Polypeptides that inhibit the binding of RANK to RANKL and their applications

Abstract

The present invention relates to the field of biomedicine, and particularly relates to a polypeptide that inhibits the binding of RANK and RANKL and its application. The amino acid sequence of the polypeptide has the amino acid sequence shown in SEQ ID NO: 1-3. The polypeptide provided by the present invention can effectively inhibit the binding of RANK and RANKL, and thus can prevent and / or treat diseases related to its signaling pathway.

Description

Technical Field

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

[0002] RANK (receptor activator of NF-κB), also known as nuclear factor-κB receptor activator, belongs to the tumor necrosis factor TNF (tumor necrosis factor) superfamily. RANK contains 616 amino acids and consists of a C-terminal cytoplasmic region, an N-terminal extracellular region, a signal peptide, and a transmembrane region. It is mainly expressed on the cell surfaces of pre-osteoclasts, mature osteoclasts, dendritic cells, etc. With the in-depth study, the presence of RANK has also been detected on the surfaces of some cancer cells (such as breast cancer and prostate cancer).

[0003] RANKL (receptor activator of NF-κB ligand) is the ligand of nuclear factor-κB receptor activator. It is a type II transmembrane protein and exists in the form of a type II membrane protein or a soluble form released by extracellular protease hydrolysis.

[0004] The specific binding of RANK and RANKL, and the signal pathway mediated by them plays a core role in many physiological and pathological processes. Under normal physiological conditions, the binding of RANK and RANKL is crucial for the differentiation, maturation of osteoclasts, and the fine regulation of bone remodeling, precisely regulating and maintaining the dynamic balance of bone metabolism. For example, during the growth and development stage of children's bones, this pathway coordinates the construction and remodeling of bone tissue to ensure normal bone growth and morphological shaping.

[0005] However, when this pathway is abnormally activated, a series of serious bone-related diseases will be triggered. In osteoporosis, the expression of RANKL is significantly up-regulated, over-stimulating the activity of osteoclasts, resulting in bone resorption far exceeding bone formation, causing a sharp loss of bone mass, damage to the microstructure of bone tissue, and greatly increasing the risk of fractures. According to statistics, about 200 million women worldwide are affected by osteoporosis, and the incidence rate increases significantly with age. In addition, during the bone metastasis process of various malignant tumors, tumor cells secrete a large amount of RANKL, inducing the over-activation of osteoclasts, destroying bone tissue, and at the same time, the released bone matrix growth factors further promote the proliferation and migration of tumor cells, forming a vicious cycle. For solid tumors such as breast cancer and prostate cancer, about 70% of advanced patients will develop bone metastasis, seriously affecting the quality of life and prognosis of patients.

[0006] At present, the main treatment methods targeting the RANK / RANKL pathway are monoclonal antibody drugs, such as the marketed Denosumab. Although such drugs have achieved certain curative effects in clinical applications, can effectively inhibit the activity of RANKL, and reduce bone resorption, there are also many limitations. First of all, monoclonal antibody drugs have a large molecular weight, are difficult to penetrate tissue barriers, and the drug concentration reaching the lesion site is limited, affecting the treatment effect. Their production cost is high, making it difficult for many patients to afford in the long term, which limits their wide clinical application. Secondly, the administration methods of monoclonal antibodies are usually subcutaneous or intravenous injections, which are inconvenient to use and require professional medical staff to operate, increasing the medical burden on patients. In addition, as exogenous proteins, monoclonal antibodies have potential immunogenicity and may trigger immune responses in the body, leading to serious adverse reactions, such as allergic reactions and infusion reactions, and some patients have to interrupt treatment due to intolerance.

[0007] In view of the deficiencies of existing treatment methods, there is an urgent clinical need to develop a class of polypeptide drugs that can efficiently inhibit the binding of RANK and RANKL. Polypeptide drugs have advantages such as relatively small molecular weight, good tissue penetration, strong specificity, and low immunogenicity, and are expected to overcome the disadvantages of monoclonal antibody drugs and provide a safer, more effective, and more economical treatment plan for the treatment of bone-related diseases.

[0008] Therefore, developing a new type of drug that can specifically inhibit the binding of RANK and RANKL has important clinical significance and market value. Summary of the Invention

[0009] The object of the present invention is to provide a drug polypeptide that can inhibit the binding of RANK and RANKL, and this drug polypeptide has a high affinity for RANK and / or RANKL.

[0010] To achieve the above object, the first aspect of the present invention provides a polypeptide that inhibits the binding of RANK and RANKL or a pharmaceutically acceptable salt thereof, and this polypeptide is a polypeptide having an amino acid sequence shown in any one of SEQ ID NO: 1-3 or a variant thereof.

[0011] The second aspect of the present invention provides a gene, and the nucleotide sequence of this gene is a nucleotide sequence capable of encoding the amino acid sequence of the polypeptide that inhibits the binding of RANK and RANKL described in the first aspect above.

[0012] The third aspect of the present invention provides a vector, and this vector contains the gene described in the second aspect above.

[0013] The fourth aspect of the present invention provides a host cell, and this host cell contains the vector described in the third aspect above.

[0014] The fifth aspect of the present invention provides a pharmaceutical composition, which contains a therapeutically effective amount of the polypeptide that inhibits the binding of RANK to RANKL described in the first aspect above or a pharmaceutically acceptable salt thereof.

[0015] The sixth aspect of the present invention provides the use of at least one of the polypeptide that inhibits the binding of RANK 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, the host cell described in the fourth aspect above, and the pharmaceutical composition described in the fifth aspect above in the preparation of a drug for preventing and / or treating diseases related to the signaling pathway associated with the binding of RANK to RANKL.

[0016] The polypeptide provided by the present invention is obtained by screening, and it can effectively inhibit the binding of RANK to RANKL, thereby being able to prevent and / or treat diseases related to its signaling pathway.

[0017] Other features and advantages of the present invention will be described in detail in the following specific implementation section. Brief Description of the Drawings

[0018] Figure 1 It is a diagram of the ELISA result of the polypeptide with the amino acid sequence shown in SEQ ID NO: 1 - 3 provided by the present invention inhibiting the binding of RANK to RANKL;

[0019] Figure 2 It is a diagram of the ELISA result of the polypeptide containing the amino acid sequence shown in SEQ ID NO: 4 - 11 provided by the present invention inhibiting the binding of RANK to RANKL. Detailed Description of the Invention

[0020] As disclosed herein, the endpoints and any values within the disclosed ranges are not limited to the exact ranges or values. 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 individual point values, and between individual 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.

[0021] 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.

[0022] As described above, the first aspect of the present invention provides a polypeptide that inhibits the binding of RANK to RANKL or a pharmaceutically acceptable salt thereof, and the polypeptide is a polypeptide with the amino acid sequence shown in any one of SEQ ID NO: 1 - 3 or a variant thereof.

[0023] In the present invention, the variant includes a polypeptide having an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 1-3; it also includes a polypeptide obtained by linking a tag protein or modification to the amino terminus and / or carboxyl terminus of the amino acid sequence shown in any one of SEQ ID NOs: 1-3; and further includes a polypeptide having the same function obtained by substitution, deletion, and / or addition to the amino acid sequence shown in any one of SEQ ID NOs: 1-3.

[0024] In the present invention, the "sequence identity" refers to the degree to which two sequences (such as amino acids) have the same residues at the same positions after alignment. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" means that the amino acid sequence has X% identity to SEQ ID NO: Y, and it is described that X% of the residues in the amino acid sequence are the same as the residues in the sequence disclosed in SEQ ID NO: Y. Generally, such calculations are performed using computer programs. Exemplary computer programs for comparing and aligning sequence pairs can be ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984). In addition, when determining the degree of sequence identity between two amino acid sequences, those skilled in the art can consider "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, and which has little or no effect on the function, activity, or other biological properties of the polypeptide. Such "conservative" amino acids can be amino acids known in the art.

[0025] It should be noted that the present invention does not particularly limit the type of the tag protein, and those skilled in the art can select it according to the known technical means in the art. The tag protein does not affect the activity of the polypeptide provided by the present invention. In actual application, those skilled in the art can choose whether to link the tag protein to the amino terminus and / or carboxyl terminus of the amino acid sequence according to the need.

[0026] In the present invention, the modification includes amination modification, hydroxylation modification, carboxylation modification, carbonylation modification, amidation modification, alkylation modification, phosphorylation modification, glycosylation modification, cyclization modification, biotinylation modification, acetylation modification, esterification modification, fluorescent group modification, polyethylene glycol modification, and immobilization modification.

[0027] The amino acid sequence shown in any one of SEQ ID NO: 1-3 in the present invention is a polypeptide detected by high-throughput screening technology from a polypeptide library and having the effect of inhibiting the binding of RANK to RANKL. The discovery steps of this polypeptide include: dissolving and diluting the polypeptide library to obtain a mixed solution; then successively mixing the mixed solution with reactant I containing RANKL and its label, reactant II containing RANK, and reactant III capable of undergoing a color reaction with the label to obtain a detection solution; finally, using an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance of the detection solution at a specific wavelength, and obtaining the target polypeptide according to the inhibition rate calculated from the absorbance.

[0028] It should be noted that the present invention has no particular limitation on the methods of dissolution and dilution. Those skilled in the art can choose according to the known technical means in the art. An exemplary preferred specific implementation manner is provided in the following text of the present invention, and those skilled in the art should not understand it as a limitation of the present invention.

[0029] In the present invention, the polypeptide library is a polypeptide library containing nearly 73,000 80-amino acid polypeptides constructed by Hunan Zhongcheng Peptide Biotechnology Co., Ltd. using the PICT (Peptide Information Compression Technology) technology. The specific construction method can be specifically referred to CN107849737A and CN111727194A.

[0030] It should be noted that the present invention has no particular limitation on the synthesis method of the polypeptide. Those skilled in the art can choose according to the known technical means in the art. Exemplarily, a preferred polypeptide synthesis method is provided in the following text of the present invention, and those skilled in the art should not understand it as a limitation of the present invention.

[0031] Preferably, the polypeptide further includes a polypeptide having an amino acid sequence with 20-40 amino acids disassembled from the amino acid sequence shown in any one of SEQ ID NO: 1-3.

[0032] More preferably, the disassembled polypeptide is a polypeptide having an amino acid sequence shown in any one of SEQ ID NO: 4-11.

[0033] According to a particularly preferred embodiment, the first amino acid and the last amino acid in the amino acid sequence of the polypeptide form a ring through a peptide bond.

[0034] Preferably, the pharmaceutically acceptable salt is selected from at least one of trifluoroacetate, acetate, hydrochloride, and phosphate.

[0035] In the present invention, the amino acid sequences shown in SEQ ID NO: 1 - SEQ ID NO: 11 are shown in Table 1.

[0036] Table 1

[0037]

[0038] As described above, the second aspect of the present invention provides a gene, and the nucleotide sequence of this gene is a nucleotide sequence capable of encoding the amino acid sequence of the polypeptide that inhibits the binding of RANK to RANKL described in the first aspect above.

[0039] As described above, the third aspect of the present invention provides a vector, and this vector contains the gene described in the second aspect above.

[0040] As described above, the fourth aspect of the present invention provides a host cell, and this host cell contains the vector described in the third aspect above.

[0041] As described above, the fifth aspect of the present invention provides a pharmaceutical composition, and this pharmaceutical composition contains a therapeutically effective amount of the polypeptide that inhibits the binding of RANK to RANKL described in the first aspect above or a pharmaceutically acceptable salt thereof.

[0042] Preferably, the composition further contains an excipient.

[0043] It should be noted that the present invention does not particularly limit the type of the excipient, and those skilled in the art can select according to the known technical means in the art, as long as the polypeptide provided in the present invention can target and act on RANK and / or RANKL.

[0044] As described above, the sixth aspect of the present invention provides the use of at least one of the polypeptide that inhibits the binding of RANK 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, the host cell described in the fourth aspect above, and the pharmaceutical composition described in the fifth aspect above in the preparation of a drug for preventing and / or treating diseases related to the signaling pathway associated with the binding of RANK to RANKL.

[0045] Preferably, the diseases related to the signaling pathway associated with the binding of RANK to RANKL are selected from at least one of anaphylactic shock, skin allergic reaction, respiratory allergic reaction, digestive tract allergic reaction, pathological cardiac remodeling and dysfunction.

[0046] More preferably, the diseases related to the signaling pathway associated with the binding of RANK to RANKL are selected from at least one of osteoporosis, bone cancer, osteolytic tumor, rheumatoid arthritis.

[0047] Further 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 metastatic carcinoma, giant cell tumor of bone, and multiple myeloma.

[0048] Particularly preferably, the bone metastatic carcinoma includes breast cancer, lung cancer, and prostate cancer.

[0049] The present invention will be described in detail below by way of examples.

[0050] In the following examples, unless otherwise specified, the raw materials used are commercially available.

[0051] In the following examples, unless otherwise specified, the room temperature means 25 ± 5°C.

[0052] Experimental reagents:

[0053] Reagent 1: Biotinylated Human TNFSF11 / RANKL Protein, His, AvitagTM, activetrimer (purchased from Beijing Protein Innovation Co., Ltd.);

[0054] Reagent 2: Human RANK / TNFSF11A Protein, Mouse IgG2a Fc Tag (purchased from Beijing Protein Innovation Co., Ltd.);

[0055] Reagent 3: Streptavidin-Eu (purchased from AAT Bioquest);

[0056] Reagent 4: Goat Anti-mouse IgG Fc-Alexa 647 (purchased from Jackson ImmunoResearch Laboratories);

[0057] Reagent 5: Streptavidin, HRP Conjugated (purchased from Thermo).

[0058] Example 1: TR-FRET screening of polypeptides

[0059] Dissolution of the polypeptide library: Place the polypeptides in the polypeptide library in a 96-well deep-well plate, centrifuge at 4000 rpm for 3 min, then add 200 μL / well of ultrapure water (polypeptide concentration is 50 μM); then seal with a silica gel lid and heat in a water bath at 95°C for 5 min and then centrifuge at 4000 rpm for 3 min;

[0060] Dilution of the polypeptide library: The polypeptides after the above centrifugation were transferred to a 384-well plate using a workstation, and the polypeptide concentration was diluted to the experimental concentration (10 μM) with loading buffer (loading buffer: Tris-HCl buffer, pH = 7.4) to obtain each mixture;

[0061] Screening of polypeptides: In a 384-well plate, 4 μL / well of each of the above mixtures at 2 μM, 1 nM of reagent 1, and 1 nM of reagent 2 were added in sequence, followed by instantaneous centrifugation to remove air bubbles. Then, 8 μL / well of the premixed solution of reagent 3 and reagent 4 (mixed at a volume ratio of 1:1 at room temperature for 1 h) was added, followed by instantaneous centrifugation to remove air bubbles; then incubated in the dark at room temperature for 2 h. Detection was performed using a microplate reader (model cytation5), with an excitation light of 320 nm, and the fluorescence signal values of each well were detected at two wavelengths of 665 nm and 620 nm. After calculating the inhibition rate of each polypeptide concentration according to the formula, the IC50 values of each polypeptide were calculated by plotting using GraphPad prism software, and 3 target polypeptides were screened, namely polypeptides with amino acid sequences as shown in SEQ ID NO: 1-3; among them, the formula for calculating the inhibition rate is:

[0062] Inhibition% = (1 - (signal value of the sample - signal value of the background) / (signal value of the positive - signal value of the background)) × 100%; where

[0063] Sample: Refers to adding a polypeptide to the reaction system;

[0064] Positive: Refers to not adding a polypeptide to the reaction system;

[0065] Background: Refers to not adding a polypeptide, reagent I, and reagent II to the reaction system;

[0066] Signal value: Refers to the ratio of the fluorescence signal value of each well at 665 nm to the fluorescence signal value of the corresponding well at 620 nm.

[0067] Example 2: Synthesis of polypeptides

[0068] Polypeptides with amino acid sequences containing 5 - 80 amino acids were designed based on the 3 target polypeptides obtained in Example 1 and synthesized according to the following method:

[0069] Step 1: Gene construction

[0070] The DNA sequences of the target polypeptides were designed as overlapping oligonucleotide primers and synthesized. The full-length target DNA sequences were obtained by PCR reaction, and the sequences were ligated into the expression vector pET15b-sumo for fusion expression with an intein. Then, the aforementioned vector was transferred into Escherichia coli for culture, the plasmid was extracted and sequenced, and the sequencing results were analyzed to ensure consistency with the designed target sequence.

[0071] Step 2: Polypeptide Expression

[0072] The correctly constructed bacterial solution above was inoculated into the self-inducing medium. After adding 1 / 1000 of ampicillin (stock solution concentration: 100 mg / mL), it was cultured overnight with shaking at 37 °C and 200 rpm for induced expression.

[0073] Step 3: Polypeptide Purification

[0074] The overnight-induced bacterial solution was poured into urea and dissolved in a water bath at 60 °C for 5 min. Then 6 M NaOH was added for ultrasonic disruption (disrupting for 3 s, interval of 8 s, time for 3 min, power 60%). Then 6 M HCl was added for neutralization, and then it was incubated with 25 mL of nickel magnetic beads for 1 h. After strongly magnetically adsorbing and removing the supernatant, it was resuspended, washed 3 times, and then eluted with 500 mM imidazole. The obtained eluate was self-cleaved overnight at 35 °C. The cleavage solution was loaded onto a pre-equilibrated C18 reverse-phase column (600 mg), washed with 5 wt% acetonitrile + 1 wt% formic acid, and then eluted with 50 wt% acetonitrile. The obtained eluate was detected by SDS-PAGE electrophoresis. The molecular weight of the polypeptide was detected by mass spectrometry, and the purity of the polypeptide was detected by HPLC.

[0075] Example 3: Further ELISA Screening of Polypeptides

[0076] Reagent 2 was diluted to 0.125 μg / mL with coating buffer (0.05 M carbonate buffer (pH = 9.6)) and then added to a 384-well plate at a volume of 25 μL / well and coated overnight at 4 °C. Then it was washed 4 times with washing buffer (a mixture of Tween-20 and TBS buffer (pH = 7.4) prepared according to a mass-to-volume ratio of 0.05%). Then blocking solution (a mixture of BSA and TBS buffer (pH = 7.4) prepared according to a mass-to-volume ratio of 2%) was added for blocking at 37 °C. After washing 4 times with the above washing buffer, the polypeptides to be tested diluted in gradients, a mixture of reagent 1 and reagent 5 at 0.06 μg / mL mixed at a volume ratio of 1:1 were added in sequence. After incubating at 37 °C for 1 h, the unbound part was washed away with the above washing buffer, and then 25 μL of TMB (manufacturer: Solarbio, catalog number: PR1210) was added to each well and incubated at 37 °C for 30 min for color development. Finally, 25 μL of termination solution (1 M HCl) was added to each well to terminate the reaction. The absorbance of each well at 450 nm was read using a microplate reader (model: cytation5). After calculating the inhibition rate of each polypeptide concentration according to the formula, the IC50 values of each polypeptide were calculated by plotting with GraphPad prism software, and 8 target polypeptides were screened, namely polypeptides with amino acid sequences as shown in SEQ ID NO: 4 - 11; among them, the formula for calculating the inhibition rate is:

[0077] Inhibition% = (1 - (Absorbance of sample - Absorbance of background) / (Absorbance of positive - Absorbance of background)) × 100%; where,

[0078] Sample: refers to adding the polypeptide into the reaction system;

[0079] Positive: refers to not adding the polypeptide into the reaction system;

[0080] Background: refers to not adding the polypeptide and Reagent II into the reaction system.

[0081] Table 2: IC50 values of some polypeptides

[0082]

[0083] Figure 1 and Figure 2 is the ELISA result graph showing that the polypeptides with the amino acid sequences shown in SEQ ID NO: 1 - 11 provided by the present invention inhibit the binding of RANK and RANKL. Among them, Concentration in the graph represents the concentration of the polypeptide, and Inhibition represents the inhibition rate. The IC50 values of each polypeptide can be calculated from the graph, and some results are shown in Table 2.

[0084] It can be seen from the results in Table 2 that the polypeptides provided by the present invention can effectively inhibit the binding of RANK and RANKL.

[0085] 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 inhibits the binding of RANK 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: 1, 4-9.

2. The polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The first amino acid and the last amino acid in the amino acid sequence of the polypeptide form a ring through a peptide bond.

3. A gene, characterized in that, The nucleotide sequence of the gene is a nucleotide sequence capable of encoding the amino acid sequence of the polypeptide that inhibits the binding of RANK to RANKL as described in claim 1 or 2.

4. A carrier, characterized in that, The vector contains the gene as described in claim 3.

5. A host cell, characterized in that, The host cell contains the vector as described in claim 4.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains a therapeutically effective amount of the polypeptide that inhibits the binding of RANK to RANKL as described in claim 1 or 2 or a pharmaceutically acceptable salt thereof.

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