KR20220065450A - Peptide For Stem Sell Migration That Binds To The Formyl Peptide Receptor - Google Patents

Abstract

A peptide of the present invention is a novel chemotactic peptide designed based on the interaction principle between the WKYMVm peptide and the FPR receptor, and can be used for therapeutic purposes such as induction of stem cell migration and tissue regeneration and others using the same.

Description

Peptide for Stem Cell Migration that Binds to Formyl Peptide Receptor {Peptide For Stem Sell Migration That Binds To The Formyl Peptide Receptor}

The present invention relates to a peptide for stem cell migration that binds to a formyl peptide receptor.

Mesenchymal stem cells (MSCs) are stromal cells with multipotential (multipotent). MSCs can differentiate into various cells such as osteoblasts, chondrocytes, adipocytes, and muscle cells, so research on using them to treat diseases requiring tissue regeneration is active.

The movement of MSCs circulating in the blood or bone marrow to the damaged tissue is called homing. Homing is known to be induced by chemotactic substances such as human stromal cell-derived factor (SDF-1a) or immune response-inducing cytokines.

For regeneration of damaged tissues, it is important to promote migration and angiogenesis of mesenchymal stem cells (MSCs). FPR (formyl peptide receptor), a G-protein-coupled receptor, is known to play an important signal transduction role in the defense mechanism and inflammatory response of leukocytes and phagocytes. It is known to act on the movement of various cells, such as (endothelial cells). However, the effect on the migration of mesenchymal stem cells is still unknown.

Republic of Korea Publication No. 10-2012-0102501 (2012.09.18)

According to one embodiment, a novel peptide capable of inducing the migration (homing) of stem cells is provided.

One aspect provides a peptide for stem cell migration consisting of an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 6.

The peptide consisting of the sequence of SEQ ID NOs: 1 to 6 is a novel chemotactic peptide designed by the present inventors based on the interaction principle between the WKYMVm peptide and the FPR receptor using a machine learning-based protein design algorithm. The peptide consisting of the sequence of SEQ ID NO: 1 is FRA1, the peptide consisting of the sequence of SEQ ID NO: 2 is FRA2, the peptide consisting of the sequence of SEQ ID NO: 3 is FRA3, the peptide consisting of the sequence of SEQ ID NO: 4 is FRA4, the sequence of SEQ ID NO: 5 The peptide consisting of FRA5, and the peptide consisting of the sequence of SEQ ID NO: 6 was designated as FRA6. According to one embodiment, FRA2 and FRA4 may have a strong interaction with the formyl peptide receptor (FPR), and may activate stem cell migration or homing. As a result of in vitro experiments, it was confirmed that FRA2 and FRA4 had a superior effect on inducing stem cell migration than WKYMVm.

The "WKYMVm" peptide is known as a selective agonist of FPR1, FPR2 (EC50 = 75 pM), and FPR3 (EC50 = 3 nM) expressed in immune cells. In addition to this, "WKYMVm" peptide is known to induce Ca ²⁺ mobilization, superoxide production, and chemotaxic migration in monocytes and neutrophils, and PKC-dependent pathways, It plays a role in promoting the survival of monocytes through a PI3-kinase-dependent pathway and an Akt-dependent pathway. The "WKYMVm" peptide is known by its sequence name.

The term “migration” refers to a migratory activity in which stem cells move from the site of transplantation or administration to the site of injury or a site requiring the movement of stem cells, and may also refer to homing.

The term "chemotaxis" refers to a positive (toward the stimulus) or negative (a direction away from the stimulus) migration of stem cells induced by chemical stimuli, and refers to a chemotactic substance, such as a chemokine. ) and is mediated by the corresponding cell surface receptors.

According to one embodiment, the stem cell may be a stem cell capable of inducing migration by the WKYMVm peptide consisting of the sequence of SEQ ID NO: 7 or a stem cell expressing a formyl peptide receptor (FPR). According to one embodiment, the FRA peptide can have a stronger interaction with the same principle as the mechanism by which WKYMVm binds to FPR and activates signal transduction. Therefore, the FRA1 to FRA6 peptides of the present invention may have a strong homing effect on stem cells induced by WKYMVm or stem cells expressing the formyl peptide receptor (FPR). According to one embodiment, the stem cell is not particularly limited as long as it is a stem cell capable of inducing migration by the WKYMVm peptide or a stem cell expressing a formyl peptide receptor (FPR), for example, a multipotent mesenchymal Stem cells, pluripotent hematopoietic stem cells, pluripotent neural stem cells, pluripotent endothelial stem cells, pluripotent epithelial stem cells, pluripotent epidermal stem cells, dedifferentiated stem cells, pluripotent muscle stem cells, pluripotent adult stem cells, Pluripotent pancreatic stem cells, pluripotent myocardial stem cells, pluripotent vascular stem cells, pluripotent hair follicle stem cells, pluripotent corneal stem cells, pluripotent adipose stem cells, pluripotent cord/umbilical cord blood stem cells, pluripotent dental stem cells, It may be one selected from the group consisting of pluripotent stem cells, and pluripotent cancer stem cells.

According to one embodiment, the first amino acid of the peptide consisting of the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 may be formylated methionine.

According to one embodiment, the methionine of the peptide may be an L-amino acid or a D-amino acid.

According to one embodiment, the peptide consisting of the sequence of SEQ ID NO: 1 may be Formyl-mKFYIm, the peptide consisting of the sequence of SEQ ID NO: 2 may be Formyl-mMFIPW, and the peptide consisting of the sequence of SEQ ID NO: 3 is It may be WPMYPm, the peptide consisting of the sequence of SEQ ID NO: 4 may be Formyl-mKYPYm, the peptide consisting of the sequence of SEQ ID NO: 5 may be Formyl-mVMYKm, and the peptide consisting of the sequence of SEQ ID NO: 5 is Formyl -MPLIYM, wherein m is D-form methionine.

According to one embodiment, the peptide for stem cell migration may be one obtained by amidation of the C-terminus (C-terminus amidation). The amidated C-terminus is the C-terminal form of peptide-C(=O)-NH ₂ . It is known to those skilled in the art that C-terminal amidation of peptides can generally increase the activity, stability, shelf life of peptides. When the C-terminus of the peptide for stem cell migration is amidated, the FRA receptor helps to recognize the peptide, and the activity can be increased.

Another aspect provides a composition for moving stem cells comprising the peptide for moving stem cells.

Since the peptide for stem cell migration can exert a strong homing effect on stem cells, the inflow of mesenchymal stem cells can be promoted with high efficiency when a composition for stem cell migration comprising the same is administered to a damaged tissue.

When the composition is prepared as a pharmaceutical composition for therapeutic purposes, it may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are those commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, poly vinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but is not limited thereto. When the composition is prepared as a pharmaceutical composition, the composition may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like, in addition to the above components.

The composition may be prepared in unit dose form or may be prepared by introduction into multi-dose containers. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in oil or an aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

The composition for stem cell migration may be administered parenterally, for example, it may be administered by injection into a damaged tissue. The composition for stem cell migration may be administered before the administration of the stem cell therapeutic agent, together with the stem cell therapeutic agent, or after the administration of the stem cell therapeutic agent.

The composition for stem cell migration may be administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, activity of the drug, and the type of disease in the patient; Sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs and other factors well known in the medical field may be determined. The composition may be administered in combination with other cellular therapeutics. Taking all of the above factors into consideration, it is important to administer an amount capable of obtaining the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art. Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption of the active ingredient into the body, inactivation rate, excretion rate, disease type, and drugs used in combination, the route of administration, It may increase or decrease according to the severity of obesity, sex, weight, age, and the like.

Another aspect provides a cell therapeutic agent comprising the peptide for stem cell migration.

Since the peptide for stem cell migration can exert a strong homing effect on stem cells, the inflow of mesenchymal stem cells can be promoted with high efficiency when a cell therapeutic agent containing the same is administered to a damaged tissue.

The composition and administration of the cell therapy agent can be understood with reference to the above-described composition for stem cell migration, and description thereof is omitted to avoid excessive overlap.

Diseases to which the cell therapeutic can be applied may be bone damage, joint damage, nerve damage, bone marrow cancer, lymphoma, or skin damage. The bone damage and joint damage may include damage to all skeletal parts of the body, rheumatoid arthritis, osteoporosis, achondroplasia, chondroma, disc herniation, accidental damage, autoimmune disease damage, cancer damage, diabetes It may include, but is not limited to, damage caused by The nerve damage includes both pathological or physical nerve tissue damage, for example, Parkinson's disease, Alzheimer's, multiple sclerosis, neurotrophic sclerosis (ALS), cerebral ischemia, cerebral hemorrhage, spinal cord injury, motor neuron disease, demyelinating disease. , an auditory nerve injury, or Huntington's, but is not limited thereto. The skin damage may be, but is not limited to, burns, abrasions, frostbite, penetrating wounds, bruises, skin ulcers, pressure sores, damage caused by cancer, or damage caused by an accident. The cell therapeutic may be used for hearing regeneration in patients with hearing loss. For example, the peptide can induce auditory progenitor cells to migrate to a desired location, and can be used for hearing regeneration therapy by differentiating into hair cells at a location requiring treatment.

Another aspect provides a support for tissue regeneration comprising the peptide for stem cell migration.

Since the peptide for stem cell migration can exert a strong homing effect on stem cells, transplantation of a tissue regeneration scaffold comprising the same into damaged tissue can promote the inflow of mesenchymal stem cells with high efficiency.

The scaffold for tissue regeneration may be referred to as a bioactive or bioactive scaffold. The bioactivity or bioactivity may mean biocompatible and/or biodegradable. The bioactivity or bioactivity may mean that the scaffold exhibits an appropriate reaction with the tissue of the insertion site of the host transplanted to perform the expected function, and can exist without harming the host due to toxicity or the like.

The scaffolds may be biodegradable scaffolds. Specifically, it may be a three-dimensional structure containing the peptide for stem cell migration as a chemotactic factor. The scaffold may induce the migration of stem cells when transplanted into damaged tissue requiring regeneration, and may provide physical support for the differentiated stem cells to form a tissue. The support may be a porous support.

The support is, for example, poly(lactic-coglycolic acid) (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), poly-ε-caprolactone (PCL), poly(amino acid) (PAA), polyanhydride lides, polyosoesters, derivatives, copolymers thereof, and mixtures thereof; Biodegradable material consisting of hyaluronic acid, chitosan, collagen, polyvinyl alcohol sponge, polysaccharide (eg, alginate), polyphosphazene, polyacrylate and polyethylene oxide-polypropylene glycol blot copolymer It may be selected from, but is not limited thereto.

The tissue requiring regeneration may be, for example, bone tissue, joint tissue, nerve tissue, or skin tissue.

The tissue regeneration scaffold may be used for hearing regeneration in a patient with hearing loss. For example, it can be induced to move auditory progenitor cells to a desired location by a peptide contained in the support, and can be used for hearing regeneration therapy by differentiating into hair cells at a location requiring treatment.

Since the peptide according to one embodiment has excellent stem cell homing effect, it can be used for the purpose of treating damaged tissues.

The peptide according to one embodiment can be used for the purpose of treating damaged tissue by applying it to a cell therapy agent or a support for tissue regeneration.

1 shows a method for confirming stem cell migration by a 2D stencil method.
2 is a result confirming the stem cell migration effect of FRA2 and FRA4 by the 2D stencil method.
Figure 3 shows a method for confirming the stem cell migration by the 3D spheroid method.
4 is a result confirming the stem cell migration effect of FRA2 and FRA4 by the 3D spheroid method.
Figure 5 shows a method of confirming the stem cell migration using Transwell ^® .
6 is a result confirming the stem cell migration effect of FRA2 by transwell.
7 is a result confirming the stem cell migration effect of FRA4 by transwell.

Hereinafter, one or more specific embodiments will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.

Example 1: Peptide Design and Selection

It is known that the WKYMVm peptide (SEQ ID NO: 7) can induce vascular endothelial cell migration by interacting with formal peptide receptor 1 (FPR1), FPR2, and FPR3 expressed in immune cells. Based on the structure of the WKYMVm peptide (SEQ ID NO: 7), new peptides capable of binding and interacting with FPR1, FPR2, or FPR3 were prepared.

The structure of the FPR2 complex to which the WKYMVm peptide was bound was obtained from Protein Data Bank (PDB ID: 6LW5). Energy minimization was performed using the MOE software, and potential interactions of the minimized WKYMVm and FPR2 complex structures were analyzed, and new peptides with improved binding affinity were discovered using this information.

The C-terminus of the WKYMVm peptide penetrates into the binding space while occupying a deeper site within the transmembrane helix bundle of FRA2. Upon closer examination of the ligand-binding pocket, two hydrophobic clusters were found that play important roles in ligand recognition and receptor activation of FRA2. Therefore, residues containing the two hydrophobic clusters were selected as binding pockets of the WKYMVm peptide, and a new peptide was designed in consideration of the characteristics of these residues and their interaction with the WKYMVm peptide. The sequence of the WKYMVm peptide was modified with appropriate amino acids to increase binding affinity and stability with FRA. The choice of amino acids was based on the charge and hydrophobicity of the receptor surface and the effects of the modifications and each modified amino acid were confirmed using the MOE module protein builder and protein design.

In this way, using the MOE protein builder and protein design module, peptides in which each residue was mutated to another amino acid in the amino acid sequence of WKYMVm were prepared, and their affinity for FPR (dAffinity score) and safety (dStability score) were confirmed. . Peptides FRA1 (SEQ ID NO: 1), FRA2 (SEQ ID NO: 2), FRA3 (SEQ ID NO: 3), FRA4 (SEQ ID NO: 4), FRA5 (SEQ ID NO: 5), and FRA6 (SEQ ID NO: 6) was selected. (See Table 1 below) An amine group was added (amidated) to the C-terminus of the peptides FRA1 to FRA6.

SEQ ID Peptide Name Sequence One FRA1 Formyl-mKFYIm-NH ₂ 2 FRA2 Formyl-mMFIPW-NH ₂ 3 FRA3 WPMYPm-NH ₂ 4 FRA4 Formyl-mKYPYm-NH ₂ 5 FRA5 Formyl-mVMYKm-NH ₂ 6 FRA6 Formyl-MPLIYM-NH ₂ 7 WKYMVm WKYMVm-NH ₂

In Table 1, a lowercase letter m is D-form methionine. The N-terminal methionine to which the formyl group is attached is N-formylmethionine in which the nitrogen of methionine is formylated.

Example 2: Cell Migration Analysis In Vitro

2-1. Stem cell migration analysis using 2D stencil

The stencil mold is 13mm in diameter, and there are two areas in which cells are to be seeded, and the interval between them is 1mm. A stencil was manufactured by pouring PDMS into the stencil mold. The produced PDMS stencil was dried after high-temperature steam sterilization. After placing the dried stencil on the bottom of the 12 well one day before cell seeding, the medium was filled so that the stencil was completely submerged, and incubated for one day to remove bubbles. After removing the media, 1x10 ⁵ cells/well of human mesenchymal stem cells (hMSCs) were seeded on the stencil, and the stencil was removed after waiting until the stencil was completely filled with cells. The wells from which the stencil was removed were carefully washed with 1x PBS. For the control group, WKYMVm, and the experimental group, 2 ml of DMEM serum free media containing FRA2 or FRA4 peptide at a concentration of 50 nM were added, and microscopic images were taken after 0 and 24 hours, respectively.

To measure the amount of cell migration, normalize the area occupied by cells at 0 hours and the area occupied by cells after 24 hours with the image j program (0 hour: 0 hour area/0 hour area)x100, 24 hours: (24 hour area/0) Time area) x 100) to observe the movement of cells to the area where the stencil was removed, and quantitative analysis was performed. (See Fig. 1)

As a result, the number of cells that migrated to the stencil-removed area was 10% higher in the experimental group administered with FRA2 or FRA4 than the control group. (See Fig. 2)

2-2. Stem cell migration analysis using 3D spheroid

After preparing about 2x10 ⁵ human mesenchymal stem cells (hMSCs) based on the total amount, 10 ml of DMEM basal media was added and 25 μl of droplets were made on the dish lid using a multi-pipette. The dish lid was turned over and cultured for 24 hours by a hanging drop method to form a spheroid (size: 150-200㎛) consisting of about 500 cells per drop. The formed mesenchymal stem cell spheroids were mixed with a collagen solution at a concentration of 2 mg/ml, and then gelated to place the spheroids in the collagen gel. After that, DMEM basal media containing 10 μM concentration of control, FRA2 or FRA4 peptide was placed on collagen gel containing spheroid, incubated for 16 hours, then photographed using a microscope, and the length of cells extending from the spheroid using the imageJ program. was measured to obtain the sprouting length. (See Fig. 3)

As a result of the experiment, the FRA2 administration group and the FRA4 administration group moved about 10 μm further than the control WKYMVm, so it was confirmed that the homing effect was more excellent by 10% or more. (See Fig. 4)

2-3. Stem Cell Migration Analysis Using Transwell

Cells were detached using 0.25% Trypsin-EDTA. Human Mesenchymal Stem Cells (hMSC) 1x10 ⁵ cells/100ul cell suspension was added to the filter membrane and incubated for 10 minutes. A medium mixed with a chemo-attractant peptide (FRA2 or FRA4) at a concentration of 50 nM was put into the bottom lower chamber of the Transwell transmigration plate by 600 μl. After 24 hours, the inserted plate was removed, and the media was carefully removed with a cotton swab. 600 μl of 4% PFA was added and fixed for 10 minutes. After 10 minutes, the remaining PFA was removed with a cotton swab. 600 μl of 0.1% crystal violet was added and reacted at RT for 5 to 10 minutes. Crystal violet solution was removed with a cotton swab and washed several times with DW. Membrane was separated, mounted on a slide glass, and cells that passed through micropores and migrated toward chemokines were observed under a microscope. (See Fig. 5)

As a result of the experiment, the number of cells that migrated to the lower chamber increased by more than 10% in the FRA2 experimental group and the FRA4 experimental group than the control group. (refer to FIGS. 6 and 7)

<110> Korea University Research and Business Foundation AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Peptide For Stem Sell Migration That Binds To The Formyl Peptide receptor <130> PN200328 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> FRA1 <400> 1 Met Lys Phe Tyr Ile Met 1 5 <210> 2 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> FRA2 <400> 2 Met Met Phe Ile Pro Trp 1 5 <210> 3 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> FRA3 <400> 3 Trp Pro Met Tyr Pro Met 1 5 <210> 4 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> FRA4 <400> 4 Met Lys Tyr Pro Tyr Met 1 5 <210> 5 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> FRA5 <400> 5 Met Val Met Tyr Lys Met 1 5 <210> 6 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> FRA6 <400> 6 Met Pro Leu Ile Tyr Met 1 5 <210> 7 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> WKYMVm <400> 7 Trp Lys Tyr Met Val Met 1 5

Claims (7)

A peptide for stem cell movement consisting of an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 6. According to claim 1,
The stem cells are stem cells capable of inducing migration by the WKYMVm peptide,
Peptide for stem cell migration. According to claim 1,
The stem cells are stem cells expressing the formyl peptide receptor (FPR),
Peptide for stem cell migration. 3. The method of claim 2,
The stem cells are pluripotent mesenchymal stem cells, pluripotent hematopoietic stem cells, pluripotent neural stem cells, pluripotent endothelial stem cells, pluripotent epithelial stem cells, pluripotent epidermal stem cells, immunized stem cells, and pluripotent muscle stem cells. Cells, pluripotent adult stem cells, pluripotent pancreatic stem cells, pluripotent myocardial stem cells, pluripotent vascular stem cells, pluripotent hair follicle stem cells, pluripotent corneal stem cells, pluripotent adipose stem cells, pluripotent cord/umbilical cord blood stems Which is selected from the group consisting of cells, pluripotent dental stem cells, pluripotent stem cells, and pluripotent cancer stem cells,
Peptide for stem cell migration. According to claim 1,
The first amino acid of the peptide consisting of the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 is formylated methionine,
Peptide for stem cell migration. A composition for stem cell movement comprising the peptide for stem cell movement of claim 1. A support for tissue regeneration comprising the peptide for stem cell migration of claim 1.

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