KR100400637B1 - Biologically useful peptide - Google Patents

Abstract

PURPOSE: Provided is a biologically useful peptide which conjugates with an erythropoietine receptor molecule, and contains the whole or a partial sequence of V-W-Q-G-L-A-L-L-S-E. CONSTITUTION: A biologically useful peptide characteristically conjugates with an erythropoietine receptor molecule, and contains the whole or a partial sequence of V-W-Q-G-L-A-L-L-S-E. At least one of amino acids thereof has D-form. The peptide is of a multimer formed by binding at least two peptide molecules by NH-NH, NH-COOH or COOH-COOH.

Description

Biologically Useful Peptides

The present invention relates to a peptide having an activity of conjugating with an erythropoietin receptor molecule and containing all or part of a sequence of the peptide V-W-Q-G-L-A-L-L-S-E.

Erythropoietin is a glycoprotein with a molecular weight of 30,000 to 34,000 Daltons, a hematopoietic factor that promotes the production and differentiation of red blood cells. The protein begins by conjugating to receptors on erythrocyte progenitor cells, causing an increase in calcium ions, an increase in DNA biosynthesis, and hemoglobin production stimulation. Therefore, the stimulatory factor erythropoietin can be used as a therapeutic agent for anemia in nephropathy, anemia in premature infants, anemia caused by hypothyroidism, and anemia caused by malnutrition.

As with most physiologically active substances, erythropoietin is thought to have only a small fraction of the total 165 amino acid sequences that perform the above-mentioned actions in connection with receptors. It was thought that a peptide having a very small molecular weight could be obtained. In addition, since the peptide of such a small molecular weight generally shows in vivo stability, absorbency, residual concentration, drug efficacy, etc. completely different from the existing macromolecules, a significant improvement in function may be achieved depending on the design direction.

Based on this idea, the present inventors have concentrated on a series of experiments to find peptide sequences having the above-mentioned properties, and consequently consisted of specific amino acid sequences that can be specifically conjugated with erythropoietin receptor molecules. The peptide was discovered and the present invention was completed.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention relates to a peptide having an activity of conjugating with an erythropoietin receptor molecule and containing all or part of a sequence of the peptide V-W-Q-G-L-A-L-L-S-E. More preferably, the present invention relates to a peptide having an activity of conjugating with an erythropoietin receptor molecule and essentially containing the entire sequence of peptide V-W-Q-G-L-A-L-L-S-E.

The present inventors first performed inhibition experiments using several kinds of antibodies against erythropoietin to find the site of conjugation with the receptor molecule in the amino acid sequence of erythropoietin. That is, in vitro analysis of the activity of erythropoietin using monoclonal antibodies CFC-6, CFC-9 and polyclonal antibody anti-erythropoietin rabbit antibody showed that monoclonal antibody CFC-6 inhibited the activity of erythropoietin. As a result, it was found that monoclonal antibody CFC-6 blocked the receptor conjugation site in erythropoietin.

Therefore, when the erythropoietin and the CFC-6 conjugate were identified, the receptor conjugate of the erythropoietin was automatically identified. Thus, the following experiment was performed to clarify the amino acid sequence of the CFC-6 conjugate.

A solution composed of several peptides produced by digesting erythropoietin protein molecules with proteolytic enzyme Glu-C (Endoproteinase Glu-C, sequencing grade, BM Biochemical) was separated into individual peptides using an HPLC C18 column. Western blot was performed on the isolated peptides using monoclonal antibodies CFC-6, CFC-9 and polyclonal antibody anti-erythropoietin rabbit antibodies as primary antibodies, respectively. 53 showed a very strong positive reaction. The peptide having a positive reaction was isolated and analyzed by its amino acid sequence, and found to be 10mer peptide of V ₆₃ -WQGLALLSE ₇₂ .

As can be seen from FIG. 1, the sequence is contained in the second helix portion of four helix bundle structures characteristic of cytokine-based molecules, and the receptor junction of erythropoietin reported so far is Amino acid sequence Nos. 99-129 is reported to be a different site than the sequence (see A. J. Sytkowski, JBC 262, 1161-1165, 1987). However, in the case of many cytokines, including growth hormones, the activation of receptors is initiated by homo-dimerization, in which two growth hormones are attached to one growth hormone. It has two receptor junctions. That is, considering that different amino acid sequences in hormones that are bound even to the junction to the same receptor are related (De Vos et al., Science 255, 306-312, 1992). Erythropoietin is also believed to activate receptors by this mechanism (Watowich S., et al., PNAS 89, 2140-2144, 1992). Thus, the peptide sequence found by the inventors is presumed to be the second receptor junction that is not known until now.

Since the peptide sequence is expected to be specifically conjugated with the erythropoietin receptor molecule, the present inventors artificially synthesize the peptide and couple it with a keyhole limpet hemocyanin (KLH), a carrier protein, to form a peptide-KLH complex. Formed. As a result of the in vitro titer test of the complex, ³ H-thymidine uptake was increased in the cell as in the case of erythropoietin. Therefore, it was confirmed that the peptide according to the present invention specifically conjugated to the receptor molecule in the same manner as erythropoietin.

As described above, since it was confirmed that the 10mer peptide of V ₆₃ -WQGLALLSE ₇₂ has a specific conjugation to the erythropoietin receptor molecule, not only the peptide consisting of the sequence but also a peptide containing the sequence as part of the erythropoietin can be substituted. The road to industrial development has opened. In addition, in the development of an alternative substance for erythropoietin according to the object of the present invention, by applying the peptide sequence as a main axis in various ways, a part of the sequence to other amino acids equivalent to the same within the range showing the same function It is understood that even if substituted, other amino acids are inserted or partially deleted in the sequence, it is within the scope of the present invention.

In addition, in the process of synthesizing the peptide according to the present invention, one or more of the amino acids can be inserted into the D-form for the purpose of increasing the stability and absorption in vivo; It may also be made of a multimer in which two or more peptides are bound, wherein the binding between peptide molecules consists of NH-NH, NH-COOH or COOH-COOH; Peptides can be used in combination with carrier proteins such as Keyhole limpet hemocyanin (KLH), obval-umin or bovine serum albumin (BSA); Peptides may also be chemically modified.

In chemically modifying one or more of the amino acids constituting the peptide according to the present invention, all known methods may be applied for the purpose. However, when using only the 10mer peptide itself, N- using Traut's reagent may be used. A terminal -SH group derivatization method or a blocking method of N-terminal amino groups using Sulfo-NHS-acetate and cystaconic anhydride reagent can be applied.

One or more of the above-mentioned methods may be selected and applied to enhance the in vivo function of the peptides according to the present invention. In applying such methods, those known to those skilled in the art to which the present invention pertains may be applied. Conventional methods can be used.

Hereinafter, the present invention will be described in more detail based on the following examples. However, these examples are only for the understanding of the present invention, and the scope of the present invention in any sense is not limited to these examples.

Example 1: In vitro assay of erythropoietin

Cells were isolated by extracting the spleen three days after treatment with phenyl hydrazine (60 mg / kg) in a hybrid first generation mouse (weight 16-22 g) of Bal b / c and C57 BL / 6. 50 μl of erythropoietin was added to 100 μl of the extracted cell solution ( ⁶ × 10 ⁶ cells / ml) within a concentration range of 0.005 to 1.0 U / ml, followed by incubation for 22 hours in a CO ₂ incubator. 50 µl of dimethyl- ³ H-thymidine (20 µl / ml) was added thereto and left for 2 hours in a CO ₂ incubator. Cells were attached to glass fibers and then radioactivity was measured in a β-counter to obtain a standard curve of the potency test as shown in FIG.

Example 2: Confirmation of Inhibition Activity of Monoclonal Antibody CFC-6

In vitro titer as described in Example 1 was performed with increasing concentrations of monoclonal antibodies CFC-6, CFC-9 and polyclonal antibody anti-erythropoietin rabbit antibody. The monoclonal antibodies CFC-6 and CFC-9 used in the present invention have been deposited under the Treaty of Budapest by the Korean Cell Line Research Foundation Director of Seoul National University Medical University on October 26, 1994. The accession number of -6 is KCLRF-BP-00008 and the accession number of CFC-9 is KCLRF-BP-00009.

At this time, the concentration of erythropoietin was fixed at 0.2 units, and the test result showed that the monoclonal antibody CFC-6 inhibited the activity of erythropoietin. 3).

Example 3: Peptide Mapping

Proteolytic enzyme Glu-C (Endoproteinase Glu-C, sequencing grade, BM Biochemical) was dissolved in distilled water, and 40 μl of erythropoietin was dissolved in 0.1 ml of ammonium carbonate buffer (ammonium carbonate buffer, 25 mM, pH 7.8). An enzyme dilution was added to the erythropoietin solution to bring the enzyme amount to 2 μg, followed by incubation at 25 ° C. for 18 hours. The digested solution was separated into individual peptides using an HPLC reversed phase C ₁₈ column (4.6 × 250 mm). At this time, tertiary distilled water containing 0.1% trifluoroacetic acid (TFA) was used as the solvent A, and acetonitrile containing 0.1% trifluoroacetic acid was used as the solvent B. The solvent B was eluted with a linear thickener for 120 minutes at a flow rate of 0.8 ml / min so that the concentration of solvent B was 5% to 80%, and the separation profile was as shown in FIG.

Example 4: Western Blots for Peptides

After attaching the peptide isolated according to Example 3 to the nitrocellulose membrane, Western blot method using monoclonal antibody CFC-6, monoclonal antibody CFC-9 and anti-erythropoietin rabbit antibody of polyclonal antibody as the primary antibody, respectively Was performed. As a secondary antibody, horse radish peroxidase was used. Experimental results showed that the peptide fraction No. Since 53 shows a strong positive reaction (see FIG. 5), this peptide was found to contain a junction to the erythropoietin receptor.

Example 5: Amino Acid Sequence Analysis of Peptides (No. 53)

No. found to contain a junction to the erythropoietin receptor in Example 4 according to the Edman degradation. The amino acid sequence of 53 peptides was analyzed. As a result, the peptide was a 10mer peptide having the V ₆₃ -WQGLALLSE ₇₂ sequence.

Example 6: Coupling of Peptides and Carrier Proteins

The peptide whose amino acid sequence was determined in Example 5 was coupled to Keyhole limpet hemocyanin (KLH) according to a known method (Reichlin, M., 1980, Methods in Enzymology 70, 157-165). That is, 5 mg of the peptide was dissolved in 500 µl of distilled water to prepare a 10 mg / ml solution. KLH was selected as a carrier protein, and 12.5 mg of this protein was dissolved in 100 µl of 50% glycerol solvent to 125 mg / ml. A solution of was prepared. 80 µl of the prepared KLH solution was taken, 520 µl of distilled water was added thereto, and 100 µl of 1M phosphate buffer solution (pH 7.0) was added, followed by mixing of 300 µl of the prepared peptide solution. In addition, 10 µl of 25% glutaraldehyde was dissolved in 1 ml of 0.1 M phosphate buffer solution (pH 7.0) to give a final concentration of 21 mM. 0.5 ml of the diluted glutaraldehyde solution was slowly added to 1 ml of a fabtide / KLH mixed solution, and then left at room temperature for 16 hours. As a result of performing the in vitro titer test described in Example 1 for each peptide concentration on the peptide-KLH complex thus produced, it was observed that ³ H-thymidine uptake was increased in the cell as in the case of erythropoietin.

Figure 1 shows the entire amino acid sequence of erythropoietin, where the expected receptor junction of the second helix is shown in bold circles.

Figure 2 shows the standard curve of in vitro assay of erythropoietin.

3 shows that thymidine uptake is inhibited when monoclonal antibody CFC-6 is added.

4 is a peptide map that appears when erythropoietin is treated with endoproteinase Glu-C and separated by HPLC.

FIG. 5 shows the results of Western blot when each peptide isolated by HPLC is used as an antigen and an anti-erythropoietin rabbit antibody, which is a CFC-6, CFC-9 or polyclonal antibody, is used as a primary antibody, respectively.

Claims (8)

A peptide having an activity of conjugating with an erythropoietin receptor molecule, the peptide containing the sequence of peptide V-W-Q-G-L-A-L-L-S-E. The peptide of claim 1, wherein at least one of the amino acids is a D-form. The peptide of claim 1, wherein the peptide is present as a multimer in which two or more peptides are bound. 4. The peptide according to claim 3, wherein the bond between peptide molecules consists of NH-NH, NH-COOH or COOH-COOH. The peptide of claim 1, wherein the peptide is coupled with a carrier protein. The peptide of claim 5, wherein the carrier protein is one selected from KLH (Keyhole limpet hemocyanin), Ovalbumin, and Bovine Serum Albumin (BSA). The peptide of claim 1, wherein at least one of the amino acids constituting the peptide is chemically modified. 8. The peptide according to claim 7, wherein the peptide is modified by at least one method selected from blocking of the peptide N-terminal amino group and derivatization of the -SH group.