA_position / cDNA_len: Indicates the location of the mutated cDNA (complementary DNA) and the total length of the cDNA. For example, "998 / 1041" means that the total length of the cDNA is 1041 bp and the mutation occurred at the 998th position.
[0126] - CDS_position / CDS_len: Indicates the position and total length of the CDS (coding sequence) where the mutation occurred. For example, "1373 / 2721" means that the total length of the coding sequence is 2721 bp and the mutation occurred at the 1373rd coding sequence position.
[0127] - Protein_position / Protein_len: Indicates the location within the protein where the mutation occurred and the total length of the protein. For example, "636 / 906" means that the total length of the protein is 906 amino acids and the mutation occurred at the 636th amino acid position of the protein.
[0128]
[0129] RefAltPosp.Gene NameDescriptionTranscript (codon_change ; protein_change ; cDNA_position / cDNA_len ; CDS_position / CDS_len ; Protein_position / Protein_len)AC18841D333ATuc2009_32Minor capsid protein 1c.998A>C ; p.Asp333Ala ; 998 / 1041 ; 998 / 1041 ; 333 / 346TC30269M458TTuc2009_48Tal2009 (tail-associated lysin)c.1373T>C ; p.Met458Thr ; 1373 / 2721 ; 1373 / 2721 ; 458 / 906AC30803E636ATuc2009_48Tal2009 (tail-associated lysin)c.1907A>C ; p.Glu636Ala ; 1907 / 2721 ; 1907 / 2721 ; 636 / 906AG32467I280VTuc2009_49BppU (upper baseplate protein)c.838A>G ; p.Ile280Val ; 838 / 969 ; 838 / 969 ; 280 / 322TA33912L145QTuc2009_51BppL (lower baseplate protein)c.434T>A ; p.Leu145Gln ; 434 / 522 ; 434 / 522 ; 145 / 173TA33930I151KTuc2009_51BppL (lower baseplate protein)c.452T>A ; p.Ile151Lys ; 452 / 522 ; 452 / 522 ; 151 / 173AG33942N155STuc2009_51BppL (lower baseplate protein)c.464A>G ; p.Asn155Ser ; 464 / 522 ; 464 / 522 ; 155 / 173CG35438L397VTuc2009_53Neck passage structurec.1189C>G ; p.Leu397Val ; 1189 / 2031 ; 1189 / 2031 ; 397 / 676
[0130]
[0131] First, the D333A mutation was identified in "Minor capsid protein 1 (MCP1)," which constitutes part of the bacteriophage capsid. Second, M458T and E636A mutations were found in "Tail-associated lysin (Tal2009)," which is responsible for the enzymatic activity of the tail. Third, the I280V mutation was identified in the BppU gene, which constitutes the baseplate of the tail, while the L145Q, I151K, and N155S mutations were identified in the BppL gene, respectively. Finally, the L397V mutation was identified in the gene constituting the "Neck passage structure (NPS)," the connection between the capsid and the tail. These missense mutations are predicted to have caused changes in the protein structure and function of the bacteriophage through single amino acid substitutions. For example, the D333A mutation in MCP1 may affect capsid stability, while mutations in Tal2009 (M458T, E636A) have the potential to enhance tail enzymatic activity and host cell wall degradation ability. Mutations in BppU and BppL (I280V, L145Q, I151K, N155S) may strengthen the structural stability of the tail baseplate or increase host binding efficiency. Additionally, the L397V mutation found in NPS has the potential to have a positive effect on the connection strength and assembly efficiency between the capsid and the tail.
[0132] The results described above suggest that genetic variations generated during the repeated administration-isolation-proliferation-re-administration process may have improved the in vivo characteristics (e.g., stability, host adaptability, and infectivity) of the bacteriophages. In particular, variations within structural protein genes are suggested to have acted as important factors contributing to the stability and functional enhancement of the bacteriophages.
[0133] The mutations disclosed in the present invention can be used as a key strategy to improve pharmacokinetic properties in designing therapeutic bacteriophages.
[0134]
[0135] Representative embodiments or combinations of embodiments based on the above description are as follows:
[0136] [Example of Implementation 1]
[0137] A Lactococcus phage Tuc2009 mutant strain with improved pharmacokinetic properties comprising at least one missense mutation in one or more structural protein genes selected from the group consisting of (a) to (e) below: (a) the Tuc2009_32 gene consisting of the nucleotide sequence of SEQ ID NO. 2; (b) the Tuc2009_48 gene consisting of the nucleotide sequence of SEQ ID NO. 4; (c) the Tuc2009_49 gene consisting of the nucleotide sequence of SEQ ID NO. 6; (d) the Tuc2009_51 gene consisting of the nucleotide sequence of SEQ ID NO. 8; and (e) the Tuc2009_53 gene consisting of the nucleotide sequence of SEQ ID NO. 10.
[0138] [Example of Implementation 2]
[0139] In the above-described embodiment, the missense mutation results in one or more amino acid substitutions selected from the group consisting of (i) to (viii) below: (i) substitution of the 333rd aspartic acid (D) with alanine (A) based on the amino acid sequence of SEQ ID NO. 3; (ii) substitution of the 458th methionine (M) with threonine (T) based on the amino acid sequence of SEQ ID NO. 5; (iii) substitution of the 636th glutamic acid (E) with alanine (A) based on the amino acid sequence of SEQ ID NO. 5; (iv) substitution of the 280th isoleucine (I) with valine (V) based on the amino acid sequence of SEQ ID NO. 7; (v) substitution of the 145th leucine (L) with glutamine (Q) based on the amino acid sequence of SEQ ID NO. 9; (vi) substitution of the 151st isoleucine (I) with lysine (K) based on the amino acid sequence of SEQ ID NO. 9; (vii) the 155th asparagine (N) in the amino acid sequence of SEQ ID NO. 9 is substituted with serine (S); and (viii) the 397th leucine (L) in the amino acid sequence of SEQ ID NO. 11 is substituted with valine (V).
[0140] [Example of Implementation 3]
[0141] In any one of the embodiments described above, the wild type of Lactococcus phage Tuc2009 is a mutant strain comprising a whole-length genome sequence consisting of the nucleotide sequence of SEQ ID NO. 1.
[0142] [Example of Implementation 4]
[0143] In any one of the embodiments described above, the pharmacokinetic characteristics are one or more selected from the group consisting of resistance to immune response, pH resistance, and temperature resistance, in a mutant strain.
[0144] [Example 5 of Implementation]
[0145] A mutant strain characterized in that, in any one of the embodiments described above, the mutant strain has a half-life in vivo that is at least twice as long as that of the wild type.
[0146] [Example 6 of Implementation]
[0147] In any one of the aforementioned embodiments, the mutant strain is characterized by being stable for at least one hour in an environment of pH 3.5 to pH 12.
[0148] [Example 7]
[0149] In any one of the aforementioned embodiments, the mutant strain is characterized by being stable for at least one hour in an environment of 4°C to 50°C.
[0150] [Example of Implementation 8]
[0151] An engineered phage with improved pharmacokinetic properties by introducing at least one missense mutation into one or more structural protein genes selected from the group consisting of (a) to (e): (a) Minor capsid protein 1 gene; (b) Tail-associated lysin gene; (c) Upper baseplate protein (BppU) gene; (d) Lower baseplate protein (BppL) gene; and (e) Neck passage structure (NPS) gene.
[0152] [Example of Implementation 9]
[0153] An engineered phage in any one of the embodiments described above, wherein the pharmacokinetic properties are one or more selected from the group consisting of resistance to immune response, pH resistance, and temperature resistance.
[0154] [Example of Implementation 10]
[0155] An engineered phage, wherein in any one of the embodiments described above, the engineered phage is characterized by having an increased in vivo duration compared to before the introduction of at least one missense mutation.
[0156] [Example of Implementation 11]
[0157] In any one of the embodiments described above, the engineered phage is an engineered phage based on Lactococcus phage Tuc2009.
[0158] [Example 12]
[0159] In any one embodiment of the above-described embodiments, the engineered phage comprises a protein encoded by one or more structural protein genes selected from the group consisting of (a) to (e), wherein the structural protein gene has at least one missense mutation introduced: (a) a Tuc2009_32 gene consisting of the nucleotide sequence of SEQ ID NO. 2; (b) a Tuc2009_48 gene consisting of the nucleotide sequence of SEQ ID NO. 4; (c) a Tuc2009_49 gene consisting of the nucleotide sequence of SEQ ID NO. 6; (d) a Tuc2009_51 gene consisting of the nucleotide sequence of SEQ ID NO. 8; and (e) a Tuc2009_53 gene consisting of the nucleotide sequence of SEQ ID NO. 10.
[0160] [Example 13]
[0161] In any one embodiment of the above-described embodiments, the engineered phage comprises one or more structural proteins selected from the group consisting of (i) to (v): (i) a structural protein comprising the amino acid sequence of SEQ ID NO. 3 comprising a D333A mutation; (ii) a structural protein comprising the amino acid sequence of SEQ ID NO. 5 comprising M458T and / or E636A mutations; (iii) a structural protein comprising the amino acid sequence of SEQ ID NO. 7 comprising an I280V mutation; (iv) a structural protein comprising the amino acid sequence of SEQ ID NO. 9 comprising one or more mutations selected from the group consisting of L145Q, I151K and N155S; and (v) a structural protein comprising the amino acid sequence of SEQ ID NO. 11 comprising an L397V mutation.
[0162] [Example 14]
[0163] An engineered phage, characterized in that, in any one of the embodiments described above, the engineered phage has a different host range or an extended host range compared to the wild-type Lactococcus phage Tuc2009.
[0164] [Example 15]
[0165] In any one of the embodiments described above, the engineered phage is an engineered phage comprising a payload.
[0166] [Example 16]
[0167] In any one embodiment of the above-described embodiments, the payload comprises an engineered phage 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.
[0168] [Example 17]
[0169] A pharmaceutical composition for the prevention or treatment of disease comprising an engineered phage according to any one of the embodiments described above as an active ingredient.
[0170] [Example 18]
[0171] Use of a composition comprising an engineered phage according to any one of the aforementioned embodiments as an active ingredient for the prevention or treatment of disease.
[0172] [Example of Implementation 19]
[0173] Use for producing a drug for the prevention or treatment of disease of an engineered phage according to any one of the embodiments described above.
[0174] [Gu Hyeon-ye 20]
[0175] A method for preventing or treating a disease comprising the step of administering a composition containing an engineered phage as an active ingredient according to any one of the embodiments described above to an individual in need thereof.
[0176] [Example of Implementation 21]
[0177] A vector comprising a polynucleotide encoding an engineered phage according to any one of the aforementioned embodiments, operably connected to a promoter.
[0178] [Example of Implementation 22]
[0179] A method for preparing a phage with improved pharmacokinetic properties, comprising the step of inducing or introducing at least one missense mutation into one or more structural protein genes selected from the group consisting of (a) to (e): (a) Minor capsid protein 1 gene; (b) Tail-associated lysin gene; (c) Upper baseplate protein (BppU) gene; (d) Lower baseplate protein (BppL) gene; and (e) Neck passage structure (NPS) gene.
[0180] [Example of Implementation 23]
[0181] A method in any one of the embodiments described above, wherein the phage is Lactococcus phage Tuc2009 and the missense mutation results in one or more amino acid substitutions selected from the group consisting of (i) to (viii) below: (i) substitution of the 333rd aspartic acid (D) with alanine (A) based on the amino acid sequence of SEQ ID NO. 3; (ii) substitution of the 458th methionine (M) with threonine (T) based on the amino acid sequence of SEQ ID NO. 5; (iii) substitution of the 636th glutamic acid (E) with alanine (A) based on the amino acid sequence of SEQ ID NO. 5; (iv) substitution of the 280th isoleucine (I) with valine (V) based on the amino acid sequence of SEQ ID NO. 7; (v) substitution of the 145th leucine (L) with glutamine (Q) based on the amino acid sequence of SEQ ID NO. 9; (vi) the 151st isoleucine (I) based on the amino acid sequence of SEQ ID NO. 9 is substituted with lysine (K); (vii) the 155th asparagine (N) based on the amino acid sequence of SEQ ID NO. 9 is substituted with serine (S); and (viii) the 397th leucine (L) based on the amino acid sequence of SEQ ID NO. 11 is substituted with valine (V).
[0182]
[0183] 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. comprising at least one missense mutation in one or more structural protein genes selected from the group consisting of (a) to (e) below Lactococcus phage Tuc2009 mutant strain with improved pharmacokinetic properties: (a) The Tuc2009_32 gene consisting of the nucleotide sequence of SEQ ID NO. 2; (b) The Tuc2009_48 gene consisting of the nucleotide sequence of SEQ ID NO. 4; (c) The Tuc2009_49 gene consisting of the nucleotide sequence of SEQ ID NO. 6; (d) The Tuc2009_51 gene consisting of the nucleotide sequence of SEQ ID NO. 8; and (e) The Tuc2009_53 gene consisting of the nucleotide sequence of SEQ ID NO.
10.
2. In Paragraph 1, The above missense mutation results in one or more amino acid substitutions selected from the group consisting of (i) to (viii) below, a mutant strain: (i) Based on the amino acid sequence of SEQ ID NO. 3, the 333rd aspartic acid (D) is substituted with alanine (A); (ii) Based on the amino acid sequence of SEQ ID NO. 5, the 458th methionine (M) is substituted with threonine (T); (iii) Based on the amino acid sequence of SEQ ID NO. 5, the 636th glutamic acid (E) is substituted with alanine (A); (iv) Based on the amino acid sequence of SEQ ID NO. 7, the 280th isoleucine (I) is substituted with valine (V); (v) Based on the amino acid sequence of SEQ No. 9, the 145th leucine (L) is substituted with glutamine (Q); (vi) Based on the amino acid sequence of SEQ ID NO. 9, the 151st isoleucine (I) is substituted with lysine (K); (vii) The 155th asparagine (N) in the amino acid sequence of SEQ ID NO. 9 is substituted with serine (S); and (viii) Based on the amino acid sequence of SEQ ID NO. 11, the 397th leucine (L) is substituted with valine (V).
3. In Paragraph 1, The wild type of the above-mentioned Lactococcus phage Tuc2009 is a mutant strain comprising a whole-genome sequence consisting of the nucleotide sequence of SEQ ID NO.
1.
4. In Paragraph 1, A mutant strain in which the above pharmacokinetic characteristics are any one or more selected from the group consisting of resistance to immune response, pH resistance, and temperature resistance.
5. In Paragraph 1, The above mutant strain is characterized by having a half-life in vivo that is at least twice as long as that of the wild type.
6. In Paragraph 1, The above mutant strain is characterized by being stable for at least one hour in an environment of pH 3.5 to pH 12.
7. In Paragraph 1, The above mutant strain is characterized by being stable for at least one hour in an environment of 4°C to 50°C.
8. An engineered phage with improved pharmacokinetic properties in which at least one missense mutation is introduced into one or more structural protein genes selected from the group consisting of (a) to (e) below: (a) Minor capsid protein 1 gene; (b) Tail-associated lysin gene; (c) Upper baseplate protein (BppU) gene; (d) Lower baseplate protein (BppL) gene; and (e) Neck passage structure (NPS) gene.
9. In Paragraph 8, The above pharmacokinetic properties are any one or more selected from the group consisting of resistance to immune response, pH resistance, and temperature resistance, of an engineered phage.
10. In Paragraph 8, The above-mentioned engineered phage is characterized by having an increased in vivo duration compared to before the introduction of at least one missense mutation.
11. In Paragraph 8, The above-mentioned engineered phage is an engineered phage based on Lactococcus phage Tuc2009.
12. In Paragraph 11, The above-mentioned engineered phage comprises a protein encoded by one or more structural protein genes selected from the group consisting of (a) to (e) below, and The above structural protein gene is an engineered phage into which at least one missense mutation has been introduced: (a) The Tuc2009_32 gene consisting of the nucleotide sequence of SEQ ID NO. 2; (b) The Tuc2009_48 gene consisting of the nucleotide sequence of SEQ ID NO. 4; (c) The Tuc2009_49 gene consisting of the nucleotide sequence of SEQ ID NO. 6; (d) The Tuc2009_51 gene consisting of the nucleotide sequence of SEQ ID NO. 8; and (e) The Tuc2009_53 gene consisting of the nucleotide sequence of SEQ ID NO.
10.
13. In Paragraph 11, The above-mentioned engineered phage comprises one or more structural proteins selected from the group consisting of (i) to (v) below: (i) A structural protein composed of the amino acid sequence of SEQ ID NO. 3 containing the D333A mutation; (ii) A structural protein consisting of the amino acid sequence of SEQ ID NO. 5 containing M458T and / or E636A variants; (iii) A structural protein composed of the amino acid sequence of SEQ ID NO. 7 containing an I280V mutation; (iv) a structural protein comprising the amino acid sequence of SEQ ID NO. 9, comprising one or more variants selected from the group consisting of L145Q, I151K and N155S; and (v) A structural protein consisting of the amino acid sequence of SEQ ID NO. 11 containing the L397V mutation.
14. In Paragraph 11, The above-mentioned engineered phage is characterized by having a different host range or an extended host range compared to the wild-type Lactococcus phage Tuc2009.
15. In Paragraph 8, The above-mentioned engineered phage is an engineered phage including a payload.
16. In Paragraph 8, The above payload comprises an engineered phage 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 agent; or a combination thereof.
17. A pharmaceutical composition for the prevention or treatment of disease comprising an engineered phage according to any one of claims 8 to 16 as an active ingredient.
18. A vector comprising a polynucleotide encoding an engineered phage according to any one of claims 8 through 16, operably connected to a promoter.
19. A method for preparing a phage with improved pharmacokinetic properties, comprising the step of inducing or introducing at least one missense mutation into one or more structural protein genes selected from the group consisting of (a) to (e) below: (a) Minor capsid protein 1 gene; (b) Tail-associated lysin gene; (c) Upper baseplate protein (BppU) gene; (d) Lower baseplate protein (BppL) gene; and (e) Neck passage structure (NPS) gene.
20. In Paragraph 19, The above phage is Lactococcus phage Tuc2009, and A method in which the above missense mutation results in one or more amino acid substitutions selected from the group consisting of (i) to (viii) below: (i) Based on the amino acid sequence of SEQ ID NO. 3, the 333rd aspartic acid (D) is substituted with alanine (A); (ii) Based on the amino acid sequence of SEQ ID NO. 5, the 458th methionine (M) is substituted with threonine (T); (iii) Based on the amino acid sequence of SEQ ID NO. 5, the 636th glutamic acid (E) is substituted with alanine (A); (iv) Based on the amino acid sequence of SEQ ID NO. 7, the 280th isoleucine (I) is substituted with valine (V); (v) Based on the amino acid sequence of SEQ No. 9, the 145th leucine (L) is substituted with glutamine (Q); (vi) Based on the amino acid sequence of SEQ ID NO. 9, the 151st isoleucine (I) is substituted with lysine (K); (vii) The 155th asparagine (N) in the amino acid sequence of SEQ ID NO. 9 is substituted with serine (S); and (viii) Based on the amino acid sequence of SEQ ID NO. 11, the 397th leucine (L) is substituted with valine (V).