Polypeptide combined with RANKL and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to polypeptide combined with RANKL and application of the polypeptide. The polypeptide has an amino acid sequence as shown in a formula I. The polypeptide provided by the invention has relatively high affinity with the RANKL, and can inhibit the combination of the RANKL and a ligand thereof, so that diseases mediated by the RANKL can be prevented and / or treated.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a polypeptide binding to RANKL and applications thereof. Background Art

[0002] Osteoporosis is a systemic skeletal disease characterized by decreased bone mass and destruction of bone tissue microarchitecture, leading to increased bone fragility and increased risk of fractures, which seriously affects the quality of life and health of patients.

[0003] Receptor activator of nuclear factor κB ligand (RANKL) plays a key role in bone metabolism balance. In recent years, many important advances have been made in the study of RANKL, and the research scope is not limited to osteoporosis, but also has achieved fruitful results in other disease areas.

[0004] In terms of cancer, especially breast cancer, prostate cancer and other tumors that are prone to bone metastasis. RANKL plays a key role in the interaction between tumor cells and the bone microenvironment. Tumor cells can secrete RANKL, stimulate osteoclast activity, and lead to bone dissolution, while the growth factors released by bone tissue in turn promote the proliferation, survival and migration of tumor cells, forming a vicious cycle. Studies have shown that blocking the RANKL signaling pathway can inhibit bone destruction and tumor growth in a mouse model of breast cancer bone metastasis, providing a new target for the treatment of bone metastasis. In addition, RANKL is also related to the invasion and metastasis potential of tumor cells. Tumor cells with high expression of RANKL are more likely to break through the basement membrane and infiltrate into surrounding tissues.

[0005] In autoimmune diseases such as rheumatoid arthritis, in addition to the bone erosion problem mentioned above, RANKL is involved in the formation of an inflammatory microenvironment. Under the stimulation of inflammatory factors, fibroblast-like synoviocytes in the synovial membrane of the joints will express a large amount of RANKL, promote osteoclast formation, and aggravate joint bone destruction. At the same time, RANKL also affects the function of immune cells, regulates the differentiation and activation of T cells and B cells, and is closely related to the immune imbalance of the disease. Clinical studies have found that inhibiting RANKL can effectively reduce joint inflammation and bone damage in patients with rheumatoid arthritis, and improve patients' joint function and quality of life.

[0006] At the molecular mechanism level, studies have found that after RANKL binds to RANK, it not only activates the classic NF-κB signaling pathway, but also initiates multiple signal transduction pathways such as MAPK and PI3K, synergistically promoting the differentiation of osteoclast precursor cells into mature osteoclasts and maintaining the survival and function of osteoclasts. In animal experiments, mouse models with gene knockout of RANKL or RANK genes have confirmed the core role of the RANKL-RANK signaling pathway in bone development and maintenance of bone mass. The lack of this signaling pathway can lead to severe osteosclerosis, while excessive activation can cause severe osteoporosis phenotypes. In clinical studies, serum RANKL levels have been used as an important biomarker to assess the progression and treatment response of diseases such as osteoporosis and rheumatoid arthritis.

[0007] In the pathological processes of osteoporosis and other bone-related diseases such as bone erosion caused by rheumatoid arthritis and osteolytic lesions caused by tumor bone metastasis, the RANKL signaling pathway is overactivated, causing bone absorption to far exceed bone formation, thereby causing serious damage to the bone.

[0008] At present, the therapeutic drugs for bone-related diseases such as osteoporosis mainly include bisphosphonates, estrogen receptor modulators, calcitonin, etc. However, these traditional drugs have many limitations. Bisphosphonates may cause serious gastrointestinal adverse reactions, and long-term use may also lead to rare but serious complications such as mandibular osteonecrosis; estrogen receptor modulators increase the risk of endometrial cancer and venous thrombosis; calcitonin has relatively limited efficacy and a short duration of action. In addition, most of these drugs are small molecule or large molecule antibody drugs. Small molecule drugs lack specificity and are prone to off-target effects. Antibody drugs are difficult to penetrate tissue barriers due to their large molecular weight, and their production costs are high. The method of administration is limited (usually injection is required), and patient compliance is poor.

[0009] In view of the shortcomings of existing treatment methods, it is urgent to develop a safe, effective, highly specific and pharmacokinetic drug for the treatment of bone-related diseases. Peptide drugs have become a very promising research direction due to their unique advantages.

[0010] Peptide drugs have the advantages of moderate relative molecular weight, strong specificity, high activity, easy synthesis and modification, and low immunogenicity. If a class of peptide drugs that can specifically bind to RANKL can be developed to accurately block the RANKL-RANK signaling pathway and inhibit the excessive activation of osteoclasts, it will be expected to provide a new, efficient and safe treatment strategy for the treatment of osteoporosis and other bone-related diseases, fill the gap in current clinical treatment, and have broad market prospects and important medical value. Summary of the invention

[0011] The purpose of the present invention is to provide a drug polypeptide that can bind to RANKL, and the drug polypeptide has a high affinity to RANKL.

[0012] In order to achieve the above object, the first aspect of the present invention provides a polypeptide binding to RANKL or a pharmaceutically acceptable salt thereof, wherein the polypeptide has an amino acid sequence as shown in any one of SEQ ID NOs: 1-89.

[0013] The second aspect of the present invention provides a gene, the nucleotide sequence of which is a nucleotide sequence capable of encoding the polypeptide binding to RANKL as described in the first aspect.

[0014] The third aspect of the present invention provides a vector, which contains the gene described in the second aspect.

[0015] The fourth aspect of the present invention provides a pharmaceutical composition, which contains a therapeutically effective amount of the polypeptide binding to RANKL or a pharmaceutically acceptable salt thereof as described in the first aspect.

[0016] The fifth aspect of the present invention provides the use of at least one of the polypeptide binding to RANKL or a pharmaceutically acceptable salt thereof as described in the first aspect, the gene as described in the second aspect, the vector as described in the third aspect, and the pharmaceutical composition as described in the fourth aspect for preparing a drug for preventing and / or treating a disease mediated by RANKL, wherein the disease mediated by RANKL is at least one of osteoporosis, bone cancer, osteolytic tumors, and rheumatoid arthritis.

[0017] The polypeptide provided by the present invention is obtained by phage screening, has a high affinity with RANKL, and can effectively inhibit the binding of RANKL to its ligand, thereby preventing and / or treating diseases mediated by RANKL.

[0018] Other features and advantages of the present invention will be described in detail in the following detailed description. DETAILED DESCRIPTION

[0019] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0020] It should be noted that, unless otherwise defined herein, the scientific and technical terms used in the present invention should have the meanings commonly understood by those skilled in the art.

[0021] As mentioned above, the first aspect of the present invention provides a polypeptide binding to RANKL or a pharmaceutically acceptable salt thereof, wherein the polypeptide has an amino acid sequence as shown in any one of SEQ ID NOs: 1-89.

[0022] The amino acid sequence shown in any one of SEQ ID NOs: 1-89 in the present invention is obtained by modifying the N-terminus and C-terminus of the target polypeptide obtained by phage display technology. Specifically, it is as follows: A single-stranded DNA mutation random library was constructed, and the mutation random library was transferred into Escherichia coli strain SS320 cells infected with M13KO7 helper phage to obtain a phage display library; RANKL was used as the target protein, and the phage display library was used as the mobile phase for co-incubation, and the target polypeptide was obtained after washing, elution and amplification in sequence, and then the C-terminus of the target polypeptide was modified with a connecting peptide SAAG and the N-terminus of the target polypeptide was modified with a connecting peptide GSGS.

[0023] It should be noted that the present invention has no particular limitation on the construction methods of vectors and phages, and those skilled in the art can select them in combination with the known technical means in the art. The present invention provides a method exemplarily, which should not be construed as limiting the present invention by those skilled in the art. Specifically, reference can be made to Liu, B., et al. Improving the mutagenesis efficiency of the Kunkel method by codon optimization and annealing temperature adjustment. New Biotechnology. 56(2019).

[0024] It should be noted that the present invention has no particular limitation on the method for constructing the random library of single-stranded DNA mutations, and those skilled in the art can select it in combination with the known technical means in the art. The present invention exemplarily provides a construction method, which should not be construed as a limitation of the present invention by those skilled in the art. Specifically, reference can be made to Tonikian, R., et al. Identifying specificity profiles for peptide recognition modules from phage-displayed peptide libraries. Nature Protocol. 2.6(2007):1368-1386.

[0025] It should be noted that the present invention has no particular limitation on the above-mentioned method for obtaining the target polypeptide, and those skilled in the art can select it in combination with the known technical means in the art. The present invention provides an exemplary screening method, which should not be construed as limiting the present invention by those skilled in the art. Specifically, reference can be made to Padmanaban, G., et al. Identification of peptides that selectively bind to myoglobin by biopanning of phage displayed-peptide library. Journal of Biotechnology. 2.6(2007):1368-1386.

[0026] Preferably, the pharmaceutically acceptable salt is at least one of trifluoroacetate, acetate, hydrochloride and phosphate.

[0027] In the present invention, the amino acid sequences shown in SEQ ID NO: 1-89 are shown in Table 1 below.

[0028] Table 1

[0029]

[0030] As mentioned above, the second aspect of the present invention provides a gene, the nucleotide sequence of which is a nucleotide sequence capable of encoding the polypeptide binding to RANKL as described in the first aspect above.

[0031] As mentioned above, the third aspect of the present invention provides a vector, which contains the gene described in the second aspect.

[0032] As mentioned above, the fourth aspect of the present invention provides a pharmaceutical composition, which contains a therapeutically effective amount of the polypeptide binding to RANKL or a pharmaceutically acceptable salt thereof as described in the first aspect.

[0033] Preferably, the composition further contains a pharmaceutically acceptable carrier.

[0034] It should be noted that the present invention has no particular limitation on the type of the carrier, and those skilled in the art can select the carrier according to the technical means known in the art, as long as the polypeptide provided in the present invention can target RANKL.

[0035] As described above, the fifth aspect of the present invention provides the use of at least one of the polypeptide binding to RANKL or a pharmaceutically acceptable salt thereof as described in the first aspect, the gene as described in the second aspect, the vector as described in the third aspect, and the pharmaceutical composition as described in the fourth aspect for preparing a drug for preventing and / or treating a RANKL-mediated disease; the RANKL-mediated disease is at least one of osteoporosis, bone cancer, osteolytic tumors, and rheumatoid arthritis.

[0036] Preferably, the osteoporosis includes familial osteoporosis, senile or postmenopausal osteoporosis, osteoporosis caused by glucocorticoids, drug-induced osteoporosis and immobilization osteoporosis; the osteolytic tumors include bone metastasis, giant cell tumor of bone, and multiple myeloma.

[0037] Particularly preferably, the bone metastasis cancer includes breast cancer, lung cancer, and prostate cancer.

[0038] The present invention will be described in detail below by way of examples.

[0039] In the following examples, unless otherwise specified, all raw materials used were commercially available.

[0040] In the following examples, unless otherwise specified, the room temperature refers to 25±5°C.

[0041] Example 1: Screening of target polypeptides After resuspending and washing the magnetic beads (streptavidin T1 magnetic beads, manufacturer: Invitrogen, catalog number 65602) according to the instructions, fix 60μg of RANKL (target protein) to 105μL magnetic beads, and add 60μL of PBS, and the final total volume is 180μL. Place on a rotating mixer and mix at 4°C for 20±4h. After incubation, use a magnetic stand to precipitate the magnetic beads bound to the target protein, remove the supernatant, and wash the magnetic beads 4 times with 1mL of 0.1wt% Tween PBS (PBST). Subsequently, the magnetic beads were blocked with 0.5wt% BSA-PBS at room temperature for 30min. In the first round, 5μL of 1×10 11pfu of M13KO7 helper phage library (dissolved in PBS), gently mixed and incubated at 4°C for 1h. After incubation, unbound phages were repeatedly washed with PBST. In the next round of screening, the concentration of Tween was gradually increased from 0.1wt% to 0.4wt% at an increase of 0.1wt% / round. Phages bound to the target protein were obtained by incubation for 2min with 0.1M citrate (pH=3.1) for elution, and immediately neutralized with 1M Tris-HCl (pH=9.1). 20μL was taken for phage titer determination, and the remaining phages were amplified using Escherichia coli strain SS320. The phages were purified and used for the next round of screening. The amount of phage input in each round was 1×10 11 pfu.

[0042] Example 2: ELISA identification of target polypeptide 1. Plate coating: Dilute SA to 5 μg / mL with 50 mM sodium bicarbonate (pH=8.5), take 50 μL and coat the ELISA plate at 37°C for 2 hours; 2. Blocking: Use TBST + 20 mg / mL BSA blocking solution to block the above ELISA plate at 4°C overnight; 3. Washing: Wash the ELISA plate 3 times with 200 μL of washing solution (25 mM Tris-HCl (pH = 7.2) + 150 mM NaCl + 0.1 wt% BSA + 0.05 wt% Tween 20), each wash for 3 min; 4. Binding to target protein: 200 ng of RANKL was incubated with the above ELISA plate at 37°C for 1 hour; 5. Washing: Wash three times with 200 μL of washing solution (25 mM Tris-HCl (pH = 7.2) + 150 mM NaCl + 0.1 wt% BSA + 0.05 wt% Tween 20), each wash for 3 min; 6. Combined with phage: 1×10 9 / 1×10 10 pfu of phage supernatant was incubated with ELISA plates coated with RANKL at 37°C for 1 h; 7. Washing: Wash 5 times with 200 μL washing solution (25 mM Tris-HCl (pH = 7.2) + 150 mM NaCl + 0.1 wt% BSA + 0.05 wt% Tween 20), each wash for 3 min; 8. Binding antibody: Incubate 100 μL of 0.1 μg / mL M13K07 helper phage antibody with the phage in the above ELISA plate at 37°C for 1 h; 9. Washing: Wash 5 times with 200 μL washing solution (25 mM Tris-HCl (pH = 7.2) + 150 mM NaCl + 0.1 wt% BSA + 0.05 wt% Tween 20), each wash for 3 min; 10. Color development: Add 100 μL of TMB color development solution mixed with equal volumes of solution A and solution B, and develop the color for 5 minutes at room temperature; 11. Stop: Add 100 μL of 2M H2SO4; 12. Scanning: Use an ELISA reader to read the OD450 to obtain the target peptide with high affinity to RANKL.

[0043] Example 3: Synthesis of target polypeptide The polypeptide and its derivatives provided by the present invention adopt a solid phase synthesis method to synthesize their linear precursors, and obtain a crude target polypeptide after cutting. The solid phase carrier is 2-Chlotrityl Resin resin. In the synthesis process, the 2-Chlotrityl Resin resin is first fully swollen in N, N-dimethylformamide (DMF), and then the swollen solid phase carrier and the activated amino acid derivative are repeatedly condensed → washed → Fmoc protection → washed → the next round of amino acid condensation to achieve the desired length of the synthesized polypeptide chain, and finally a mixed solution of trifluoroacetic acid: water: triisopropylsilane: anisyl thioether (v:v:v:v=90:2.5:2.5:5) is reacted with the solid phase resin to cleave the polypeptide from the solid phase carrier, and then the target polypeptide crude product is obtained after precipitation by frozen methyl tert-butyl ether. The target polypeptide crude product is purified and separated by a C18 reverse phase preparative chromatography column in a 0.1% trifluoroacetic acid acetonitrile / water system to obtain a pure product of the polypeptide and its derivatives. The following is a specific synthesis method of SEQ ID NO: 1, wherein Table 2 lists the reagents used in the synthesis.

[0044] Table 2

[0045] Synthesis of SEQ ID NO: 1: Step 1: Coupling of the first amino acid Fmoc-Ser(tBu)-OH Fully swell 0.1mmol of 2-Chlorotrityl chloride resin in DCM for 1h. Weigh 0.08mmol of Fmoc-Ser(tBu)-OH and 0.32mmol of DIEA in 5mL of DCM, add to the swollen resin, and react at room temperature for 2h. After the reaction is completed, add 10mL of blocking solution (DCM: methanol: DIEA (v: v: v = 85: 10: 5) and block at room temperature for 10min. Wash the blocked resin with DCM 5 times and then with DMF 5 times before use.

[0046] Step 2: Synthesis of linear precursor peptide chain The linear precursor peptide chain of SEQ ID NO: 1: SAAGFTCSWYNWSMVEKCRVR-GSGS.

[0047] The resin obtained in step 1 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the second position of the carboxyl terminal to the amino terminal. Each coupling cycle was performed as follows: (1) The swollen resin was Fmoc-deprotected twice with 10 mL of a mixture of piperidine:DMF (v:v = 20:80), each time for 8 min; (2) Wash the resin with DMF 6-8 times until the pH of the liquid is neutral; (3) Dissolve 0.5 mmol of Fmoc-AA, 0.5 mmol of HCTU and 1 mmol of NMM in DMF, then add the washed resin and react at room temperature for 1 h; (4) Wash the resin 4-6 times with DMF before coupling the next amino acid; After the synthesis of the linear precursor peptide chain, the resin was washed 5 times with DMF and then 5 times with DCM, and the washed resin was dried in a vacuum for later use.

[0048] Step 3: Cleavage of the linear precursor peptide chain Add 10 mL of a freshly prepared mixture of trifluoroacetic acid: water: triisopropylsilane: thioanisole (v:v:v:v = 90:2.5:2.5:5) to the resin obtained in step 2 and shake for 2 h at room temperature. After the reaction is completed, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate with 4 times the volume of frozen MTBE to obtain a crude polypeptide. Wash the crude polypeptide with MTBE 3 times and then dry it in a vacuum for later use.

[0049] Step 4: Purification of crude peptide The crude peptide was dissolved in 20 vol% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and separated using a reversed-phase high-performance liquid chromatography system. The buffer was phase A (0.1 wt% trifluoroacetic acid aqueous solution) and phase B (0.1 wt% trifluoroacetic acid acetonitrile solution). The chromatographic column was a BR-C18 (Saifen Technology) reversed-phase column. During the purification process, the chromatograph detection wavelength was set to 230 nm, the flow rate was 15 mL / min, and the gradient was phase B changed from 20% to 50% within 0-40 min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions > 95% were combined and freeze-dried to obtain the pure peptide.

[0050] Step 5: Detection and characterization methods The purity of the peptide obtained in step 4 was determined by analytical HPLC and LC / MS.

[0051] Other polypeptides of the present invention can be synthesized by referring to the synthesis method of SEQ ID NO:1.

[0052] Example 4: Determination of affinity of target polypeptide to RANKL by ELISA After RANK protein (concentration of 0.125μg / mL) was fixed on a 384-well plate at 25μL / well, the plate was washed 4 times with TBS solution containing 0.05wt% Tween-20, and then blocked with TBS solution containing 2wt% BSA at 37℃. Then, the above target peptide solutions were added to the 384-well plate at 12.5μL / well using a workstation, and then the premix (RANKL protein (concentration of 0.06μg / mL) and horseradish peroxidase-labeled streptavidin (concentration of 0.2μg / mL) were mixed at room temperature for 1h at a volume ratio of 1:1) in the 384-well plate at 12.5μL / well), centrifuged to remove bubbles, and then placed in a 37℃ incubator for 1h. After discarding the liquid in the well, 80μL of 1×TBST washing solution with pH=7.4 was added to wash the plate 4 times, each time for 4min. The 384-well plate was drained, and 25 μL of TMB colorimetric solution was added to each well. The plate was then placed in an incubator and incubated at 37°C for 30 min. Finally, 25 μL of stop solution (1M HCl) was added to each well to terminate the reaction. The absorbance of each well at 450 nm was read using an ELISA reader. The inhibition rate of each target peptide concentration was calculated according to formula (1), and the IC50 value of each peptide was calculated using GraphPad prism software. The specific results are shown in Table 3. The inhibition rate was calculated as follows: Inhibition%=(1-(absorbance of sample-absorbance of background) / (absorbance of positive-absorbance of background))×100%; where, Sample: refers to adding peptides into the reaction system; Positive: refers to the absence of peptide in the reaction system; Background: refers to the reaction system without adding peptide and RANKL protein.

[0053] Table 3: IC50 values ​​of target peptides

[0054]

[0055] Example 5: Determination of the inhibitory effect of the target polypeptide on RANKL activation of RANK cells The day before the experiment, HEK293 / NF-KB-AP-1 / RANK cells (purchased from GenScript) were seeded in 384-well plates at 12,000 cells / well and cultured overnight in an incubator at 37°C and 5% CO2. On the second day, 10 μL / well of the target peptide and 10 μL / well of 8 μg / mL of RANKL protein were added and culture continued for 6 hours. 250 μL / well of one-lite Luciferase Assay kit (purchased from Novizan) chemiluminescent detection reagent was added, and the chemiluminescent signal was detected after 15 minutes of oscillation in the dark. The inhibition rate was calculated, and the IC50 value of the inhibitory effect of the target peptide on RANK cell activation was calculated using GraphPad prism. The specific results are shown in Table 4. Among them, the calculation formula for the inhibition rate is: Inhibition%=(1-(sample signal value-background signal value) / (positive signal value-background signal value))×100%; where, Sample: refers to adding peptides into the reaction system; Positive: refers to the absence of peptide in the reaction system; Background: refers to the reaction system without adding peptide and RANKL protein.

[0056] Table 4

[0057] It can be seen from the results in Table 3 and Table 4 that the polypeptide provided by the present invention has good inhibitory activity on the binding of RANKL and RANK at both the molecular level and the cellular level.

[0058] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A polypeptide binding to RANKL or a pharmaceutically acceptable salt thereof, characterized in that: The polypeptide has an amino acid sequence as shown in any one of SEQ ID NOs: 1-89.

2. A gene, characterized in that The nucleotide sequence of the gene is a nucleotide sequence capable of encoding the polypeptide binding to RANKL as claimed in claim 1.

3. A carrier, characterized in that The vector contains the gene according to claim 2.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition contains a therapeutically effective amount of the polypeptide binding to RANKL according to claim 1 or a pharmaceutically acceptable salt thereof.

5. Use of at least one of the polypeptide binding to RANKL according to claim 1 or a pharmaceutically acceptable salt thereof, the gene according to claim 2, the vector according to claim 3, and the pharmaceutical composition according to claim 4 in the preparation of a medicament for preventing and / or treating a RANKL-mediated disease; the RANKL-mediated disease is at least one of osteoporosis, bone cancer, osteolytic tumors, and rheumatoid arthritis.

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