A polypeptide having binding affinity for il-6 and uses thereof

Abstract

The application discloses a polypeptide with binding affinity to IL-6 and application thereof. The amino acid sequence of the polypeptide comprises one or more of sequences shown in SEQ ID NO. 1-6. The application obtains the polypeptide with affinity to IL-6 through screening, which can specifically bind to IL-6, so that IL-6 is enriched, and then IL-6 is adsorbed at an inflammation infection site, thereby providing more choices for treatment of diseases related to removal of inflammatory factors and IL-6 overabundance, and the polypeptide can be used for IL-6 detection and preparation of drugs for targeted binding to IL-6 or treatment of diseases related to IL-6.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more particularly to a polypeptide with binding affinity for IL-6 and its uses. Background Technology

[0002] Phage display is a technique that uses genetic engineering to insert exogenous polypeptide or protein DNA sequences into the genome of a bacteriophage, thereby displaying these molecules on the phage surface. By constructing phage libraries containing multiple random polypeptide sequences, specific targets can be screened, allowing for the study of interactions between polypeptides and their targets. This technique is widely used in biology to investigate the interactions between polypeptides and molecules such as proteins and DNA, as well as the targeting of polypeptides 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 a crucial role in cytokine storms. When the body experiences severe infection or other severe stimuli, immune cells release large amounts of IL-6, leading to an uncontrolled inflammatory response and the massive release of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), thus triggering a cytokine storm. Cytokine storms can lead to severe complications such as multiple organ dysfunction syndrome and shock, and can even be life-threatening. Therefore, controlling cytokine storms is of great significance for the treatment of infectious and other inflammatory diseases, and IL-6, as one of the key regulators, has become an important target for related therapeutic strategies.

[0004] Currently, biotechnology drugs targeting the IL-6 signaling pathway have become a hot research topic. In the international market, the FDA has approved three drugs: Tocilizumab (trade names: Actemra / RoActemra), Siltuximab (trade name: Sylvant), and Sarilumab (trade name: Kevzara). In China, only Tocilizumab is available on the market. Summary of the Invention

[0005] This invention provides a polypeptide with binding affinity for IL-6 and its uses.

[0006] The specific technical solution is as follows:

[0007] The first aspect of this invention protects a polypeptide having binding affinity for IL-6, the amino acid sequence of which comprises one or more of the sequences shown in SEQ ID NO. 1-6.

[0008] A second aspect of the present invention protects a fusion protein comprising a polypeptide as described above and a portion fused to said polypeptide; said portion being selected from any of the following: antigen, antibody or antigen-binding portion thereof, ligand, receptor, cytokine, transcription factor, fluorescent protein, and enzyme.

[0009] A third aspect of the present invention protects a conjugate comprising a polypeptide as described above and a portion conjugated to or non-covalently linked to the polypeptide, wherein the portion is selected from any of the following: proteins, peptides, nucleic acids, antibiotics, anti-inflammatory drugs, antitumor drugs, neuroprotective agents, chemotherapeutic agents, cytotoxins, radioisotopes, fluorescent markers, luminescent substances, chromogenic substances, and enzymes.

[0010] A fourth aspect of this invention protects an isolated polynucleotide encoding a polypeptide or a fusion protein as described above.

[0011] A fifth aspect of this invention protects a recombinant vector comprising the polynucleotides described above.

[0012] A sixth aspect of the present invention protects a host cell comprising a recombinant vector as described above, or a genome incorporating polynucleotides as described above.

[0013] The seventh aspect of this invention protects the use of the polypeptide, fusion protein, conjugate, polynucleotide, recombinant vector, or host cell described above in at least one of the following:

[0014] 1) Used to prepare drugs that target and bind to IL-6;

[0015] 2) Used to detect IL-6;

[0016] 3) Reagents used to prepare for the detection of IL-6;

[0017] 4) Used for the preparation or screening of drugs for the treatment of IL-6-related diseases.

[0018] The eighth aspect of this invention protects a pharmaceutical composition comprising a polypeptide or a fusion protein as described above, and a pharmaceutically acceptable carrier.

[0019] The ninth aspect of this invention protects a detection reagent or detection kit comprising the polypeptide, fusion protein, conjugate, or pharmaceutical composition as described above.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention yields a polypeptide with specific affinity for IL-6, targeting the IL-6 protein. Addressing the limitations of existing biological agents, such as limited efficacy and significant side effects, the polypeptide of this invention possesses the ability to specifically bind to the IL-6 protein without accumulating in other sites, thus improving therapeutic efficacy and reducing side effects. This provides a new option for reducing excess IL-6 in the body and treating IL-6 deficiency-related diseases. Attached Figure Description

[0022] Figure 1 This is a graph showing the logarithmic results of the output and input ratio of each round of screening for IL-6 protein using the Ph.D.-12TM phage polypeptide library in Example 1.

[0023] Figure 2 These are a series of high-frequency sequences obtained after analyzing and translating the DNA sequencing results in Example 2.

[0024] Figure 3 In Example 3, an enzyme-linked immunosorbent assay (ELISA) was used to test the affinity of phages with six different polypeptide sequences for the IL-6 protein. Detailed Implementation

[0025] This invention utilizes a Ph.D.-12TM phage peptide library to screen for IL-6 protein, obtaining six phages with specific affinity for IL-6 protein. DNA sequencing was then performed on these phages to obtain the amino acid sequences of their peptides. Enzyme-linked immunosorbent assay (ELISA) revealed that the affinity of all six peptides for IL-6 protein was higher than that of wild-type phages and the PBS negative control group. Based on these findings, this invention was completed.

[0026] The first aspect of this invention protects a polypeptide having binding affinity for IL-6, the amino acid sequence of which comprises one or more of the sequences shown in SEQ ID NO. 1-6.

[0027] DGLHWNPRLWYR (SEQ ID NO.1)

[0028] GTNWSIHENNMG (SEQ ID NO.2)

[0029] TSGTMQTNPLPV (SEQ ID NO.3)

[0030] TIPNLTRVSNIV (SEQ ID NO.4)

[0031] SWSNASDYHIGA (SEQ ID NO.5)

[0032] YSSGFDQMPALP (SEQ ID NO.6)

[0033] A second aspect of the present invention protects a fusion protein comprising a polypeptide as described above and a portion fused to said polypeptide; said portion being selected from any of the following: antigen, antibody or antigen-binding portion thereof, ligand, receptor, cytokine, transcription factor, fluorescent protein, and enzyme.

[0034] A third aspect of this invention protects a conjugate comprising a polypeptide as described above and a portion conjugated to or non-covalently linked to the polypeptide, said portion being selected from any of the following: proteins, peptides, nucleic acids, antibiotics, anti-inflammatory drugs, antitumor drugs, neuroprotective agents, chemotherapeutic agents, cytotoxic agents, radioisotopes, fluorescent markers, luminescent substances, chromogenic substances, and enzymes. The conjugate may also include a compound covalently linked to the polypeptide; or a mixture of polymers linking the polypeptide.

[0035] A fourth aspect of this invention protects an isolated polynucleotide encoding a polypeptide or a fusion protein as described above.

[0036] A fifth aspect of this invention protects a recombinant vector comprising the polynucleotides described above.

[0037] In some embodiments, the recombinant vector can be constructed by inserting the polynucleotides described above into the multiple cloning site of the expression vector. The recombinant vector can be transformed, transduced, or transfected into host cells, enabling the expression of its carried genetic material elements within the host cells.

[0038] In some 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 λ phage or M13 phage, and animal viruses. Viral vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). The vector may contain various elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain a replication initiation site. The vector may also include components that facilitate its entry into the cell, including but not limited to viral particles, liposomes, or protein coats.

[0039] A sixth aspect of the present invention protects a host cell comprising a recombinant vector as described above, or a genome incorporating polynucleotides as described above.

[0040] In some embodiments, the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.

[0041] In some embodiments, the host cell includes many cell types, such as prokaryotic cells like Escherichia coli or Bacillus subtilis, fungal cells like yeast or Aspergillus, insect cells like S2 ​​Drosophila or Sf9, or animal cells like fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0042] The seventh aspect of this invention protects the use of the polypeptide, fusion protein, conjugate, polynucleotide, recombinant vector, or host cell described above in at least one of the following:

[0043] 1) Used to prepare drugs that target and bind to IL-6;

[0044] 2) Used to detect IL-6;

[0045] 3) Reagents used to prepare for the detection of IL-6;

[0046] 4) Used for the preparation or screening of drugs for the treatment of IL-6-related diseases.

[0047] In some implementations, the IL-6-related diseases are selected from inflammatory diseases, autoimmune diseases, and cancer.

[0048] In some 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, and aortitis.

[0049] In some specific implementations, the autoimmune disease is selected from systemic lupus erythematosus and psoriasis.

[0050] The polypeptide of this invention has a high affinity for IL-6 protein, specifically recruiting IL-6 and adsorbing excess IL-6, thereby inhibiting and reducing inflammation. It shows broad application prospects in the treatment of various inflammatory diseases, such as arthritis and cytokine release syndrome.

[0051] In some embodiments, the method for detecting IL-6 includes contacting a test sample with the peptide or conjugate or composition described above, and determining the presence or amount of IL-6 in the test sample.

[0052] The eighth aspect of this invention protects a pharmaceutical composition comprising a polypeptide or a fusion protein as described above, and a pharmaceutically acceptable carrier.

[0053] The ninth aspect of this invention protects a detection reagent or detection kit comprising the polypeptide, fusion protein, conjugate, or pharmaceutical composition as described above.

[0054] In some embodiments, the detection reagent may further include a peptide marker, and the types of markers that can be selected include, but are not limited to, one or more combinations of fluorescent markers, radioactive markers, enzyme-linked immunosorbent assay (ELISA) markers, and chemiluminescent markers. Depending on the detection principle of the kit, the kit may also contain one or more reagents required for the detection. Furthermore, the kit may, as needed, include: containers, controls (negative or positive controls), buffers, and auxiliary agents, which can be selected by those skilled in the art according to specific circumstances.

[0055] In some embodiments of the present invention, the kit is an ELISA, Western blot, immunofluorescence, immunoprecipitation, immunohistochemistry, flow cytometry, or in vivo immunoimaging kit.

[0056] In another aspect, the present invention also provides a method for preparing the aforementioned polypeptide, comprising the following steps: culturing the host cells described above under conditions suitable for expressing the polypeptide, thereby expressing the polypeptide, and purifying and isolating the polypeptide.

[0057] The advantages of the polypeptides of this invention are: (1) short sequence, easy to prepare, and low synthetic production cost; (2) stable polypeptide fragment structure, reducing the cost and difficulty of transportation and preservation; (3) can be administered in multiple ways. It can be used for IL-6 detection, providing a new approach for preparing drugs that target and bind to IL-6 or treat IL-6-related diseases. The invention will be further described below with reference to specific embodiments. The following examples are only specific embodiments of the invention, but the scope of protection of the invention is not limited thereto.

[0058] Example 1: Four rounds of screening were performed on IL-6 protein using the Ph.D. 12TM phage peptide library.

[0059] 1) Coating IL-6 protein: Add 150 μL of 4 μg / mL IL-6 protein solution to a 24-well plate, place the plate in a humidified chamber, and incubate overnight on a shaker at 4 °C.

[0060] 2) Blocking: Absorb the liquid from the plate, invert the plate onto a sterile paper towel and shake vigorously to remove any remaining solution. Then fill the well plate with 5% BSA blocking solution and gently shake at room temperature for 2 hours;

[0061] 3) Background removal: Take a new 24-well plate, add the Ph.D.12™ phage peptide library, and incubate in a shaker at room temperature for 1 hour;

[0062] 4) Washing: Aspirate the blocking solution, invert the plate onto a sterile paper towel and shake vigorously to remove any remaining solution. Then wash the plate rapidly 6 times with PBST buffer, rotating it each time to ensure the bottom and edges of the wells are washed. Pour off the buffer solution, invert the plate onto a sterile paper towel and shake vigorously to remove any remaining solution.

[0063] 5) Binding: Add the phage solution from step 3) to the well plate treated in step 4), and incubate on a shaker for 1 hour;

[0064] 6) Washing: Discard any unbound phages, invert the plate onto a sterile paper towel and shake vigorously to remove any remaining solution. Wash the plate 10 times with PBST buffer as described in step 4), gently shaking for 10 minutes each time, and shake with a clean paper towel after each wash to avoid cross-contamination;

[0065] 7) Elution: First add 100 μL of elution buffer, shake gently at room temperature for 8 minutes, then add 15 μL of neutralization buffer, and collect the mixture into an EP tube;

[0066] 8) Plate Spreading: Preheat the prepared LB / IPTG / Xgal plates in a 37°C incubator. Dilute a portion of the phage solution collected in step 7) to an appropriate concentration. Take 10 μL of the diluted phage solution and incubate it with 200 μL of activated E. coil and ER2738 bacterial suspension for 15 minutes. Transfer the incubated phage / bacteria mixture to LB / IPTG / Xgal plates, spread evenly with a disposable sterile spreader, and incubate overnight at 37°C. Count the number of blue phage spots the next day.

[0067] 9) Amplification to form a daughter library: Add the remaining phage solution from step 7) to 20 mL of E. coil and ER2738 bacterial culture in the logarithmic growth phase. Incubate for 20 minutes, then place in a shaker at 37°C and shake at 220 rpm for 4.5 hours. After centrifuging to remove bacteria from the phage / bacteria mixture, add one-fifth volume of PEG / NaCl solution to the supernatant and allow to settle overnight at 4°C. The next day, centrifuge again, retain the precipitate, and resuspend the precipitate in sterile PBS to complete one round of phage purification. Repeat the purification process twice; the resulting phage daughter library will be used for the next round of amplification.

[0068] 10) Repeat steps 1) to 9) above 4 times to ensure that the amount of phage input is consistent in each round and complete the screening process.

[0069] Input-output test: The input-output ratio of each round of screening can be obtained by dividing the number of phage blue spots counted in step 8) by the number of phages input.

[0070] The number of blue phage spots typically refers to the number of phage spots formed on bacterial colonies after binding to a specific target in phage display technology. This number reflects the binding affinity of the displayed peptide or protein to the target. During screening, counting the number of blue phage spots can assess the affinity and specificity of the displayed peptide or protein. The input-output ratio reflects the screening efficiency; a higher ratio indicates higher screening efficiency and more effective screening of phages that bind to the target.

[0071] Take the logarithm of the input-output ratio to form a column. Figure 1 .

[0072] from Figure 1 It can be seen that the input-output ratio increases with each round, indicating that the phages that have a specific affinity for the IL-6 protein have been significantly enriched.

[0073] Example 2

[0074] DNA sequencing was performed on the bacteriophage obtained in Example 1 that exhibits specific affinity for the IL-6 protein to obtain a polypeptide that specifically binds to IL-6. The process included the following steps:

[0075] 1) Preservation and amplification of phages with specific affinity for IL-6: 80 blue spots were randomly selected from the plates from the fourth round of screening and added to shake tubes containing LB medium for amplification. After 24 hours, a portion of the bacterial culture was stored at -80°C, and the remaining culture was used for DNA sequencing.

[0076] The DNA sequences of bacteriophages that exhibit specific affinity for the IL-6 protein are as follows:

[0077] DGLHWNPRLWYR (SEQ ID NO.1)

[0078] GTNWSIHENNMG (SEQ ID NO.2)

[0079] TSGTMQTNPLPV (SEQ ID NO.3)

[0080] TIPNLTRVSNIV (SEQ ID NO.4)

[0081] SWSNASDYHIGA (SEQ ID NO.5)

[0082] YSSGFDQMPALP (SEQ ID NO.6)

[0083] 2) Sequencing Result Analysis: In the DNA sequencing results, the inserted foreign gene sequence was identified, the codons were translated into amino acid sequences, and the repetitive polypeptide sequences and their frequencies were statistically analyzed. The results are as follows: Figure 2 As shown.

[0084] from Figure 2 It was found that the frequency of occurrence of the six phage sequences varied, with the DGLHWNPRLWYR sequence appearing most frequently, followed by GTNWSIHENNMG, TSGTMQTNPLPV, TIPNLTRVSNIV, SWSNASDYHIGA, and YSSGFDQMPALP sequences. This indicates that the enrichment degree of each sequence of phages varied during the screening process, with the DGLHWNPRLWYR sequence exhibiting the strongest enrichment ability.

[0085] Example 3

[0086] In this embodiment, the IL-6 affinity of the phages obtained through screening was tested using an enzyme-linked immunosorbent assay (ELISA).

[0087] 1) Amplification and purification of IL-6 affinity phage: Five phage samples and wild-type phage samples preserved from each round of screening in Example 1 were thawed at room temperature. 200 μL of each sample was added to 20 mL of E. coil and ER2738 bacterial cultures in logarithmic growth phase. After incubation for 20 minutes, the mixture was shaken at 220 rpm for 4.5 hours at 37°C. After centrifugation to remove bacteria, one-fifth volume of PEG / NaCl solution was added to the supernatant, and the mixture was allowed to settle overnight at 4°C. The next day, the precipitate was centrifuged again and resuspended in sterile PBS, completing one stage of phage purification. The purification process was repeated twice, and the resulting phage solution was used for IL-6 affinity verification.

[0088] 2) Coating IL-6 protein: Add 50 μL of IL-6 protein at a concentration of 4 μg / mL to a 96-well microplate, place the microplate in a humidified chamber, and incubate overnight on a shaker at 4°C.

[0089] 3) Blocking: Aspirate the liquid from the wells, invert the plate onto a sterile paper towel, and shake vigorously to remove any remaining solution. Add 200 μL of 5% BSA blocking buffer to the wells and incubate for 1 hour.

[0090] 4) Washing: Discard the blocking solution, invert the ELISA plate on a sterile paper towel and shake vigorously to remove any remaining solution. Add 200 μL of PBST buffer and wash the plate 6 times, incubating on a shaker for 6 minutes each time. After each wash, shake vigorously on a sterile paper towel to remove any remaining solution.

[0091] 5) Binding: Add 50 μL of the phage amplified in step 1), maintaining a consistent concentration for each phage. Incubate at room temperature for 1 hour. Also, design groups containing PBS and wild-type phage for control purposes.

[0092] 6) Washing: Aspirate the phage solution and wash 6 times with PBST buffer as in step 4);

[0093] 7) Add primary antibody: Add 100 μL of antiphage capsid protein g8p antibody diluted to the appropriate multiple and incubate at room temperature for 1 hour;

[0094] 8) Washing: Aspirate the primary antibody and wash three times with PBST buffer as per step 4);

[0095] 9) Add secondary antibody: Add 100 μL of HRP-conjugated goat anti-mouse secondary antibody diluted to the appropriate multiple, and incubate at room temperature for 1 hour;

[0096] 10) Washing: Aspirate the secondary antibody and wash three times with PBST buffer as per step 4);

[0097] 11) Add TMB colorimetric solution: Add 100 μL of TMB colorimetric solution and incubate at 37°C for 10-20 minutes;

[0098] 12) Add stop solution: After step 11), when the color turns blue, add 100 μL of stop solution 2MH2SO4 to stop the reaction;

[0099] 13) Measure absorbance: Measure absorbance at 450 nm using an ELISA reader.

[0100] At the same time, a negative control group was set up, that is, 200 μL PBS was used to replace the phage sample in step 1) of this embodiment, and the subsequent steps were the same.

[0101] ELISA (Enzyme-Linked Immunosorbent Assay) is a commonly used experimental technique for detecting the content of biomolecules such as proteins, antibodies, and antigens. ELISA utilizes 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 coat protein g8p of bacteriophages. By detecting the absorbance at 450 nm, the binding amount of bacteriophages displaying different polypeptide sequences to the IL-6 protein can be reflected. A higher absorbance value indicates a greater number of bacteriophages with specific polypeptides that bind strongly to the IL-6 protein, and a stronger affinity for that polypeptide.

[0102] The absorbance values ​​for each group are shown below. Figure 3 WT represents wild-type phage, and PBS represents the negative control group.

[0103] from Figure 3 It was found that the binding affinity of the six selected phages to IL-6 protein was higher than that of the wild-type phage and the PBS negative control group. The binding affinity of the phages with IL-6 protein varied among the different sequences. The phage displaying the YSSGFDQMPALP sequence showed a significantly stronger affinity for IL-6 protein than the other phages, exhibiting a significant difference from the two control groups, being more than 6 times that of the PBS group and the wild-type phage group (p < 0.0001). The affinity for IL-6 protein, from highest to lowest, was as follows: YSSGFDQMPALP sequence, TSGTMQTNPLPV sequence, SWSNASDYHIGA sequence, DGLHWNPRLWYR sequence, GTNWSIHENNMG sequence, and TIPNLTRVSNIV sequence.

Claims

1. A polypeptide with binding affinity for IL-6, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO. 3, 5 or 6.

2. A fusion protein, characterized in that, The fusion protein comprises the polypeptide as described in claim 1 and a portion fused to the polypeptide; the portion is selected from any one of the following: fluorescent protein.

3. A conjugate, characterized in that, The conjugate comprises the polypeptide as described 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: a fluorescent label, a luminescent substance, or a chromogenic substance.

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

5. A recombinant vector, characterized in that, It contains the polynucleotide as described in claim 4.

6. A host cell, characterized in that, It includes the recombinant vector as described in claim 5, or the polynucleotide as described in claim 4 is integrated into the genome.

7. The use of the polypeptide of claim 1, the fusion protein of claim 2, the conjugate of claim 3, the polynucleotide of claim 4, the recombinant vector of claim 5, or the host cell of claim 6 in at least one of the following: 1) Used for the detection of IL-6, the use of which is for non-diagnostic purposes; 2) Used to prepare detection reagents for detecting IL-6.

8. A detection reagent or detection kit, characterized in that, It comprises the polypeptide as described in claim 1, the fusion protein as described in claim 2, or the conjugate as described in claim 3.

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