Polypeptide with binding affinity to IL-6 (interleukin-6) and application thereof

Abstract

The invention discloses a polypeptide with binding affinity to IL-6 (interleukin-6) and application of the polypeptide. The amino acid sequence of the polypeptide comprises one or more of the sequences as shown in SEQ ID NO.1-6. The polypeptide compatible with IL-6 is obtained through screening, the polypeptide can be specifically compatible with IL-6, IL-6 is enriched, then IL-6 is adsorbed at an inflammation infection part, more choices are provided for absorption and removal of inflammatory factors and treatment of diseases related to too high IL-6, and the polypeptide can be used for IL-6 detection and preparation of drugs for targeted combination of IL-6 or treatment of Il-6 related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a polypeptide having binding affinity to IL-6 and uses thereof. Background Art

[0002] Phage display technology is a method of inserting DNA sequences of foreign peptides or proteins into the phage genome through genetic engineering to display these molecules on the phage surface. By constructing a phage library of multiple random peptide sequences, specific targets can be screened to study the interaction between peptides and targets. This technology is widely used in the field of biology to explore the interaction between peptides and molecules such as proteins and DNA, as well as the targeting of peptides to specific tissues or organs.

[0003] Interleukin-6 (IL-6) is a multifunctional cytokine with both pro-inflammatory and anti-inflammatory properties. IL-6 plays an important role in cytokine storms. When the body is severely infected or subjected to other severe stimulation, immune cells release a large amount of IL-6, leading to an uncontrolled inflammatory response and a large release of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), etc., thereby inducing a cytokine storm. Cytokine storms may lead to severe complications such as severe multiple organ dysfunction syndrome and shock, and may even be life-threatening. Therefore, controlling the occurrence of cytokine storms is of great significance for the treatment of infectious diseases and other inflammatory diseases, and IL-6, as one of the key regulatory factors, has become one of the important targets of related treatment strategies.

[0004] At present, biotechnology drugs targeting the IL-6 signaling pathway have become a hot spot for research and development. In the international market, the FDA has approved three drugs for marketing, namely Tocilizumab (trade name: Actemra / RoActemra), Siltuximab (trade name: Sylvant), and Sarilumab (trade name: Kevzara). In China, only Tocilizumab is on the market. Summary of the invention

[0005] The present invention provides a polypeptide having binding affinity to IL-6 and uses thereof.

[0006] The specific technical solutions are as follows: The first aspect of the present invention protects a polypeptide having binding affinity to IL-6, wherein the amino acid sequence of the polypeptide comprises one or more sequences shown in SEQ ID NO. 1-6. The second aspect of the present invention protects a fusion protein comprising a polypeptide as described above and a portion fused to the polypeptide; the portion is selected from any one of the following: an antigen, an antibody or its antigen-binding portion, a ligand, a receptor, a cytokine, a transcriptional regulator, a fluorescent protein and an enzyme.

[0007] The third aspect of the present invention protects a conjugate comprising a polypeptide as described above and a portion conjugated to the polypeptide or a portion non-covalently linked to the polypeptide, wherein the portion is selected from any one of the following: proteins, peptides, nucleic acids, antibiotics, anti-inflammatory drugs, anti-tumor drugs, neuroprotectants, chemotherapeutic agents, cytotoxins, radioactive isotopes, fluorescent markers, luminescent materials, color-developing substances and enzymes.

[0008] The fourth aspect of the present invention protects an isolated polynucleotide encoding the polypeptide as described above or the fusion protein as described above.

[0009] The fifth aspect of the present invention protects a recombinant vector comprising the polynucleotide as described above.

[0010] The sixth aspect of the present invention protects a host cell, comprising the recombinant vector as described above, or a host cell in which the polynucleotide as described above is integrated into its genome.

[0011] The seventh aspect of the present invention protects the use of the polypeptide as described above, or the fusion protein as described above, or the conjugate as described above, or the polynucleotide as described above, or the recombinant vector as described above, or the host cell as described above in at least one of the following: 1) For preparing drugs that target and bind to IL-6; 2) Used to detect IL-6; 3) Used to prepare a detection reagent for detecting IL-6; 4) Used for preparing or screening drugs for treating IL-6 related diseases.

[0012] The eighth aspect of the present invention protects a pharmaceutical composition comprising the polypeptide as described above or the fusion protein as described above, and a pharmaceutically acceptable carrier.

[0013] The ninth aspect of the present invention protects a detection reagent or a detection kit, comprising the polypeptide as described above, or the fusion protein as described above, or the conjugate as described above, or the pharmaceutical composition as described above.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses IL-6 protein as a target to obtain a polypeptide with specific affinity to IL-6. In view of the shortcomings of existing biological preparations such as limited efficacy and obvious side effects, the polypeptide involved in the present invention has the ability to specifically affinity to IL-6 protein without enrichment in other parts, thereby improving the therapeutic effect and reducing side effects, and providing a new option for reducing excessive IL-6 in the body and treating diseases related to IL-6 deficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a logarithmic result graph of the output-to-input ratio in each round of screening during the four-round screening of IL-6 protein using the Ph.D.-12TM phage polypeptide library in Example 1.

[0016] Figure 2 These are a series of sequences with higher frequencies obtained after analyzing and translating the DNA sequencing results in Example 2.

[0017] Figure 3 The affinity of phages displaying 6 different polypeptide sequences to IL-6 protein was tested by enzyme-linked immunosorbent assay in Example 3. DETAILED DESCRIPTION

[0018] The present invention uses Ph.D.-12TM phage polypeptide library to screen IL-6 protein, obtains 6 phages that can specifically bind to IL-6 protein; then DNA sequencing is performed on the phages that can specifically bind to IL-6 protein to obtain the amino acid sequence of the polypeptide; enzyme-linked immunosorbent assay shows that the binding affinity of the 6 polypeptides to IL-6 protein is higher than that of the wild-type phage and the PBS negative control group. On this basis, the present invention is completed.

[0019] The first aspect of the present invention protects a polypeptide having binding affinity to IL-6, wherein the amino acid sequence of the polypeptide comprises one or more sequences shown in SEQ ID NO. 1-6.

[0020] DGLHWNPRLWYR (SEQ ID NO.1) GTNWSIHENNMG (SEQ ID NO. 2) TSGTMQTNPLPV (SEQ ID NO. 3) TIPNLTRVSNIV (SEQ ID NO. 4) SWSNASDYHIGA (SEQ ID NO. 5) YSSGFDQMPALP (SEQ ID NO. 6) The second aspect of the present invention protects a fusion protein comprising a polypeptide as described above and a portion fused to the polypeptide; the portion is selected from any one of the following: an antigen, an antibody or its antigen-binding portion, a ligand, a receptor, a cytokine, a transcriptional regulator, a fluorescent protein and an enzyme.

[0021] The third aspect of the present invention protects a conjugate, comprising a polypeptide as described above and a part conjugated to the polypeptide or a part non-covalently linked to the polypeptide, wherein the part is selected from any one of the following: proteins, peptides, nucleic acids, antibiotics, anti-inflammatory drugs, anti-tumor drugs, neuroprotective agents, chemotherapeutic agents, cytotoxins, radioisotopes, fluorescent markers, luminescent substances, color-developing substances and enzymes. Compounds covalently linked to the polypeptide; polymer mixtures linked to the polypeptide.

[0022] The fourth aspect of the present invention protects an isolated polynucleotide encoding the polypeptide as described above or the fusion protein as described above.

[0023] The fifth aspect of the present invention protects a recombinant vector comprising the polynucleotide as described above.

[0024] In certain embodiments, the recombinant vector can be constructed by inserting the polynucleotide described above into the multiple cloning site of an expression vector. The recombinant vector can be transformed, transduced or transfected into a host cell so that the genetic material elements it carries can be expressed in the host cell.

[0025] In certain embodiments, the recombinant vector is a viral vector or a non-viral vector. For example, non-viral vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses, etc. Viral vectors include: retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). The vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site. The vector may also include components that assist it in entering the cell, including, but not limited to, viral particles, liposomes or protein shells.

[0026] The sixth aspect of the present invention protects a host cell, comprising the recombinant vector as described above, or a host cell in which the polynucleotide as described above is integrated into its genome.

[0027] In certain embodiments, the host cell can be a prokaryotic cell, such as a bacterial cell, etc.; or a lower eukaryotic cell, such as a yeast cell, etc.; or a higher eukaryotic cell, such as a mammalian cell, etc.

[0028] In certain embodiments, the host cell includes many cell types, such as prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0029] The seventh aspect of the present invention protects the use of the polypeptide as described above, or the fusion protein as described above, or the conjugate as described above, or the polynucleotide as described above, or the recombinant vector as described above, or the host cell as described above in at least one of the following: 1) For preparing drugs that target and bind to IL-6; 2) Used to detect IL-6; 3) Used to prepare a detection reagent for detecting IL-6; 4) Used for preparing or screening drugs for treating IL-6 related diseases.

[0030] In certain embodiments, the IL-6-related disease is selected from an inflammatory disease, an autoimmune disease, and cancer.

[0031] In certain specific embodiments, the inflammatory disease is selected from rheumatoid arthritis, polyarticular juvenile idiopathic arthritis (pJIA), systemic juvenile idiopathic arthritis (sJIA), giant cell arteritis (GCA), cytokine release syndrome (CRS), Castleman's disease, Takayasu arteritis In certain specific embodiments, the autoimmune disease is selected from systemic lupus erythematosus and psoriasis.

[0032] The polypeptide of the present invention has a high affinity for IL-6 protein, can specifically recruit IL-6, adsorb excess IL-6, and then inhibit and eliminate inflammation. It has broad application prospects in the treatment of various inflammatory diseases, such as arthritis, cytokine release syndrome, etc.

[0033] In certain embodiments, the method for detecting IL-6 comprises: contacting a test sample with the polypeptide described above or the conjugate or composition described above, and determining the presence or amount of IL-6 in the test sample.

[0034] The eighth aspect of the present invention protects a pharmaceutical composition comprising the polypeptide as described above or the fusion protein as described above, and a pharmaceutically acceptable carrier.

[0035] The ninth aspect of the present invention protects a detection reagent or a detection kit, comprising the polypeptide as described above, or the fusion protein as described above, or the conjugate as described above, or the pharmaceutical composition as described above.

[0036] In certain embodiments, the detection reagent may also include a polypeptide marker, and the types of markers that can be selected include, but are not limited to, a combination of one or more of a fluorescent marker, a radioactive marker, an enzyme-labeled marker, a chemiluminescent marker, etc. According to the detection principle of the kit, the kit may also generally include one or more reagents required for detection. In addition, the kit may also include, as required, a container, a control (negative or positive control), a buffer, an adjuvant, etc., which may be selected by a person skilled in the art according to the specific circumstances.

[0037] In certain embodiments of the present invention, the kit is an ELISA, Western blot, immunofluorescence, immunoprecipitation, immunohistochemistry, flow cytometry or in vivo immune imaging kit.

[0038] Another aspect of the present invention also provides a method for preparing the polypeptide, comprising the following steps: culturing the host cell described above under conditions suitable for expressing the polypeptide to express the polypeptide, and purifying and isolating the polypeptide.

[0039] The advantages of the polypeptide of the present invention are: (1) the sequence is short, easy to prepare, and the synthetic production cost is low; (2) the structure of the polypeptide fragment is stable, which reduces the cost and difficulty of transportation and storage; (3) it can be administered in a variety of ways. It can be used for IL-6 detection and can provide a new idea for the preparation of drugs that target IL-6 or treat IL-6 related diseases. The present invention is further described below in conjunction with specific embodiments. The following are only specific embodiments of the present invention, but the protection scope of the present invention is not limited to them.

[0040] Example 1: Four rounds of screening were performed on IL-6 protein using the Ph.D. 12TM phage polypeptide library.

[0041] 1) Coating with IL-6 protein: Add 150 μL of 4 μg / mL IL-6 protein solution into a 24-well plate, place the plate in a wet box, and incubate on a shaker at 4°C overnight; 2) Blocking: Aspirate the liquid in the plate, turn the plate upside down on a sterile paper towel and shake it vigorously to remove the residual solution. Then fill the well plate with 5% BSA blocking solution and shake it gently at room temperature for 2 hours; 3) Background removal: Take a new 24-well plate, add Ph.D. 12TM phage peptide library, and incubate in a shaker at room temperature for 1 hour; 4) Washing: Remove the blocking solution by aspirating, invert the plate on a sterile paper towel and shake vigorously to remove the residual solution, then quickly wash the plate 6 times with PBST buffer, rotating each time to ensure that the bottom and edges of the plate are washed. Pour out the buffer, invert the plate on a sterile paper towel and shake vigorously to remove the residual solution; 5) Binding: Add the phage solution in step 3) to the well plate treated in step 4) and incubate on a shaker for 1 hour; 6) Washing: Pour off unbound phages, invert the plate on a sterile paper towel and shake vigorously to remove residual solution. Wash the plate 10 times with PBST buffer according to the method described in step 4), gently shake it on a shaker for 10 minutes each time, and shake it with a clean paper towel after each wash to avoid cross contamination; 7) Elution: first add 100 μL of elution solution, shake gently at room temperature for 8 minutes, then add 15 μL of neutralization solution, and collect the mixed solution into an EP tube; 8) Plate coating: Preheat the prepared LB / IPTG / Xgal plate in a 37°C incubator in advance. Dilute a portion of the phage solution collected in step 7) to an appropriate multiple, take 10 μL of the diluted phage solution and incubate with 200 μL of activated E.coil and ER2738 bacterial solution for 15 minutes. Transfer the incubated phage / bacteria mixture to the LB / IPTG / Xgal plate, spin coat evenly with a disposable sterile coating rod, and culture in a 37°C incubator overnight. Count the number of phage blue plaques the next day; 9) Amplify to form a sub-library: Add the remaining phage solution in step 7) to 20 mL of E.coil and ER2738 bacterial solution in the logarithmic growth phase, incubate for 20 minutes, and then place in a shaker at 37°C and shake at 220 rpm for 4.5 hours. After centrifuging and sterilizing the phage / bacteria mixture, add one-fifth volume of PEG / NaCl solution to the supernatant and let it settle overnight at 4°C. The next day, retain the precipitate after centrifugation and resuspend the precipitate with sterile PBS to complete the purification of the phage. Repeat the purification process twice, and the formed phage sub-library is used for the next round of amplification; 10) The above steps 1) to 9) were repeated 4 times to ensure that the amount of phage input in each round was consistent to complete the screening process.

[0042] Input-output test: The number of phage blue plaques counted in step 8) is divided by the number of phages input to obtain the input-output ratio of each round of screening.

[0043] The number of phage blue plaques usually refers to the number of phage plaques formed on bacterial colonies after binding to a specific target in phage display technology. This number can reflect the binding ability of the phage-displayed peptide or protein to the target. In the screening process, the affinity and specificity of the phage-displayed peptide or protein can be evaluated by counting the number of phage blue plaques. The input-output ratio can reflect the screening efficiency. The higher the ratio, the higher the screening efficiency, and the more effectively the phages that bind to the target can be screened.

[0044] Taking the logarithm of the input-output ratio makes the column form Figure 1 .

[0045] from Figure 1 It can be seen that the input-output ratio of each round increases, indicating that phages with specific affinity to IL-6 protein are significantly enriched.

[0046] Example 2 DNA sequencing is performed on the phage obtained in Example 1 that has specific affinity with the IL-6 protein to obtain a polypeptide that specifically binds to IL-6. The method comprises the following steps: 1) Preservation and amplification of phages with specific affinity for IL-6: Randomly pick 80 blue spots from the plate coated with the fourth round of screening and add them to a shaking tube containing LB medium for amplification. After 24 hours, take a portion of the bacterial solution and store the strain in a -80℃ refrigerator, and the remaining bacterial solution is used for DNA sequencing; After DNA sequencing, the phage that has specific affinity with IL-6 protein has the following sequence: DGLHWNPRLWYR (SEQ ID NO.1) GTNWSIHENNMG (SEQ ID NO. 2) TSGTMQTNPLPV (SEQ ID NO. 3) TIPNLTRVSNIV (SEQ ID NO. 4) SWSNASDYHIGA (SEQ ID NO. 5) YSSGFDQMPALP (SEQ ID NO. 6) 2) Analysis of sequencing results: In the DNA sequencing results, find the inserted foreign gene sequence, translate the codons into amino acid sequences, and count the repeated polypeptide sequences and their frequencies. The results are as follows: Figure 2 shown.

[0047] from Figure 2It can be seen that the frequencies of occurrence of phages of the six sequences are different, among which the DGLHWNPRLWYR sequence has the highest frequency, followed by the GTNWSIHENNMG sequence, TSGTMQTNPLPV sequence, TIPNLTRVSNIV sequence, SWSNASDYHIGA sequence and YSSGFDQMPALP sequence. This shows that the enrichment degree of phages of each sequence is different during the screening process, and the DGLHWNPRLWYR sequence has the strongest enrichment ability.

[0048] Example 3 In this example, the IL-6 affinity of the screened phages was tested by enzyme-linked immunosorbent assay.

[0049] 1) Amplification and purification of IL-6 affinity phage: After the five phage samples and wild-type phage samples preserved in each round of screening in Example 1 were melted at room temperature, 200 μL were taken and added to 20 mL of E.coil and ER2738 bacterial solution in the logarithmic growth phase. After incubation for 20 minutes, it was placed in a shaker at 37°C and shaken at 220 rpm for 4.5 hours. After the phage / bacteria mixture was centrifuged and sterilized, one-fifth volume of PEG / NaCl solution was added to the supernatant and precipitated overnight at 4°C. The next day, the precipitate was retained after centrifugation and resuspended with sterile PBS to complete the purification of the phage. The purification process was repeated twice, and the obtained phage solution was used for IL-6 affinity verification.

[0050] 2) Coating with IL-6 protein: Add 50 μL of 4 μg / mL IL-6 protein into a 96-well ELISA plate, place the ELISA plate in a wet box, and incubate on a shaker at 4°C overnight; 3) Blocking: Aspirate the liquid in the well plate, turn the ELISA plate upside down on a sterile paper towel and shake it vigorously to remove the residual solution. Then add 200 μL of 5% BSA blocking solution to the well plate and incubate for 1 hour; 4) Washing: Discard the blocking solution, invert the ELISA plate on a sterile paper towel and shake it vigorously to remove the residual solution. Add 200 μL PBST buffer and wash the plate 6 times. After each incubation on a shaker for 6 minutes, shake it vigorously on a sterile paper towel to remove the residual solution. 5) Binding: Add 50 μL of the phage amplified in step 1), keeping the concentration of each phage the same. Incubate at room temperature for 1 hour, and design a group with PBS and wild-type phage for control; 6) Washing: Aspirate the phage solution and repeat washing 6 times with PBST buffer according to step 4); 7) Add primary antibody: add 100 μL of anti-bacteriophage coat protein g8p antibody diluted to an appropriate multiple and incubate at room temperature for 1 hour; 8) Washing: Aspirate the primary antibody and repeat washing 3 times with PBST buffer according to step 4); 9) Add secondary antibody: add 100 μL of HRP-conjugated goat anti-mouse secondary antibody diluted to an appropriate multiple and incubate at room temperature for 1 hour; 10) Washing: Aspirate the secondary antibody and repeat washing 3 times with PBST buffer according to step 4); 11) Add TMB colorimetric solution: Add 100 μL of TMB colorimetric solution and incubate at 37°C for 10-20 minutes; 12) Add stop solution: Wait until the color turns blue after step 11), then add 100 μL of stop solution 2MH2 SO4 to terminate the reaction; 13) Measure absorbance: Measure the absorbance at 450 nm using an enzyme reader.

[0051] Meanwhile, a negative control group was set up, that is, 200 μL PBS was used to replace the phage sample in step 1) of this Example 1, and the subsequent steps were the same.

[0052] ELISA (Enzyme-Linked Immunosorbent Assay) is an experimental technique commonly used to detect the content of biological molecules such as proteins, antibodies, and antigens. ELISA technology uses the principle of enzyme labeling and antibody binding to quantitatively or qualitatively detect the presence of specific molecules in a sample. In this experiment, antibodies are used to bind to the phage coat protein g8p, and by detecting the absorbance at 450nm, the amount of phage binding to IL-6 protein that displays different polypeptide sequences can be reflected. The higher the absorbance value, the more specific polypeptide phages that bind firmly to the IL-6 protein, and the stronger the affinity of the polypeptide.

[0053] The absorbance values ​​of each group are shown in Figure 3 Among them, WT represents wild-type phage, and PBS represents negative control group.

[0054] from Figure 3It can be seen that the binding affinity of the six phages screened to IL-6 protein is higher than that of the wild-type phage and the PBS negative control group. The binding affinity of each sequence phage to IL-6 protein is different. Among them, the phage displaying the YSSGFDQMPALP sequence has a significantly stronger affinity to IL-6 protein than other phages, which is significantly different from the two control groups and is more than 6 times that of the PBS group and the wild-type phage group (****p<0.0001). The affinity to IL-6 protein is from large to small: YSSGFDQMPALP sequence, TSGTMQTNPLPV sequence, SWSNASDYHIGA sequence, DGLHWNPRLWYR sequence, GTNWSIHENNMG sequence and TIPNLTRVSNIV sequence.

Claims

1. A polypeptide having binding affinity for IL-6, characterized in that The amino acid sequence of the polypeptide comprises one or more sequences shown in SEQ ID NO.1-6.

2. A fusion protein, characterized in that: The method comprises the polypeptide according to claim 1 and a part fused to the polypeptide; the part is selected from any one of the following: an antigen, an antibody or an antigen-binding portion thereof, a ligand, a receptor, a cytokine, a transcriptional regulator, a fluorescent protein and an enzyme.

3. A conjugate, characterized in that The method comprises a polypeptide as claimed in claim 1 and a portion conjugated to the polypeptide or a portion non-covalently linked to the polypeptide, wherein the portion is selected from any one of the following: proteins, peptides, nucleic acids, antibiotics, anti-inflammatory drugs, anti-tumor drugs, neuroprotective agents, chemotherapeutic agents, cytotoxins, radioactive isotopes, fluorescent markers, luminescent substances, color-developing substances and enzymes.

4. An isolated polynucleotide, characterized in that Encodes the polypeptide according to claim 1 or the fusion protein according to claim 2.

5. A recombinant vector, characterized in that: Comprising the polynucleotide as claimed in claim 4.

6. A host cell, characterized in that The recombinant vector of claim 5 is included, or the polynucleotide of claim 4 is integrated into the genome.

7. Use of the polypeptide according to claim 1, the fusion protein according to claim 2, the conjugate according to claim 3, the polynucleotide according to claim 4, the recombinant vector according to claim 5, or the host cell according to claim 6 in at least one of the following: 1) For preparing drugs that target and bind to IL-6; 2) Used to detect IL-6; 3) Used to prepare a detection reagent for detecting IL-6; 4) Used for preparing or screening drugs for treating IL-6 related diseases.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the polypeptide according to claim 1 or the fusion protein according to claim 2; and a pharmaceutically acceptable carrier.

9. A detection reagent or a detection kit, characterized in that: Comprising the polypeptide according to claim 1, or the fusion protein according to claim 2, or the conjugate according to claim 3, or the pharmaceutical composition according to claim 8.

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