WO2023128507A1 - Hexameric n-formyl peptide library and uses thereof - Google Patents

Abstract

The present invention provides a hexameric N-formyl peptide library and uses thereof, wherein the hexameric N-formyl peptide library is for discovering a formyl-peptide receptor activating active substance for the development of a therapeutic agent for inflammatory diseases using a synthetic peptide with increased affinity to a specific receptor.

Description

Hexameric N-formyl peptide library and uses thereof

The present invention relates to a hexameric N-formyl peptide library, and more particularly, to a hexameric N-formyl peptide library for discovering an effective substance for activating a formyl peptide receptor and its use.

N-formyl peptide is a peptide derivative in which a formyl group is added to the N-terminus of an amino acid. will be located In the past, N-formyl peptides were considered to exist only in proteins produced by bacteria, and were thought to be useful in recognizing foreign substances in the human body and inducing an immune response. However, it has recently been found that N-formyl peptides also exist in proteins produced by mitochondria, and it is no longer thought that they can simply be used to distinguish foreign substances in the human body. Instead, proteins or oligopeptides containing N-formyl peptides appear to come from bacteria or from the mitochondria of damaged tissue, which are pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns, respectively. (DAMP, damage-associated molecular pattern).

A representative N-formyl peptide-containing oligopeptide is N-formylmethionine-leucine-phenylalanine (fMLF), which induces/activates immune cells such as neutrophils and monocytes. These functions are related to the formyl-peptide receptor (FPR), one of the G Protein-Coupled Receptors (GPCRs) in which N-formyl peptide mediates various cell functions through sub-signaling systems. It has been shown to occur in combination. The receptor name is also known as a receptor that recognizes N-formyl peptide and was named FPR. FPR mainly includes FPR1, which induces an acute inflammatory response by binding to bacterial-derived peptides, FPR2, which controls inflammatory responses by binding to endogenous ligands such as peptides, proteins, and lipids, and FPR3, whose function is not yet well known. The recently known new function of FPR is that when an influx of external substances or infection occurs, a defense mechanism occurs through an acute inflammatory response of innate immunity, and then an inflammatory action occurs, controlling the inflammatory response to an appropriate level and inducing tissue recovery. do. In addition, it induces the removal of apoptotic cells and damaged tissues by macrophages, reduces M1 macrophages that induce inflammatory responses, and increases M2 macrophages that induce anti-inflammatory responses. Therefore, using synthetic peptides with increased affinity with specific receptors to target specific receptors as molecular targets can be a new strategy for the development of therapeutic agents for inflammatory diseases. In this regard, Korean Patent Publication No. 2021-0135488 discloses a novel peptide library and a method of using the same.

However, the development of peptide anti-inflammatory substances is still limited, and various approaches and unbiased approaches are required.

The present invention is to solve various problems, including the above problems, hexamer N- It aims to provide a formyl peptide library and its use. However, these tasks are illustrative, and the scope of the present invention is not limited thereby.

According to one aspect of the present invention, a hexameric N-formyl peptide library having a formyl group added to the N-terminus composed of 6 amino acids including an amino acid sequence selected from the group consisting of Provided:

formyl-MX ₁ X ₂ X ₃ X ₄ X ₅

(At this time, any one of X ₁ to X ₅ excluding the N-terminal formyl methionine is fixed as a specific amino acid, and the rest except for the fixed amino acid is synthesized so that 19 amino acids excluding cysteine are randomly arranged) .

According to another aspect of the present invention, the culturing step of treating the peptide library with immune cells and then culturing: analyzing the level of formyl peptide receptor activation by recovering the cells that have completed the culturing step, the lysate or medium supernatant of the cells Analysis step: a first selection step of selecting candidate amino acids effective for each fixed position by selecting a peptide pool showing higher activity than the control group in the analysis step; and a second screening step of selecting a candidate group having the highest activity by combining the candidate amino acids.

With the N-formyl peptide library having the diversity and search efficiency of the present invention made as described above, it is possible to search for active substances of peptides that activate formyl peptide receptors and regulate immune cell inflammatory responses. In addition, the N-formyl peptide library can be used to develop formyl peptide pharmacological substances exhibiting efficacy in chronic inflammatory diseases or autoimmune diseases. Of course, the scope of the present invention is not limited by these effects.

1 is a schematic view of the construction of the hexameric N-formyl peptide library of the present invention and the process of searching for effective peptides using the same.

Figure 2 is a graph showing the results of analyzing intra-cellular calcium mobilization through FPR1 activation using the hexameric N-formyl peptide library of the present invention. The values in the above graph represent the results of relatively measuring the activity of other peptides with the measured value of the intracellular calcium ion concentration of the peptide having the second amino acid fixed to AA1 in the hexameric N-formyl peptide as 1.

Figure 3 is a graph showing the results of analyzing intra-cellular calcium mobilization through FPR2 activation using the hexameric N-formyl peptide library of the present invention. The values in the above graph represent the results of relatively measuring the activity of other peptides with the measured value of the intracellular calcium ion concentration of the peptide having the second amino acid fixed to AA1 in the hexameric N-formyl peptide as 1.

Definition of Terms

As used herein, the term "formyl-peptide receptor (FPR)" is a G protein-coupled receptor involved in chemotaxis. It is encoded by a separate gene called These receptors were originally identified by their ability to bind N-formyl peptides such as N-formyl methionine produced by bacteria or by degradation of host cells.

The term "peptide library" used in this document is a technology for constructing peptides of various amino acid sequences to be easily searched for, as in finding necessary books in a library. For example, when the peptide library screening technology consisting of 6 amino acids is applied, physiologically active substances that can be developed as new biologics can be derived from the diversity of 20 to the 6th power through 120 search processes and subsequent search and verification processes.

DETAILED DESCRIPTION OF THE INVENTION:

According to one aspect of the present invention, a hexameric N-formyl peptide library having a formyl group added to the N-terminus composed of 6 amino acids including an amino acid sequence selected from the group consisting of Provided:

formyl-MX ₁ X ₂ X ₃ X ₄ X ₅

(At this time, any one of X ₁ to X ₅ excluding the N-terminal formyl methionine is fixed as a specific amino acid, and the rest except for the fixed amino acid is synthesized so that 19 amino acids excluding cysteine are randomly arranged) .

In the library, the C-terminus may be amidated and may be composed of 95 pools according to the fixed amino acids, and may be for formyl peptide receptor (FPR) activation or inflammation response regulating peptide active substance search .

According to another aspect of the present invention, the culturing step of treating the peptide library with immune cells and then culturing: analyzing the level of formyl peptide receptor activation by recovering the cells that have completed the culturing step, the lysate or medium supernatant of the cells Analysis step: a first selection step of selecting candidate amino acids effective for each fixed position by selecting a peptide pool showing higher activity than the control group in the analysis step; and a second screening step of selecting a candidate group having the highest activity by combining the candidate amino acids.

In the screening method, the immune cells include neutrophils, basophils, eosinophils, mast cells, monocytes, macrophages, and dendritic cells. , It can be selected from the group consisting of T cells, B cells, and NK cells, and the level of the formyl peptide receptor activation is analyzed by calcium ion permeability analysis, cAMP production analysis, inflammation induction or inflammation relief signal analysis, or immune response analysis. It may be selected from the group consisting of, and the immune response analysis may be performed by measuring the secretion level of cytokines, chemokines or cytoplasmic granules of immune cells or measuring the phagocytosis of macrophages.

A new function of the recently known formyl peptide receptor (FPR) is the resolution of inflammation, which ends the innate immune response and promotes an appropriate adaptive immune response and proper recovery and homeostasis of damaged tissue. refers to the mechanism that induces When an influx of external substances or infection occurs, a defense mechanism occurs through an acute inflammatory response of innate immunity, and then an inflammatory action occurs to control the inflammatory response to an appropriate level and induce tissue recovery. The anti-inflammatory action limits the influx of additional immune cells into the inflamed tissue, induces apoptosis, and promotes the reduction of inflammatory substances, cytokines, and chemokines that cause an inflammatory response. In addition, it induces the removal of apoptotic cells and damaged tissues through efferocytosis of macrophages, reduces pro-inflammatory macrophages (M1, classically activated macrophages) that induce inflammatory responses, and increases anti-inflammatory macrophages (M2) with anti-inflammatory properties. , promotes the increase of alternatively activated macrophages). Appropriate inflammatory action limits excessive inflammatory response and induces a normal acquired immune response, enabling the ability to respond to repeated inflammatory situations in the future. However, insufficient inflammatory response can cause chronic inflammation due to continuous inflammatory response and various chronic inflammatory diseases or autoimmune diseases due to abnormal acquired immune response. Therefore, the inflammatory response induced by FPR does not simply terminate the immune response, but plays a role in helping our body maintain the correct immune system. The resolution of inflammation occurs when various types of anti-inflammatory factors (SPMs, specialized pro-resolving mediators) such as lipids, proteins, and peptides bind to specific receptors of immune cells. Recently, as the specific mechanism of inflammation relief and major action factors have been identified, strategies for treating inflammatory diseases through induction of inflammation relief have attracted attention. Until now, drug development attempts using lipid inflammatory factors such as LXA4 (lipoxin A4) or Resolvin have been mainly made, but low solubility and stability of the lipids may be a problem. Therefore, it can compensate for the disadvantages of the lipid anti-inflammatory factors mentioned above, and using synthetic peptides with increased affinity with specific receptors to target specific receptors as molecular targets can be a new strategy for the development of therapeutic agents for inflammatory diseases. Accordingly, the inventors of the present invention completed the present invention by developing an N-formyl peptide library for discovering an effective substance for activating a formyl peptide receptor in order to discover an effective substance for treating inflammatory diseases (FIG. 1).

In conclusion, the N-formyl peptide library of the present invention compensates for the disadvantages of lipid anti-inflammatory factors such as low solubility and stability of lipids and improves inflammation through induction of anti-inflammatory by using synthetic peptides with increased binding force with specific receptors. It can be used to develop new strategies for the development of disease treatments.

Hereinafter, the present invention will be described in more detail through examples. However, the present invention is not limited to the embodiments disclosed below, but can be implemented in various different forms. It is provided to fully inform you.

Example 1: Construction of N-formyl peptide library

The present invention relates to a hexameric N-formyl peptide library and is composed of peptides described as formyl-MX ₁ X ₂ X ₃ X ₄ X ₅ -NH ₂ . At this time, one amino acid is fixed at each X position, and 20 kinds of amino acids or 19 kinds of amino acids excluding cysteine are randomly arranged at the remaining positions. Therefore, the N-formyl peptide library consists of a pool of 100 to 95 amino acids fixed at each X position.

In the specific synthesis procedure, Rink amide MBHA resin is reacted with DMF, and a 20% Piperidine/DMF solution is added to the reactor and stirred. After that, Fmoc-A.A-OH and HBTU/N-Methylmorpholine/DMF dissolved in DMF are put into a reactor with resin and reacted. Subsequently, the 20% Piperidine/DMF solution was put into a reactor with resin, stirred, washed with DMF, and the preceding process was repeated for each amino acid at each position to synthesize a hexameric peptide. At this time, synthesis is performed by mixing 20 or 19 amino acids (A.A) and treating them as one amino acid. Thereafter, formic acid dissolved in DMF is introduced into a reactor containing resin, and HOBT/DMF and DIC/DMf are added, stirred, drained, and washed. Thereafter, the obtained resin is stirred at room temperature after adding a cleavage solution, drained, washed with a small amount of TFA, crystallized in ether, and filtered to obtain a peptide mixture.

Example 2: Search method for formyl peptide receptor activating active substance from N-formyl peptide through intra-cellular calcium mobilization

Formyl peptide receptor (FPR), a type of G protein-coupled receptor (GPCR), induces various intracellular changes, such as changes in calcium ion permeability, when activated by ligand binding. Changes in calcium ion permeability can be measured using a fluorescent substance called Fura-2 AM. As the concentration of calcium ions inside the cell changes, Fura-2 AM shows a change in fluorescence color and changes in the activity of formyl peptides. can be visualized, and the activation of the peptide ligand is confirmed using the digitized fluorescence color change and the efficacy is evaluated by calculating the EC ₅₀ .

Specifically, immune cells such as neutrophils, eosinophils, or macrophages are treated with Fura-2AM and then cultured for several hours. At this time, the hydrophobic AM of Fura-2 AM penetrated into the cell is cleaved by the intracellular enzyme and cannot go out of the cell again. In the peptide library constructed in Example 1, after treating cells with each peptide pool in which a specific amino acid is fixed at a specific position, the fluorescence change of Fura-2, which is changed by calcium binding, is measured at 340 nm and 380 nm for several seconds Repeat the measurement at intervals. After that, the change in the ratio of 340 nm/380 nm is calculated using the measured values for each time obtained, and the efficacy of each pool is evaluated through 'maximum value-minimum value'. Through this, after selecting amino acids effective for each fixed position, a sequence is created by combining the selected amino acids, and a change in calcium ion permeability is measured and compared using peptides obtained through synthesis, and the best sequence candidates are selected. In addition, the cell line used in this example is a cell line that overexpresses only FPR2 or only FPR1 in cells that do not express FPR1 and FPR2, and FPR2-induced calcium ion concentration change activity (EC ₅₀ for FPR2) was observed using a cell line in which FPR2 was overexpressed. Calcium ion concentration change activity (EC ₅₀ for FPR1) by FPR1 was measured using a cell line in which FPR1 was overexpressed.

As a result of searching for intra-cellular calcium mobilization through FPR activation using the hexameric N-formyl peptide library of the present invention, in the first screening, a screen using a pool of 95 (19 X 5) amino acids was placed at each position Sequence information was obtained to determine whether excellent activity was shown for FPR1 and FPR2 when doing so (FIGS. 2 and 3). Based on the sequence information obtained from the N-formyl peptide library search, a peptide candidate group having specific activity for FPR1 or FPR2, or both FPRs at the same time, was designed. Activity analysis values for each formyl peptide receptor (FPR) of synthesized peptide candidates are summarized in Table 1 below.

Activity Assay for Formyl Peptide Receptor (FPR)

Peptide#	EC50
	FPR2 (nM)	FPR1 (uM)
Peptide 1	685.60	0.0218
peptide2	504.60	0.0144
Peptides3	449.60	0.0012
Peptides 4	22.80	0.0362
Peptide 5	29.12	0.0281
Peptide 6	49.09	0.0631
Peptide 7	1.24	0.3046
Peptide 8	1.03	0.1572
Peptides9	1.18	0.2342
Peptide 10	0.93	0.1526
Peptide 11	4.07	>50
Peptide 12	1.18	>50
Peptide 13	2.94	0.1168
Peptide 14	3.13	0.0792
Peptides 15	4.05	3.3320
Peptide 16	5.62	0.0295
Peptides 17	4.32	0.0991
Peptides 18	3.12	13.8500
Peptides 19	5.13	>50
Peptides20	1.63	>50

Example 3: Method for searching formyl peptide receptor activating active substances from N-formyl peptide through cAMP production analysis

G protein-coupled receptors mediate cell signaling by activating or inhibiting cAMP production. cAMP (Adenosine 3', 5'-cyclic monophosphate, cyclic adenosine monophosphate) is a secondary messenger involved in the cell signaling pathway, and is adenylate cyclase located inside the cell membrane. It activates protein kinase A (PKA) and causes signal amplification through a chain reaction of signaling proteins that follow it. Therefore, the activation of G protein-coupled receptors, including formyl peptide receptors, can be assessed through changes in the production of cAMP.

In the peptide library constructed from Example 1, each peptide pool in which a specific amino acid is fixed at a specific position is treated with immune cells such as neutrophil, eosinophil or macrophage, cultured, and then cell lysate (lysate) is recovered. Then, when alkaline phosphatase-conjugated cAMP and cAMP antibody are added to the lysate and incubated, cAMP and alkaline phosphatase-conjugated cAMP in the lysate competitively bind to the cAMP antibody. At this time, when pNpp substrate is added, it reacts catalytically with alkine phosphatase and turns yellow. The color change in each lysate is measured by optical density (OD) and the cAMP concentration is determined through a colorimetric method that compares with a standard solution of known concentration. quantify it. Through this, the change of cAMP by the treatment of each peptide pool is compared and evaluated to select amino acids effective for each fixed position. Thereafter, a sequence is generated by combining the selected amino acids, and the cAMP change value is measured and compared using the peptide obtained through synthesis, and the best sequence candidate group is selected.

Example 4: Method for searching for effective substances for activating formyl peptide receptors from N-formyl peptides through signal transduction system analysis

Intracellular signaling transmitted through G protein-coupled receptors is largely divided into two representative pathways, one is G-protein mediated signaling and the other is beta-arrestin mediated signaling (β-arrestin). arrestin mediated signaling). In general, G protein-coupled receptors can be activated in various ways by multiple ligands, which can activate or inhibit multiple signal transduction systems. At this time, the function of the cell is regulated through a specific signal transduction pathway or a combination thereof. In the case of the formyl peptide receptor (FPR), W-peptide (WKYMVm) known as a full agonist is known to strongly regulate cAMP, Ca ²⁺ , MAPK and β-arrestin. On the other hand, it is known that the inflammation-inducing signal regulates only β-arrestin, and the anti-inflammatory signal regulates cAMP and Ca ²⁺ . Therefore, even for formyl peptides that activate the same receptors, their characteristics can be distinguished or inferred through analysis of the sub-signal transduction system. can be selected

In the peptide library constructed from Example 1, each peptide pool in which a specific amino acid is fixed at a specific position is treated with immune cells such as neutrophil, eosinophil or macrophage, cultured, and then cell lysate (lysate) is recovered. If necessary, it can be treated with an immune-inducing substance to explore the efficacy of inhibiting the inflammation-inducing signaling system. Thereafter, the lysate was quantified for protein, separated according to size through SDS-PAGE, and changes in various signal transduction systems were analyzed through western blotting, and inflammation induction or anti-inflammatory signals were strongly An inducible peptide pool is selected to select amino acids effective for each fixed position. After that, a sequence is generated by combining the selected amino acids, and the best sequence candidates are selected by analyzing and verifying the signal transduction system using the peptides obtained through synthesis.

Example 5: Method for searching for effective substances for activating formyl peptide receptors from N-formyl peptides through immune response analysis of immune cells

Immune cells mediate an inflammatory response by secreting proteins such as cytokines and chemokines out of cells. In particular, inflammatory cytokines and chemokines play a role in defending against foreign antigens by triggering an inflammatory response and regulating the innate immune response. However, an excessive inflammatory reaction that is not properly controlled can cause fever, inflammation, and tissue destruction, which can aggravate the disease itself or the symptoms associated with the disease. TNFα (tumor necrosis factor α), IL-6 (interleukin 6), IL-1β (interleukin 1β) and the like are typical pro-inflammatory cytokines. Therefore, since an excessive amount of pro-inflammatory cytokines have rather detrimental effects, they must be reduced at an appropriate time after an inflammatory response occurs. Therefore, by analyzing the amount of cytokine and chemokine gene expression and secreted protein of immune cells caused by the inflammatory response, the inflammation control and anti-inflammatory efficacy of the formyl peptide can be evaluated.

To this end, in immune cells such as neutrophils, eosinophils, or macrophages, along with inflammatory substances such as lipopolysaccharide (LPS) or zymosan, a specific amino acid at a specific position in the peptide library constructed in Example 1 of the present invention Each of these fixed peptide pools are treated together and cultured. After several hours, cell lysates or medium supernatants are recovered and analyzed for cytokines and chemokines. Specifically, RT-qPCR (Real-time quantitative Reverse transcription PCR) is performed to confirm expression changes at the gene level, and enzyme-linked immunosorbent assay is performed to analyze the level of cytokine protein secretion. (ELISA, Enzyme-linked immunosorbent assay) is performed. In the case of real-time quantitative reverse transcription polymerase chain reaction, cDNA is synthesized using revese transcriptase after RNA extraction using TRIzol from control and peptide-treated cells to measure gene expression. Thereafter, the expression level is quantitatively analyzed in real time using primers of the cytokine or chemokine gene to be analyzed. In addition, in the case of enzyme-linked immunosorbent assay (ELISA), the supernatant of the medium is recovered, the medium is put on a plate coated with an antibody that recognizes the cytokine or chemokine to be analyzed, and incubated for several hours so that the antibody can bind well. Then, after adding a peroxidase-coupled antibody, a substrate is added, and the amount of the corresponding cytokine or chemokine is quantitatively analyzed through a colorimetric method. Subsequently, a peptide pool capable of suppressing cytokine or chemokine secretion of immune cells is selected to select amino acids effective for each fixed position. Thereafter, a sequence is generated by combining the selected amino acids, and a best sequence candidate group is selected using the peptide obtained through synthesis.

Example 6: Method for searching for effective substance for activating formyl peptide receptor from N-formyl peptide through granule cell immune response analysis

Granulocytes, such as basophils, neutrophils, eosinophils, and mast cells, form cytoplasmic granules that contain many inflammatory mediators, such as histamine, heparin, cytokines, chemokines, and several proteases. Involves in the inflammatory response through secretion. Therefore, the degree of activation of granulocytes can be evaluated by measuring tryptase for degranulation. The Tryptase (tetrameric serine proteinase) accounts for 20% of the total protein of mast cells derived from lung, colon and skin tissues, and is a well-known activation indicator as a major component of mast cell granules and treatment of allergic diseases. A large amount of tryptase as an intervention target indicates that excessive degranulation occurs, and is proportional to the intensity of the inflammatory response.

In the peptide library constructed in Example 1, each of the peptide pools in which a specific amino acid is fixed at a specific position in the granulocyte cells are treated together and cultured. When the culture supernatant is recovered and tosyl-gly-pro-lys-pNA is added as a substrate, p-nitroaniline (pNA) is produced by tryptase, and the color development is detected with a spectrophotometer to analyze tryptase activity. Through this, a peptide pool capable of inhibiting granulocyte degranulation is selected, and amino acids effective for each fixed position are selected. Thereafter, a sequence is generated by combining the selected amino acids, and a best sequence candidate group is selected using the peptides obtained through synthesis.

Example 7: Method for searching for effective substances for activating formyl peptide receptors from N-formyl peptides through macrophage immune response analysis

Representative immune responses of macrophages are phagocytosis, apoptotic cell recognition, and efferocytosis. The phagocytosis is the main mechanism used to remove pathogens and cell debris, and the ingested material is digested in the phagosome. In addition, the apoptotic cell recognition and phagocytosis is a process in which macrophages surround apoptotic cells and absorb large vesicles filled with liquid containing dead cells in a manner similar to macropinocytosis, and the ingested vesicles are phagosomes and They are called efferosomes. In particular, apoptotic cell recognition and phagocytosis are known as typical actions of resolution of inflammation by clearing dead immune cells and damaged tissues.

In order to evaluate the phagocytosis-inducing ability, fluorescently labeled and apoptotic cells are provided to macrophages as phagocytic targets. At this time, each peptide pool in which a specific amino acid is fixed at a specific position in the peptide library constructed in Example 1 is treated together and cultured. Thereafter, the cells are recovered and the macrophages phagocytosing the apoptotic cells are analyzed by flow cytometry (Fluorescence-activated cell sorting) or immunofluorescence. Through this, a peptide pool capable of inducing phagocytosis of macrophages is selected, and amino acids effective for each fixed position are selected. Thereafter, sequences are generated by combining the selected amino acids, and phagocytosis is re-analyzed using peptides obtained through synthesis to select the best sequence candidates.

Although the present invention has been described with reference to the above-described embodiments, these are only examples, and those skilled in the art will understand that various modifications and equivalent other embodiments are possible therefrom. Therefore, the true technical protection scope of the present invention should be determined by the technical spirit of the appended claims.

Claims (8)

1. A hexameric N-formyl peptide library with a formyl group added to the N-terminus composed of 6 amino acids containing an amino acid sequence selected from the group consisting of:

   formyl-MX ₁ X ₂ X ₃ X ₄ X ₅

   (At this time, any one of X ₁ to X ₅ excluding the N-terminal formyl methionine is fixed as a specific amino acid, and the rest except for the fixed amino acid is synthesized so that 19 amino acids excluding cysteine are randomly arranged) .
2. According to claim 1,

   A library wherein the C-terminus is amidated.
3. According to claim 1,

   A library composed of 95 pools according to the fixed amino acids.
4. According to claim 1,

   A library for searching for effective substances for formyl peptide receptor (FPR) activation or inflammatory response modulating peptides.
5. Culturing step of culturing after treatment with the peptide library of claim 1 in immune cells:

   Analysis step of analyzing the level of formyl peptide receptor activation by recovering the cells that have completed the culturing step, the lysate of the cells or the medium supernatant:

   A first selection step of selecting candidate amino acids effective for each fixed position by selecting a peptide pool showing higher activity than the control group in the analysis step; and

   A method for screening formyl peptide receptor activating candidates comprising a second selection step of selecting a candidate group with the highest activity by combining the candidate amino acids.
6. According to claim 5,

   The immune cells include neutrophils, basophils, eosinophils, mast cells, monocytes, macrophages, dendritic cells, T cells, and B cells. , And selected from the group consisting of NK cells, the method.
7. According to claim 5,

   The formyl peptide receptor activation level is selected from the group consisting of calcium ion permeability analysis, cAMP production analysis, inflammation induction or inflammation resolution signal analysis, or immune response analysis.
8. According to claim 7,

   The immune response assay is performed by measuring the secretion level of cytokines, chemokines or cytoplasmic granules of immune cells or measuring the phagocytosis of macrophages.

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