Polypeptides that bind to RANKL and their applications

Abstract

The present invention relates to the field of biomedicine, and particularly relates to a polypeptide that binds to RANKL and its applications. The polypeptide has the amino acid sequence shown in Formula I. The polypeptide provided by the present invention has a high affinity for RANKL and can inhibit the binding of RANKL to its ligand, thereby being able to prevent and / or treat diseases mediated thereby.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to polypeptides that bind to RANKL and their applications. Background Art

[0002] Osteoporosis is a systemic skeletal disease characterized by reduced bone mass, destruction of bone tissue microstructure, increased bone fragility, and elevated fracture risk, 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 progress have been made in the research on RANKL, and the research scope is not only limited to osteoporosis, but also fruitful in other disease fields.

[0004] In terms of cancer, especially tumors such as breast cancer and prostate cancer 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, resulting in bone resorption, and 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 metastatic cancer. In addition, RANKL is also related to the invasion and metastasis potential of tumor cells, and 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 the inflammatory microenvironment. Fibroblast-like synoviocytes in the joint synovium will highly express RANKL under the stimulation of inflammatory factors, promoting osteoclastogenesis and exacerbating 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 by inhibiting RANKL, the joint inflammation and bone damage of patients with rheumatoid arthritis can be effectively reduced, and the joint function and quality of life of patients can be improved.

[0006] At the molecular mechanism level, studies have found that after RANKL binds to RANK, it not only activates the classical NF-κB signaling pathway but also initiates multiple signal transduction pathways such as MAPK and PI3K, which synergistically promote the differentiation of osteoclast precursor cells into mature osteoclasts and maintain the survival and function of osteoclasts. In animal experiments, mouse models with gene knockout of RANKL or RANK genes have confirmed the core position of the RANKL-RANK signaling pathway in bone development and bone mass maintenance. Lack of this signaling pathway leads to severe osteopetrosis, while overactivation causes severe osteoporosis phenotypes. In clinical studies, serum RANKL levels have been used as important biomarkers for evaluating the disease progression and treatment response of osteoporosis, rheumatoid arthritis, and other diseases.

[0007] In pathological processes such as 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, resulting in bone resorption far exceeding bone formation, thereby causing severe bone destruction.

[0008] Currently, 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. Bisphosphonate drugs may cause severe gastrointestinal adverse reactions, and long-term use may also lead to rare but serious complications such as osteonecrosis of the jaw; estrogen receptor modulators have the risk of increasing endometrial cancer and venous thrombosis; the efficacy of calcitonin is relatively limited and the duration of action is short. In addition, most of these drugs are small molecule or macromolecule antibody drugs. Small molecule drugs have insufficient specificity and are prone to off-target effects, while antibody drugs are difficult to penetrate tissue barriers due to their large molecular weight, and have high production costs and limited administration methods (usually require injection), resulting in poor patient compliance.

[0009] Given the deficiencies of existing treatment methods, it is urgent to develop a drug for treating bone-related diseases that is safe, effective, highly specific, and has good pharmacokinetic properties. Peptide drugs have become a 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 precisely block the RANKL-RANK signaling pathway and inhibit the overactivation 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, filling the current clinical treatment gap, and having broad market prospects and important medical value. Summary of the Invention

[0011] The object of the present invention is to provide a pharmaceutical polypeptide capable of binding to RANKL, which has a high affinity for RANKL.

[0012] To achieve the above object, a first aspect of the present invention provides a polypeptide capable of binding to RANKL or a pharmaceutically acceptable salt thereof, and the polypeptide has an amino acid sequence shown in any one of SEQ ID NO: 1-89.

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

[0014] A third aspect of the present invention provides a vector, and the vector contains the gene described in the second aspect above.

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

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

[0017] The polypeptide provided by the present invention is obtained by phage screening, has a high affinity for RANKL, can effectively inhibit the binding of RANKL to its ligand, and thus can prevent and / or treat the diseases mediated by it.

[0018] Other features and advantages of the present invention will be described in detail in the following specific implementation section. Specific Implementation Modes

[0019] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and a single point value, and between single point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[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 described above, the first aspect of the present invention provides a polypeptide that binds to RANKL or a pharmaceutically acceptable salt thereof, and the polypeptide has an amino acid sequence 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 after obtaining the target polypeptide by phage display technology. Specifically as follows:

[0023] Construct a single-stranded DNA mutant random library, and transfer the mutant random library into Escherichia coli strain SS320 cells infected with M13KO7 helper phage to obtain a phage display library; use RANKL as the target protein, and co-incubate with the phage display library as the mobile phase, and then obtain the target polypeptide after washing, elution and amplification, and then use the linker peptide SAAG to modify the C-terminus of the target polypeptide and use the linker peptide GSGS to modify the N-terminus of the target polypeptide.

[0024] It should be noted that the present invention has no particular limitation on the construction methods of the vector and the phage, and those skilled in the art can select in combination with known technical means in the art. An exemplary method is provided in the present invention, and those skilled in the art should not be construed as a limitation to the present invention. Specifically, reference can be made to the method in Liu, B., et al. Improving the mutagenesis efficiency of the Kunkel method by codon optimization and annealing temperature adjustment. New Biotechnology. 56(2019).

[0025] It should be noted that the present invention has no particular limitation on the method for constructing the single-stranded DNA mutant random library, and those skilled in the art can select in combination with known technical means in the art. An exemplary construction method is provided in the present invention, and those skilled in the art should not be construed as a limitation to the present invention. Specifically, reference can be made to the method in Tonikian, R., et al. Identifying specificity profiles for peptide recognition modules from phage-displayed peptide libraries. Nature Protocol. 2.6(2007):1368-1386.

[0026] It should be noted that the present invention has no particular limitation on the above method for obtaining the target polypeptide, and those skilled in the art can make selections in combination with known technical means in the art. An exemplary screening method is provided in the present invention, which should not be construed as a limitation to the present invention by those skilled in the art. Specifically, it can be carried out with reference to the method in 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.

[0027] Preferably, the pharmaceutically acceptable salt is at least one of trifluoroacetate, acetate, hydrochloride, and phosphate.

[0028] In the present invention, the amino acid sequences shown in SEQ ID NO: 1-89 are shown in Table 1 below.

[0029] Table 1

[0030]

[0031]

[0032] As described above, the second aspect of the present invention provides a gene, and the nucleotide sequence of the gene is the nucleotide sequence capable of encoding the polypeptide that binds to RANKL described in the first aspect above.

[0033] As described above, the third aspect of the present invention provides a vector, and the vector contains the gene described in the second aspect above.

[0034] As described above, the fourth aspect of the present invention provides a pharmaceutical composition, and the pharmaceutical composition contains a therapeutically effective amount of the polypeptide that binds to RANKL described in the first aspect above or a pharmaceutically acceptable salt thereof.

[0035] Preferably, the composition further contains a pharmaceutically acceptable carrier.

[0036] It should be noted that the present invention has no particular limitation on the type of the vector, and those skilled in the art can make selections according to known technical means in the art, as long as the polypeptide provided in the present invention can target RANKL.

[0037] As described above, the fifth aspect of the present invention provides the use of at least one of the polypeptide that binds to RANKL or a pharmaceutically acceptable salt thereof described in the first aspect above, the gene described in the second aspect above, the vector described in the third aspect above, and the pharmaceutical composition described in the fourth aspect above in the preparation of a drug for preventing and / or treating RANKL-mediated diseases; the RANKL-mediated diseases are at least one of osteoporosis, bone cancer, osteolytic tumor, and rheumatoid arthritis.

[0038] Preferably, the osteoporosis includes familial osteoporosis, senile or postmenopausal osteoporosis, osteoporosis caused by glucocorticoids, drug-induced osteoporosis, and immobilization-induced osteoporosis; the osteolytic tumors include bone metastatic cancer, giant cell tumor of bone, and multiple myeloma.

[0039] More preferably, the bone metastatic cancer includes breast cancer, lung cancer, and prostate cancer.

[0040] The present invention will be described in detail below by way of examples.

[0041] In the following examples, unless otherwise specified, the raw materials used are commercially available.

[0042] In the following examples, unless otherwise specified, the room temperature means 25 ± 5°C.

[0043] Example 1: Screening of the target polypeptide

[0044] After resuspending and washing the magnetic beads (streptavidin T1 magnetic beads, manufacturer: Invitrogen, catalog number 65602) according to the method in the instruction manual, 60 μg of RANKL (target protein) was immobilized in 105 μL of magnetic beads, and 60 μL of PBS was added. Finally, the total volume was 180 μL. It was placed on a rotary mixer and mixed at 4°C for 20 ± 4 h. After the incubation, the magnetic beads bound to the target protein were precipitated with a magnetic stand, the supernatant was removed, and the magnetic beads were washed 4 times with 1 mL of PBS containing 0.1 wt% Tween (PBST). Subsequently, the magnetic beads were blocked and blocked with 0.5 wt% BSA-PBS at room temperature for 30 min. In the first round, 5 μL of 1×10 11The helper phage library of M13KO7 in pfu (dissolved in PBS) was gently mixed at 4°C and co-incubated for 1 h. After incubation, the unbound phages were washed repeatedly with PBST. In the next round of screening, the concentration of Tween was gradually increased from 0.1 wt% to 0.4 wt% at an increment of 0.1 wt% per round. Elution was carried out by incubating with 0.1 M citrate (pH = 3.1) for 2 min to obtain the phages bound to the target protein, which were immediately neutralized with 1 M Tris-HCl (pH = 9.1). 20 μL was taken for phage titer determination, and the remaining phages were amplified using the Escherichia coli strain SS320. The purified phages were used for the next round of screening, and the phage input per round was 1×10 11 pfu.

[0045] Example 2: ELISA Identification of Target Polypeptide

[0046] 1. Coating the plate: SA was diluted to 5 μg / mL with 50 mM sodium bicarbonate (pH = 8.5), and then 50 μL was taken to coat the ELISA plate at 37°C for 2 h;

[0047] 2. Blocking: The above ELISA plate was blocked overnight at 4°C with TBST + 20 mg / mL BSA blocking solution;

[0048] 3. Washing: The above ELISA plate was washed 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), with each wash lasting 3 min;

[0049] 4. Binding the target protein: 200 ng of RANKL was incubated with the above ELISA plate at 37°C for 1 h;

[0050] 5. Washing: The plate was washed 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), with each wash lasting 3 min;

[0051] 6. Binding phages: 1×10 9 / 1×10 10 pfu of phage supernatant was incubated with the ELISA plate coated with RANKL at 37°C for 1 h;

[0052] 7. Washing: The plate was washed 5 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), with each wash lasting 3 min;

[0053] 8. Conjugation of antibody: Incubate 100 μL of M13K07 helper phage antibody at a concentration of 0.1 μg / mL with the phage in the above ELISA plate at 37 °C for 1 h;

[0054] 9. Washing: Wash 5 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), 3 min for each wash;

[0055] 10. Color development: Add 100 μL of TMB color development solution prepared by mixing equal volumes of Solution A and Solution B, and develop color at room temperature for 5 min;

[0056] 11. Termination: Add 100 μL of H2SO4 at a concentration of 2 M;

[0057] 12. Scanning: Read the OD450 value using an ELISA reader to obtain the target polypeptide with high affinity for RANKL.

[0058] Example 3: Synthesis of the target polypeptide

[0059] The polypeptide and its derivatives provided by the present invention are synthesized by solid-phase synthesis to obtain the linear precursor, and the crude target polypeptide is obtained after cleavage. Among them, the solid-phase carrier is 2-Chlotrityl Resin. During the synthesis process, first, the 2-Chlotrityl Resin is fully swollen in N,N-dimethylformamide (DMF), and then the swollen solid-phase carrier is repeatedly subjected to the operations of condensation → washing → deprotection of Fmoc → washing → condensation of the next amino acid until the required length of the polypeptide chain is reached. Finally, a mixed solution of trifluoroacetic acid: water: triisopropylsilane: benzyl methyl sulfide (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 the crude target polypeptide is obtained after precipitation with cold methyl tert-butyl ether. The crude target polypeptide is purified and separated by a C18 reversed-phase preparative chromatographic column in a system of acetonitrile / water with 0.1% trifluoroacetic acid to obtain the pure product of the polypeptide and its derivatives. The following is the specific synthesis method of SEQ ID NO: 1, where Table 2 shows the reagents used in the synthesis.

[0060] Table 2

[0061]

[0062] Synthesis of SEQ ID NO: 1:

[0063] Step 1: Couple the first amino acid Fmoc-Ser(tBu)-OH

[0064] 0.1 mmol of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. 0.08 mmol of Fmoc-Ser(tBu)-OH and 0.32 mmol of DIEA were weighed and dissolved in 5 mL of DCM, and the swollen resin was added, and the reaction was carried out at room temperature for 2 h. After the reaction was completed, 10 mL of a blocking solution (DCM: methanol: DIEA (v:v:v = 85:10:5)) was added and blocked at room temperature for 10 min. The blocked resin was washed 5 times with DCM and then 5 times with DMF and reserved for use.

[0065] Step 2: Synthesis of the linear precursor peptide chain

[0066] Linear precursor peptide chain of SEQ ID NO: 1:

[0067] S-A-A-G-F-T-C-S-W-Y-N-W-S-M-V-E-K-C-R-V-R-G-S-G-S.

[0068] The resin obtained in Step 1 was fully swollen in DMF for 1 h, and then synthesized in the order from the second position at the carboxyl terminus to the amino terminus according to the linear precursor sequence. Each coupling cycle was carried out as follows:

[0069] (1) The swollen resin was deprotected with Fmoc twice with a 10 mL mixture of piperidine: DMF (v:v = 20:80), 8 min each time;

[0070] (2) The resin was washed 6 - 8 times with DMF until the liquid pH was neutral;

[0071] (3) 0.5 mmol of Fmoc-AA, 0.5 mmol of HCTU and 1 mmol of NMM were dissolved in DMF, and then added to the washed resin, and the reaction was carried out at room temperature for 1 h;

[0072] (4) The resin was washed 4 - 6 times with DMF before coupling the next amino acid;

[0073] 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 vacuo for standby.

[0074] Step 3: Cleavage of the linear precursor peptide chain

[0075] Add 10 mL of freshly prepared trifluoroacetic acid: water: triisopropylsilane: benzyl methyl sulfide (v:v:v:v = 90:2.5:2.5:5) mixture to the resin obtained in Step 2, and react with shaking at room temperature for 2 h. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate with 4 volumes of frozen MTBE to obtain the crude polypeptide. Wash the crude polypeptide with MTBE three times and then dry it under vacuum for standby.

[0076] Step 4: Purification of the crude polypeptide

[0077] Dissolve the crude polypeptide in an aqueous solution of 20 vol% acetonitrile, filter it through a 0.45 μm membrane, and then separate it using a reverse-phase high-performance liquid chromatography system. The buffer solutions are Phase A (0.1 wt% trifluoroacetic acid aqueous solution) and Phase B (0.1 wt% trifluoroacetic acid acetonitrile solution). Among them, the chromatographic column is a BR-C18 (Sepax Technologies) reverse-phase chromatographic column. During the purification process, the detection wavelength of the chromatograph is set at 230 nm, the flow rate is 15 mL / min, and the gradient is that Phase B changes from 20% to 50% within 0 - 40 min. Collect the relevant fractions of the product, combine the fractions with a purity > 95% after HPLC identification, and then lyophilize to obtain the pure polypeptide.

[0078] Step 5: Detection and characterization methods

[0079] Determine the purity of the pure polypeptide in Step 4 by analytical high-performance liquid chromatography and liquid chromatography / mass spectrometry.

[0080] Other polypeptides of the present invention can be synthesized by referring to the synthesis method of SEQ ID NO: 1.

[0081] Example 4: Determine the affinity between the target polypeptide and RANKL by ELISA

[0082] After immobilizing the RANK protein (at a concentration of 0.125 μg / mL) at 25 μL / well on a 384-well plate; washing 4 times with a TBS solution containing 0.05 wt% Tween-20, then adding a TBS solution containing 2 wt% BSA and incubating at 37 °C for blocking; then using a workstation to add the above-mentioned target polypeptide solutions at 12.5 μL / well to the aforementioned 384-well plate, and then adding a premix (mixing RANKL protein (at a concentration of 0.06 μg / mL) and streptavidin-horseradish peroxidase at a concentration of (0.2 μg / mL) in a volume ratio of 1:1 at room temperature for 1 h) at 12.5 μL / well to the aforementioned 384-well plate, centrifuging briefly to remove air bubbles and then placing in a 37 °C incubator for 1 h. After discarding the liquid in the wells, add 80 μL of 1×TBST washing solution with pH = 7.4 to wash the plate 4 times, 4 min each time. Drain the 384-well plate, add 25 μL of TMB chromogenic solution to each well, and continue to incubate in the incubator at 37 °C for 30 min. Finally, add 25 μL of termination solution (1 M HCl) to each well to terminate the reaction. Use an enzyme-linked immunosorbent assay (ELISA) reader to read the absorbance of each well at 450 nm. After calculating the inhibition rate of the concentration of each target polypeptide according to formula (1), then use GraphPad prism software to plot a graph and calculate the IC50 value of each polypeptide. The specific results are shown in Table 3. Among them, the formula for calculating the inhibition rate is:

[0083] Inhibition% = (1 - (absorbance of sample - absorbance of background) / (absorbance of positive - absorbance of background)) × 100%; where,

[0084] Sample: refers to adding a polypeptide to the reaction system;

[0085] Positive: refers to not adding a polypeptide to the reaction system;

[0086] Background: refers to not adding a polypeptide and RANKL protein to the reaction system.

[0087] Table 3: IC50 values of target polypeptides

[0088]

[0089]

[0090] Example 5: Determination of the inhibitory effect of target polypeptides on RANKL-activated RANK cells

[0091] One day before the experiment, HEK293 / NF-κB-AP-1 / RANK cells (purchased from GenScript) were seeded into 384-well plates at a density of 12,000 cells / well and cultured overnight in an incubator at 37 °C and 5% CO2. The next day, 10 μL / well of the target polypeptide and 10 μL / well of 8 μg / mL RANKL protein were added, and the cells were further cultured for 6 h. Then, 250 μL / well of one-lite Luciferase Assay kit (purchased from Novizan) chemiluminescence detection reagent was added. After shaking in the dark for 15 min, the chemiluminescence signal was detected. The inhibition rate was calculated, and the data was plotted using GraphPad prism to calculate the IC50 value of the inhibitory effect of the target polypeptide on RANK cell activation. The specific results are shown in Table 4. The formula for calculating the inhibition rate is as follows:

[0092] Inhibition% = (1 - (signal value of sample - signal value of background) / (signal value of positive - signal value of background)) × 100%; where,

[0093] Sample: refers to the addition of the polypeptide to the reaction system;

[0094] Positive: refers to the absence of the polypeptide in the reaction system;

[0095] Background: refers to the absence of both the polypeptide and RANKL protein in the reaction system.

[0096] Table 4

[0097]

[0098] As can be seen from the results in Table 3 and Table 4, the polypeptides provided by the present invention have good inhibitory activity on the binding of RANKL to RANK at both the molecular level and the cellular level.

[0099] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A polypeptide that binds to RANKL or a pharmaceutically acceptable salt thereof, characterized in that, The amino acid sequence of the polypeptide is as shown in any one of SEQ ID NO: 3-5.

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

3. A carrier, characterized in that, The vector contains the gene as claimed in claim 2.

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

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