JP6951713B2 - IgG-binding peptide - Google Patents

Description

The present invention relates to peptides having binding to immunoglobulin G.

By specifically adsorbing and removing autoantibodies and immune complexes containing immunoglobulin G (IgG) as the main body from body fluid components such as blood and plasma to adsorbents, the progression of autoimmune diseases related to bioimmune function can be promoted. There are known ways to prevent and reduce the symptoms of the disease.

As an adsorbent that can be used for such a purpose, for example, Patent Document 1 discloses an IgG antibody adsorbent in which an IgG-binding peptide having a helix-loop-helix structure is bound to a carrier as an IgG antibody. There is. Non-Patent Document 1 also discloses an IgG-binding peptide having a helix-loop-helix structure.

However, the binding property of these IgG-binding peptides to IgG is not yet sufficient, and an IgG-type antibody having higher binding property is required.

JP-A-2015-74626

K. Kawabata, et ail., Bioorganic and Medicinal Chemistry, 22 (2014), 1845-1849

An object of the present invention is to provide an IgG-binding peptide having a helix-loop-helix structure with stronger binding.

The peptide according to the present invention has the amino acid sequence shown in SEQ ID NO: 1. Here, the amino acid X in the amino acid sequence shown in SEQ ID NO: 1 is as follows.

The peptide according to the present invention exhibits strong binding to human immunoglobulin (IgG).

FIG. 1 is a diagram showing a design of a peptide according to the present invention. FIG. (A) shows the structure of HLH, which is the basic structure, and FIG. (B) shows the structure of the designed peptide. FIG. 2 is a diagram showing screening of an IgG-binding peptide library, in which (A) shows the results of a reference IgG-binding peptide (FK60), (B) shows the results after the first round, and (C). ) Indicates the result after the second round, and (D) indicates the result after the third round. FIG. 3 is a diagram showing the activity of an IgG-binding peptide by a fluorescent plate reader. FIG. 4 is a diagram showing the frequency of occurrence of amino acids at the modification site of the selected peptide having strong IgG binding property. FIG. 5 is a CD spectrum of the obtained IgG-binding peptide.

The peptide according to the present invention has the amino acid sequence shown in SEQ ID NO: 1 (FNMQCQAEFYEILHEXXAXXGGGGGXGKXXAXXAKIAALRDAC: where X is shown below). The peptide is an acyclic peptide or the fifth cysteine from the N-terminal of the amino acid sequence shown in SEQ ID NO: 1 (C5: the number added to the amino acid code indicates the position from the N-terminal in each amino acid sequence (hereinafter, The same.) And C-terminal cysteine are cyclic peptides formed by disulfide bonds.

The peptide according to the present invention exhibits strong binding to IgG of various animals, especially humans. Binding in the present invention is evaluated with a dissociation constant for human IgG (K _D). A method for calculating the dissociation constant is known to those skilled in the art, and can be obtained by, for example, the measuring method described in Examples. Preferred dissociation constant _{(K D)} or less 3000 nM, preferably not 1000nM or less, more preferably 100nM or less and preferably 50nM or less, more preferably 10nM or less.

The peptide according to the present invention logically has a helix-loop-helix structure in which two α-helices consisting of 14 amino acid residues on the N-terminal side and 14 amino acid residues on the C-terminal side are bound at 6 or 7 glycine residues. Has (HLH structure). In this peptide having an HLH structure, the two α-helices form a stable HLH structure due to the hydrophobic interaction of the leucine side chains existing inside, and further, cysteine is introduced into the N-terminal and C-terminal. Intramolecular disulfide bridging further improves the stability of the three-dimensional structure (Suzuki, N., Fujii, I., Optimization of the loop length for folding of a helix-loop-helix peptide, Tetrahedron Lett. 40, 6013 ( 1999), Fujii I, Takaoka Y., Suzuki K., Tanaka, T., A Conformationally Purified α-Helical Peptide Library, Tetrahedron Lett. 42, 3323 (2001)). In the present invention, a peptide having such an HLH structure is preferable, but as long as it has IgG binding property, it does not necessarily have to be a peptide having such an HLH structure.

The peptide according to the present invention has the amino acid sequence shown in SEQ ID NO: 1. X16 (the 16th amino acid from the N-terminal amino acid shown in SEQ ID NO: 1) is L, M, I or F, and X17 (17 from the N-terminal amino acid shown in SEQ ID NO: 1) is X in this amino acid sequence. The second amino acid) is R, K, Y or Q, and X19 (the 19th amino acid from the N-terminal amino acid shown in SEQ ID NO: 1) and X20 (the 20th amino acid from the N-terminal amino acid shown in SEQ ID NO: 1). , X29 (29th amino acid from N-terminal amino acid shown in SEQ ID NO: 1), X30 (30th amino acid from N-terminal amino acid shown in SEQ ID NO: 1), X32 (32 from N-terminal amino acid shown in SEQ ID NO: 1) The third amino acid) and X33 (the 33rd amino acid from the N-terminal amino acid shown in SEQ ID NO: 1) are arbitrary amino acids, respectively. In addition, X26 (the 26th amino acid from the N-terminal amino acid shown in SEQ ID NO: 1) is G or E or deleted.

Among the peptides according to the present invention, X16 is preferably L, M, I or F, X17 is R, K, Y or Q, and X19 is V, A, L, M, F or I, X29 is I, A, L, V, M or F, X32 is A, L, F, M or I, and X33 is N, D, H, A, T or G. Furthermore, X16 is L or M, X17 is R or K, X19 is V, A, L or M, X29 is I, A, L, V or M and X32 is A, L. Or F, and X33 is more preferably a peptide of N, D, H, A or T. Among the above peptides, X16 is L or M, X17 is R or K, X19 is A or V, X20 is G, Q, R or A, X26 is G, and X29. Is I, A, L or V, X30 is A, T, Q, E or S, and X33 is A or N. More specifically, the peptide is preferably the peptide shown in SEQ ID NO: 2-51, and particularly preferable peptides are SEQ ID NO: 9 (FK6014: FNMQCQAEFYEILHELRAVGGGGGGGGGKVAALNAKIAALRDAC), SEQ ID NO: 21 (FK6032: FNMQCQAEFYEILHEMRAVQGGGGGGGKITALNAKIAALRDAC), SEQ ID NO: 33 It has the amino acid sequence shown in any of the SEQ ID NOs: 41 (KF6063: FNMQCQAEFYEILHEMRAVAGGGGGGGKLEAFNAKIAALRDAC) and SEQ ID NO: 43 (FK6065: FNMQCQAEFYEILHELRAVQGGGGGGGKISALNAKIAALRDAC).

Further, in SEQ ID NO: 1, the peptide of the present invention has I12 (isoleucine, which is the 12th amino acid from the N-terminal amino acid shown in SEQ ID NO: 1) and / or L39 (39th from the N-terminal amino acid shown in SEQ ID NO: 1). Leucine), which is an amino acid in the above, can also be a peptide which is an arbitrary amino acid. For example, a peptide in which the 12th amino acid and / or the 39th amino acid is alanine.

In the present invention, the amino acid constituting the peptide may be a natural amino acid, a derivative of a natural amino acid, or an unnatural amino acid as long as the peptide has a desired IgG binding property. Preferably, the amino acids constituting the peptide are natural amino acids. Examples of the derivative of the natural amino acid include, but are not limited to, hydroxyproline and hydroxylysine, which are amino acids into which a hydroxyl group has been introduced, and diaminopropionic acid, which is an amino acid into which an amino group has been introduced. Examples of unnatural amino acids are α, α-disubstituted amino acids (such as α-methylalanine), N-alkyl-α-amino acids, D-amino acids, β-amino acids, and α, whose main chain structure is different from that of the natural type. -Hydroxyic acid, etc .; Amino acids with different side chain structures (norleucine, homohistidine, etc.), homoamino acids with extra methylene in the side chain (homophenylalanine, homohistidine, etc.) and carboxylic acid functional groups in the side chain Examples include, but are not limited to, amino acids in which amino acids are replaced by sulfonic acid groups (such as cysteine acid).

Further, the peptide according to the present invention is preferably one in which cysteine C5 of the amino acid sequence shown in any of SEQ ID NO: 1 and cysteine C at the C-terminal are disulfide-bonded and cyclized. The cyclization makes the three-dimensional structure of the peptide more stable. At the time of cyclization, it has about 1 to 3 amino acids between the N-terminal cysteine used for cyclization and the N-terminal amino acid of the α-helix on the N-terminal side of the HLH structure, and has an HLH structure. About 1 to 3 amino acids can be inserted between the C-terminal amino acid of the α-helix on the C-terminal side of the above and the cysteine on the C-terminal side used for cyclization. For example, in the amino acid sequence of SEQ ID NO: 1, glutamine Q6 is placed between the N-terminal cysteine C5 used for cyclization and the N-terminal alanine A7 of the α-helix, and the C-terminal cysteine C43 used for cyclization. It has alanine A42 between the C-terminal aspartic acid D41 of the α-helix. Further, in the amino acid sequence set forth in any of SEQ ID NOs: 1 to 51, the two cysteines at the 5th and C-terminals from the N-terminal may be substituted with amino acids that bind to each other to form a cyclic peptide, respectively. , Can be cyclized by interconnecting the substituted peptides.

In the peptide according to the present invention, in the amino acid sequence set forth in any of SEQ ID NOs: 1 to 51, either or both of the cysteine at the 5th N-terminal and the cysteine at the C-terminal are arbitrary amino acids (for example, charged). It can also be a peptide substituted with no amino acid, preferably Q). The present inventors have a structure similar to that of the peptide having the amino acid sequence of SEQ ID NO: 1, and in the peptide having cysteine at the 5th N-terminal and the C-terminal, the 5th cysteine is replaced with glutamine which is also uncharged. However, it has been confirmed that there was no significant effect on the dissociation constant for IgG.

The peptide according to the present invention may also be a peptide in which 1 to several, preferably 1 to 3 amino acids are added, deleted or substituted in the amino acid sequence of SEQ ID NOs: 1 to 51. The total length of the amino acid sequence is 38 or more and 50 or less.

Among the above peptides, the acyclic peptide can be synthesized according to various known peptide synthesis methods. Examples thereof include liquid phase synthesis methods such as Fmoc solid phase synthesis method and fragment condensation method. The solid-phase synthesis method is preferably used because the operation is simple. Further, the cyclic peptide can be obtained by obtaining an acyclic peptide and then cyclizing the cyclic peptide. For example, it can be cyclized by forming a disulfide bond between two cysteines in the amino acid sequence by a known method. It can also be cyclized by forming a thioester or thioether bond in the molecule by a known method. Further, cyclization may be carried out by condensing a carboxy group and an amino group in the molecule by a known method such as a highly diluted method.

The acyclic peptide can also be produced by using a genetic engineering method, not by a chemical synthesis method such as the above-mentioned solid phase synthesis method and liquid phase synthesis method. For example, a nucleic acid having a base sequence encoding the amino acid sequence is incorporated into an appropriate expression vector, and an appropriate host cell (for example, mammalian cell, insect cell, Escherichia coli) is transformed with the obtained expression vector. Then, after culturing the host cell under conditions suitable for the expression of the peptide, the peptide may be separated from the culture.

Hereinafter, the present invention will be described in more detail based on the following examples, but it goes without saying that the present invention is not limited thereto.

[Preparation of peptide library]
The acyclic peptide having the HLH structure described in Patent Document 1 (having the amino acid sequence shown in SEQ ID NO: 52) is modified in various ways to obtain a cyclic peptide library having a cyclic structure (the amino acid sequence shown in SEQ ID NO: 1). (See FIG. 1). Here, X16, X19, X29, and X32 in the amino acid sequence shown in SEQ ID NO: 1 are any of the amino acids Phe, Leu, lle, Met, and Val, and X17, X20, X30, and X33 are found in natural proteins. Being one of 20 amino acids, X26 constructed the library to be Gly or Glu or deleted. Each peptide constituting the peptide library is via a spacer (N2, M3, Q4) consisting of two amino acids (C5 and Q6) and three amino acids for cyclizing the peptide on the N-terminal side of the α-helix moiety. Bound phenylalanine (F1) and two amino acids for cyclizing the peptide on the C-terminal side of the α-helix moiety (A42 and C43 (41st A and 42nd C if X26 is deleted) The two cysteines C5 and C43 have a disulfide bond.

(1) Construction of L-Random yeast surface peptide library
Gene fragments encoding the L-Random peptide library having the amino acid sequence designed above were prepared by overlap extension PCR and 2nd PCR, and treated with restriction enzymes NcoI and XhoI. A plasmid for the L-Random peptide library was constructed by ligating with the yeast surface presentation vector pYD11-BxXN treated in the same manner. Escherichia coli was transformed by the electroporation method and amplified, and then yeast cells were transformed by the lithium acetate method to obtain an L-Random yeast surface-presented peptide library having a library size ^{of 9.4 × 10 6.}

(2) Screening for human IgG-Fc The obtained library was screened for binding to human IgG-Fc using a cell sorter BD FACSAria® II (BD Bioscience). The peptide fibrary was cultured in SG / RCAA medium (culturing in SG / RCAA medium to induce the presentation of the peptide on the cell surface. After washing with PBS, bio-human IgG-Fc (final concentration 100 nM: Bethyl laboratory) and mouse anti- After reacting with FLAG antibody (Sigma), SA-PE (Streptavidin-R-phycoerythrin: Invitrogen) and anti-mouse-Alexa488 (Alexa Flour (trade name) 488 goat anti-mouse IgG (H + L)): Fluorescent labeled by binding (Invitrogen). Sorted 3 times using BD FACSAria ™.

At this time, in the region (P5 region) higher than the fluorescence intensity of the clone presented by the peptide (FK60) having the amino acid sequence (FNMQCQAEFYEILHESAADEGGGGGGGKEAARNAKIAALRDAC) represented by SEQ ID NO: 53, whose dissociation constant (KD) for human IgG-Fc is known. It was set and screened. This peptide FK60 was obtained by modifying a peptide having the amino acid sequence shown in SEQ ID NO: 52.

In Round 1, 1 × 10 ^{8 cells were sorted by Bulk sort mode, and 1.4 × 10 5} cells in the cell group existing in the P5 area set to 0.4% of the total were collected. In Round 2, all the cells collected in Round 1 were sorted in Burk sort mode, and the P5 area was set to 9% of the total, and 9000 cells were collected. Furthermore, in Round 3, the cells collected in Round 2 were sorted in the single-cell sort mode, the P5 area was set to 13% of the total, and 96 cells were collected in 96-well SD plate medium (see FIG. 2). The area of P5 was narrowed for each Round, and only yeast cells with high fluorescence intensity were selected. This was cultured at 30 ° C. for 2 days.

(3) Activity analysis of human IgG-Fc-binding clones 96 yeast cell clones collected on a 96-well SD plate in Round 3 of (2) above were cultured in SDCAA medium and amplified, and then presented again in SG / RCAA medium. Induction, the binding activity to each bio-human IgG-Fc was measured using a fluorescent plate reader (Varioskan). The relationship between the relative fluorescence intensity (RFU) of R-PE showing the binding activity and the relative fluorescence intensity (RFU) of Alexa-488 showing the presentation amount of the peptide measured at the same time was plotted in the figure (see FIG. 3). Among them, about 50 clones with high relative fluorescence intensity of R-PE (indicated by light black circles in the figure) were selected, and the gene fragment encoding the peptide held by each clone was amplified by colony PCR. A sequencing reaction was carried out using the obtained gene fragment as a template, and the amino acid sequence of the peptide held by the presented clone was determined by analyzing the DNA sequence of the gene fragment (Table 1). Then, when the frequency of appearance of amino acids in the peptide obtained by screening was examined using the web logo, the results shown in FIG. 4 were obtained.

(4) Binding property of human IgG-Fc binding peptide (calculation of dissociation constant)
Five randomly selected human IgG-Fc-binding peptides (FK6014, FK6032, FK6053, FK6063, FK6065) whose amino acid sequences were determined were selected, and each was chemically synthesized by the Fmoc solid-phase synthesis method on a 0.023 μmol scale using amid resin. The cells were synthesized and the binding activity to human IgG-Fc was measured using the SPR method.

Weigh 100 mg of Fmoc-PAL-PEG-PS (trade name) amidoresin (substitution rate: 0.2 mmol / g) by reaction vessel, and follow the protocol of the peptide automatic synthesizer PSSM-8 (SHIMAZU) for each Fmoc amino acid (10equiv. ), HCTU (10equiv), DIEA (20equiv), 30% piperidine, and 5% anhydrous acetic acid were prepared, and each peptide was chemically synthesized. After completion, the resin was washed with diethyl ether, dried, 2 mL of a crivage solution (TFA: EMS: Anisole: EDT = 93: 3: 3: 1) was added, and the mixture was reacted at room temperature for 4 hours. After filtering the Crivage solution, an excess amount of ice-cooled diethyl ether was added to precipitate the peptide. The mixture was allowed to stand on ice for 10 minutes, centrifuged at 6500 rpm at 4 ° C. for 10 minutes, and the supernatant was discarded. After adding ice-cooled diethyl ether to the recovered precipitate and stirring vigorously, the precipitate was washed by allowing it to stand on ice for 10 minutes and centrifuging at 6500 rpm at 4 ° C. for 10 minutes to remove the fine product. .. This cleaning action was repeated twice. The precipitate was dried under reduced pressure, dissolved in 2 mL of sterile water, and filtered through a 0.22 μm filter to obtain a peptide solution (acyclic peptide). When the peptide solution was purified by RP-HPLC and mass spectrometrically analyzed by MALDI-TOF-MS, it was confirmed that the target peptide was synthesized by obtaining the measured values almost as calculated.

Then, the peptide solution dissolved in sterilized water was added dropwise to 30 mM Tris-HCl (pH 8.5) overnight at room temperature while stirring to cause an oxidation reaction, and intramolecular disulfide cross-linking was performed. The reaction solution was purified by RP-HPLC under the same conditions as above. When mass spectrometry was performed by MALDI-TOF-MS, it was confirmed that the target peptide was synthesized by obtaining the measured values almost as calculated.

The obtained cyclic peptide was measured for binding activity at each concentration of 5 peptides using Biacore T200 (GE Healthcare) and Thiol coupling kit (GE Healthcare). HBS-EP + was used as the running buffer in all steps. A flow cell 2 was used for immobilization of human IgG-Fc on the Serise S CM5 sensor chip, and the flow rate was 10 μL / min at 25 ° C. After determining the optimum pH for immobilization of human IgG-Fc by a preliminary test, prepare a solution (1: 1 by volume ratio) of EDC and NHS in the kit in flow cell 2 and use it as a sensor chip for 7 minutes. The carboxyl group was activated by flowing. Human IgG-Fc was immobilized by preparing 5 μg / mL human IgG-Fc in 10 mM sodium acetate buffer (pH 5.0) and flowing it through activated flow cell 2 for 7 minutes. The amount of immobilization was 2000 RU. The unreacted activated carboxyl groups were all inactivated by running ethanolamine for 7 minutes. Flow cell 1 was used as a reference cell by activation with EDC and NHS and blocking with ethanolamine. Each peptide was serially diluted to 0.3125, 0.625, 1.25, 2.5, 5, 10, 20, 40 μM with HBS-EP + buffer, and their binding activities were measured at a flow rate of 30 μL / min. Measurements, in BIA evaluation 4.1 software to calculate the a dissociation constant (K _D) of the equilibrium value analysis. The results are shown in Table 2.

Five peptides Any very high avidity _{(FK6014: K D = 9nM,} FK6032: K D = 8nM, FK6053: K D = 5nM, FK6063: K D = 12nM, FK6065: K D = 8nM) showed .. Comparison with peptide FK60 (K D ₌ 21μM) and Non-Patent Document 1 has been circularized peptides according to (K D ₌ 19μM) 1000 times or more binding activity compared to is improved.

(Measurement of CD spectrum)
The CD spectrum of each peptide was measured. Peptides were prepared in 20 mM Phosphate buffer (pH 7.4) to a final concentration of 20 μM, and 20 using a circular dichroism dispersometer J-720 (JASCO) and a temperature controller Peltier PTC-423L (TOMY SEIKO). CD spectra at wavelengths from 190 nm to 260 nm at a temperature of ° C were measured at 0.2 nm intervals. The result is shown in FIG. It was found that each peptide had a clear negative maximum at 208 nm and 222 nm and retained a stable α-helix structure. By designing the hydrophobic amino acids to be placed at appropriate positions inside the α-helix, the amphipathic properties required for the formation of the α-helix structure are acquired, and a stable three-dimensional structure is formed. Guessed.

Next, several amino acids thought to be located inside the two α-helices were replaced with alanine, and the binding property to IgG was measured. In the measurement, among the peptides obtained in Example 1, FK6053, which showed the highest binding activity to IgG, was used. The binding property was compared with the case where FK6053 was used by using a thioredoxin fusion peptide in which thioredoxin was fused to the N-terminal amino acid of the peptide. The thioredoxin fusion peptide prepared according to the following method, was measured binding activity (dissociation constant K _D) for Human IgG-Fc by surface plasmon resonance according to the method described in Example 1. The amino acid sequence of the thioredoxin fusion peptide of FK6053 is shown in SEQ ID NO: 54. The substituted amino acids are isoleucine (I12) at the 12th N-terminal, valine (V19) at the 19th position, arginine (R20) at the 20th position, isoleucine (I29) at the 29th position, and glutamine at the 30th position in FK6053. Q30), the 32nd leucine (L32), the 33rd asparagine (N33), and the 39th leucine (L39). The amino acid sequence corresponding to the peptide of FK6053 after the substitution and the result of the comparative test are shown in Table 3. The results were expressed as "++++" when the binding activity was similar to that in FK6053, and as "++" when the binding activity was 1/10 to 1/100 of that. When high binding to IgG is shown, the amino acid at the position substituted with alanine has little effect on the binding to IgG due to its stable three-dimensional structure, and the amino acid at that position is replaced with an arbitrary amino acid. It is considered possible.

(Preparation of thioredoxin fusion peptide for measuring binding activity)
The Trx fusion peptide of peptide FK6053 and its mutant was synthesized by the pET system. BL21 CodonPlus (DE3) -RP was used as the host Escherichia coli, and pET32a (+) (Navagen) was used as the vector. Using the pET32a (+) vector into which the FK6053 gene was introduced as a template, mutant DNA was prepared by the megaprimer method and PCR was performed. After completion of the reaction, the mixture was separated by 3% agarose gel electrophoresis, the target band was excised, and purified by Gel Extraction Kit (QIAGEN). The purified PCR product was used as a megaprimer, and PCR was performed again. After completion of the reaction, the target PCR product was subjected to agarose gel electrophoresis in the same manner as before, and the target band was excised and purified. The purified DNA was treated with restriction enzymes (NcoI and XhoI) and ligated with pET32a (+) similarly treated with restriction enzymes to construct a vector for expressing Trx fusion peptide of FK6053 mutant. Host Escherichia coli transformed with the constructed vector was cultured in LB medium / Amp. Then, the medium was cooled to 18 ° C., 10 μL of 1M IPTG was added, and the cells were cultured with shaking at 18 ° C. overnight. After completion of the culture, the culture solution was centrifuged (4 ° C., 6000 g, 10 minutes) and the precipitate was suspended in PBS. Escherichia coli was ultrasonically crushed and centrifuged, and then the fine spirit was filtered through a filter (0.45 μm) to obtain a sample for purification. The purification sample was adsorbed on a packed column with Ni Sepharose High Performance (GE Healthcare) equilibrated with 25 mM imidazole and then eluted with 500 mM imidazole. The resulting eluate was dialyzed against a cellulose membrane (Spectra / Par MWCO: 6-8000, Spectrum Laboratories Inc.) in PBS at 4 ° C. and then concentrated using a centrifugal filter (Amicon Ultra-0.5, 3K device, Millipore). , A sample for binding activity.

The present invention provides an IgG-binding peptide having strong binding to IgG.

Claims (4)

A peptide having the amino acid sequence of any one of SEQ ID NO: 2-51 and SEQ ID NO: 55-SEQ ID NO: 62. A peptide having the amino acid sequence of any of SEQ ID NOs: 2-51 and SEQ ID NOs: 55 to 62, in which the fifth cysteine from the N-terminal of the amino acid sequence and the C-terminal cysteine of the same amino acid sequence are disulfide-bonded. A peptide having any of the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 21, SEQ ID NO: 33, SEQ ID NO: 41, and SEQ ID NO: 43. It has any of the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 21, SEQ ID NO: 33, SEQ ID NO: 41, and SEQ ID NO: 43, and the fifth cysteine from the N-terminal of the amino acid sequence and the C-terminal cysteine of the same amino acid sequence are present. A disulfide-bonded peptide.

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