A class of high-affinity small proteins targeting IL6Ra and their applications

Abstract

This invention provides a class of small proteins with ultra-high affinity targeting IL6Ra and their uses. Specifically, this invention provides a class of binding proteins that target IL6Ra and have ultra-high affinity. The proteins of this invention can competitively bind to wild-type IL6, and their affinity is much higher than that of wild-type IL6 to IL6Ra. This invention also provides a fusion protein comprising the aforementioned ultra-high affinity protein targeting IL6Ra.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and medicine, specifically relating to a class of small proteins with ultra-high affinity targeting IL6Ra and their uses. Background Technology

[0002] Interleukin 6 (IL-6) is a quintessential inflammation-related cytokine that plays a crucial role in host defense by regulating immune and inflammatory responses. After IL-6 binds to IL-6, it forms a complex with IL-6Ra (the α subunit of IL-6) and other signal transduction factors, transmitting signals, activating regenerative and anti-inflammatory signals, and promoting the immunomodulatory properties of IL-6. Therefore, the IL-6R / IL-6 signaling pathway is one of the important signaling pathways for regulating immunity and exerting immunosuppressive effects. Furthermore, abnormal activity of the IL-6 signaling pathway is closely related to the development and progression of various diseases, and blocking the IL-6Ra / IL-6 immunosuppressive signaling pathway has become one of the important strategies for current anti-tumor therapy.

[0003] In recent years, targeted therapies against the IL6Ra / IL6 signaling pathway have shown promising promise in the treatment of autoimmune diseases, inflammatory diseases, cancer, and antibacterial / viral infections. For example, anti-IL-6R monoclonal antibody drugs such as tocilizumab have been approved for marketing and are used to treat diseases such as rheumatoid arthritis, significantly improving patient prognosis. How to more effectively block the binding of IL6RA / IL6, and thus more effectively suppress cytokine storms, remains a pressing issue.

[0004] In summary, there is an urgent need in this field to develop a candidate drug that can more efficiently block IL6Ra / IL6 binding, thereby more effectively inhibiting IL6Ra / IL6 binding and cytokine storm. Summary of the Invention

[0005] The purpose of this invention is to provide a class of small proteins with ultra-high affinity for IL6Ra, which can block the binding of IL6Ra / IL6 and more effectively inhibit cytokine storm.

[0006] Another objective of this invention is to provide a class of fusion proteins based on a small protein with ultra-high affinity targeting IL6Ra and a method for preparing the same.

[0007] In a first aspect of the invention, a small protein targeting IL6Ra is provided, the amino acid sequence of said small protein being selected from the group consisting of:

[0008] (1) An amino acid sequence as shown in SEQ ID NO: 1, 3, 9 or 11;

[0009] (2) An amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO:1, 3, 9 or 11 (i.e., homology ≥90%, preferably ≥95%, more preferably ≥98%) and retains binding activity with IL6Ra (preferably, retains ≥70%, more preferably ≥80%).

[0010] In another preferred embodiment, the small protein blocks the binding of IL6Ra to IL6.

[0011] In another preferred embodiment, the amino acid sequence of the small protein is as shown in SEQ ID NO: 1, 3, 9 or 11.

[0012] The present invention also provides a recombinant protein comprising two or more small proteins of the present invention targeting IL6Ra, connected in tandem.

[0013] In a second aspect of the invention, a fusion protein is provided, the fusion protein comprising one or more polypeptides with structures as shown in Formula I or Formula II.

[0014] P-Mx-HF (Formula I)

[0015] PFH-Mx (Formula II)

[0016] in,

[0017] P represents the absence of a signal peptide sequence;

[0018] M is the IL6Ra binding region, which includes small proteins targeting IL6Ra as described in the first aspect of the present invention, or recombinant proteins of the present invention.

[0019] H represents the area without hinges;

[0020] F represents the constant region of an immunoglobulin, or a self-assembled protein, or a fragment thereof;

[0021] "-" indicates a peptide bond or linking peptide that connects the above elements;

[0022] x is a positive integer between 1 and 4.

[0023] In another preferred embodiment, the fusion protein is a monomer or a polymer.

[0024] In another preferred embodiment, the polymer is a dimer or a trimer.

[0025] In another preferred embodiment, the fusion protein is a homopolymer or a heteropolymer.

[0026] In another preferred embodiment, the fusion protein comprises three of the polypeptides and forms a trimer through self-assembly of proteins within the polypeptides.

[0027] In another preferred embodiment, the sequence of M is as shown in SEQ ID NO:1, 3, 9 or 11.

[0028] In another preferred embodiment, x is 1, 2, 3 or 4, preferably 1.

[0029] In another preferred embodiment, the amino acid sequence of the first polypeptide is selected from the group consisting of:

[0030] (i) A sequence as shown in SEQ ID NO:5, 7, 13, 15 or 17;

[0031] (ii) Based on SEQ ID NO:5, 7, 13, 15 or 17, one or more amino acid residues are replaced, deleted, altered or inserted, or 1 to 30 amino acid sequences are added to its N-terminus or C-terminus, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues, thereby obtaining an amino acid sequence.

[0032] In another preferred embodiment, the amino acid sequence of the first polypeptide is shown in SEQ ID NO:5, 7, 13, 15 or 17, and the nucleotide sequence encoding the first polypeptide is shown in SEQ ID NO:6, 8, 14, 16 or 18.

[0033] In a third aspect of the invention, a polynucleotide is provided that encodes a small protein targeting IL6Ra in the first aspect of the invention, or a recombinant protein thereof, or a fusion protein as described in the second aspect of the invention.

[0034] In another preferred embodiment, the sequence of the polynucleotide is as shown in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16 or 18.

[0035] In a fourth aspect of the invention, a carrier is provided, the carrier containing the polynucleotide described in the third aspect of the invention.

[0036] In another preferred embodiment, the vector is: pET vector, pGEM-T vector, pcDNA3.1, or a combination thereof.

[0037] In a fifth aspect of the invention, a host cell is provided, the host cell containing the vector described in the fourth aspect, or the genome integrating the polynucleotides described in the third aspect.

[0038] In a sixth aspect of the invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0039] (a) the small protein targeting IL6Ra as described in the first aspect of the invention, or a recombinant protein thereof, or the fusion protein as described in the second aspect of the invention; and

[0040] (b) The coupling part selected from the following group: detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.

[0041] In another preferred embodiment, the coupling portion is a drug or toxin.

[0042] In another preferred embodiment, the coupling portion is a detectable marker.

[0043] In another preferred embodiment, the conjugate is selected from the group consisting of: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents.

[0044] In a seventh aspect of the invention, a pharmaceutical composition is provided comprising:

[0045] (a) The small protein targeting IL6Ra described in the first aspect of the present invention or its recombinant protein, or the fusion protein described in the second aspect of the present invention, or its encoding gene; or the immunoconjugate described in the sixth aspect of the present invention; and

[0046] (b) Pharmaceutically acceptable carriers.

[0047] In another preferred embodiment, the pharmaceutical composition is used for the diagnosis or treatment of IL6Ra-related diseases.

[0048] In another preferred embodiment, the IL6Ra-related diseases are selected from the group consisting of autoimmune diseases, inflammatory diseases, tumors, or combinations thereof.

[0049] In another preferred embodiment, the autoimmune disease is rheumatoid arthritis.

[0050] In another preferred embodiment, the inflammatory disease is a viral infection or a bacterial infection.

[0051] In another preferred embodiment, the content of component (a) is 0.1-99.9 wt%, more preferably 10-99.9 wt%, and even more preferably 70%-99.9 wt%.

[0052] In another preferred embodiment, the dosage form of the pharmaceutical composition is an oral dosage form, an injection, or a topical dosage form.

[0053] In another preferred embodiment, the dosage form of the pharmaceutical composition includes tablets, granules, capsules, oral liquids, or injections.

[0054] In another preferred embodiment, the pharmaceutical composition or formulation is selected from the group consisting of suspensions, liquids, or lyophilized formulations.

[0055] In another preferred embodiment, the liquid formulation is an aqueous injection formulation.

[0056] In another preferred embodiment, the liquid preparation has a shelf life of one to three years, more preferably one to two years, and even more preferably one year.

[0057] In another preferred embodiment, the liquid preparation is stored at a temperature of 0°C-16°C, more preferably 0°C-10°C, and even more preferably 2°C-8°C.

[0058] In another preferred embodiment, the shelf life of the lyophilized preparation is six months to two years, more preferably six months to one year, and even more preferably six months.

[0059] In another preferred embodiment, the storage temperature of the lyophilized formulation is ≤42°C, more preferably ≤37°C, and even more preferably ≤30°C.

[0060] In another preferred embodiment, the pharmaceutically acceptable carrier includes surfactants, solution stabilizers, isotonic modifiers, buffer solutions, or combinations thereof.

[0061] In another preferred embodiment, the pharmaceutically acceptable carrier is selected from the group consisting of infusion carriers and / or injection carriers, and more preferably, the carrier is one or more carriers selected from the group consisting of physiological saline, glucose saline, or combinations thereof.

[0062] In another preferred embodiment, the solution stabilizer is selected from the group consisting of sugar solution stabilizers, amino acid solution stabilizers, alcohol solution stabilizers, or combinations thereof.

[0063] In another preferred embodiment, the sugar solution stabilizer is selected from the group consisting of reducing sugar solution stabilizers or non-reducing sugar solution stabilizers.

[0064] In another preferred embodiment, the amino acid solution stabilizer is selected from the group consisting of monosodium glutamate or histidine.

[0065] In another preferred embodiment, the alcohol solution stabilizer is selected from the group consisting of: triols, higher sugar alcohols, propylene glycol, polyethylene glycol, or combinations thereof.

[0066] In another preferred embodiment, the isotonic regulator is selected from the group consisting of sodium chloride or mannitol.

[0067] In another preferred embodiment, the buffer solution is selected from the group consisting of TRIS, histidine buffer, phosphate buffer, or combinations thereof.

[0068] In another preferred embodiment, the pharmaceutical composition or formulation is administered to humans or non-human animals.

[0069] In another preferred embodiment, the non-human animal includes rodents (such as rats and mice) and primates (such as monkeys).

[0070] In another preferred embodiment, the dosage of the pharmaceutical composition or preparation is 0.01-10 g / day, more preferably 0.05-5000 mg / day, and even more preferably 0.1-3000 mg / day.

[0071] In another preferred embodiment, the pharmaceutical composition or formulation is used to treat IL6Ra-related diseases.

[0072] In another preferred embodiment, the IL6Ra-related diseases are selected from the group consisting of autoimmune diseases, inflammatory diseases, tumors, or combinations thereof.

[0073] In another preferred embodiment, the autoimmune disease is rheumatoid arthritis.

[0074] In another preferred embodiment, the inflammatory disease is a viral infection or a bacterial infection.

[0075] In another preferred embodiment, the pharmaceutical composition or formulation may be administered in combination with other antiviral or anti-inflammatory drugs.

[0076] In another preferred embodiment, the other antiviral or anti-inflammatory drugs administered in combination are selected from the group consisting of: viral replication inhibitors, hormonal anti-inflammatory drugs, biological response modifiers, monoclonal antibodies, or combinations thereof.

[0077] In another preferred embodiment, the hormonal anti-inflammatory drug includes an anti-estrogen, an aromatase inhibitor, or an anti-androgen; preferably, the anti-estrogen is selected from the group consisting of tamoxifen, droloxifene, exemestane, or combinations thereof; the aromatase inhibitor is selected from the group consisting of ammoniaglutide, lantron, letrozole, reninide, or combinations thereof; and the anti-androgen is selected from the group consisting of flutamethasone RH-LH agonists / antagonists: novide, phenazone, or combinations thereof.

[0078] In another preferred embodiment, the biological response modifier includes: interferon, interleukin-2, thymopeptides, or combinations thereof.

[0079] In an eighth aspect of the invention, a method is provided for preparing a small protein targeting IL6Ra of the first aspect of the invention or a recombinant protein thereof or a fusion protein of the third aspect of the invention, comprising the steps of:

[0080] (a) Culturing the host cells described in the fifth aspect of the invention under suitable conditions to obtain a culture containing the small protein or its recombinant or fusion protein; and

[0081] (b) The culture obtained in step (a) is purified and / or separated to obtain the small protein targeting IL6Ra or its recombinant protein or fusion protein.

[0082] In a ninth aspect of the invention, the use of the small protein targeting IL6Ra described in the first aspect of the invention, or its fusion protein, or its immunoconjugate, for the preparation of pharmaceuticals, reagents, detection plates, or kits is provided; wherein the reagents, detection plates, or kits are used to: detect IL6Ra in a sample; wherein the pharmaceuticals are used to treat and / or prevent IL6Ra-related diseases.

[0083] In another preferred embodiment, the IL6Ra-related diseases are selected from the group consisting of autoimmune diseases, inflammatory diseases, tumors, or combinations thereof.

[0084] In another preferred embodiment, the autoimmune disease is rheumatoid arthritis.

[0085] In another preferred embodiment, the inflammatory disease is a viral infection or a bacterial infection.

[0086] In another preferred embodiment, the reagent is one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow cytometry reagents, cell immunofluorescence reagents, magnetic nanoparticles, and imaging agents.

[0087] In another preferred embodiment, the reagent for detecting IL6Ra in the sample is a contrast agent for (in vivo) detection of IL6Ra.

[0088] In another preferred embodiment, the detection is either in vivo or in vitro.

[0089] In another preferred embodiment, the detection includes flow cytometry, immunofluorescence assay, or a combination thereof.

[0090] In another preferred embodiment, the agent is used to block the interaction between IL6Ra and IL6.

[0091] In a tenth aspect of the invention, a method for treating and / or preventing IL6Ra-related diseases is provided, comprising the steps of: administering to a desired subject a safe and effective amount of the small protein or recombinant protein thereof that targets IL6Ra as described in the first aspect of the invention, or the fusion protein described in the second aspect, or the immunoconjugate described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect.

[0092] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0093] Figure 1 The diagram shows a simulated structure of a complex of a small protein targeting IL6Ra with human IL6Ra and binding to it with high affinity.

[0094] Figure 2 The results of thermal stability assays for the high-affinity small protein structure targeting IL6Ra are presented.

[0095] Figure 3 The results of thermal recovery assays for the high-affinity small protein structure targeting IL6Ra are shown.

[0096] Figure 4 The results of the affinity assay for high-affinity small proteins targeting IL6Ra are shown.

[0097] Figure 5 This study demonstrates the ultra-high affinity of the novel de novo design trivalent IL6Ra-5-C3-50-70 and C3-50-70-IL6Ra-5 for targeting IL6Ra.

[0098] Figure 6 The study demonstrated the protective effects of novel de novo-designed trivalent IL6Ra-5-C3-50-70 and C3-50-70-IL6Ra-5, which target IL6Ra with high affinity, in a small animal LPS sepsis model.

[0099] Figure 7 The novel de novo design demonstrates the affinity of high-affinity binding proteins IL6Ra-rd-15 and IL6Ra-rd-30 that target IL6Ra.

[0100] Figure 8 The novel de novo design with different linker lengths demonstrates the ultra-high affinity binding of trivalent IL6Ra-targeting proteins C3-70-11-GS16-IL6Ra-rd-30 and C3-70-11-GS18-IL6Ra-rd-30 to target IL6Ra. Detailed Implementation

[0101] Through extensive and in-depth research, the inventors, based on the structure of the wild-type IL6Ra / IL6 protein, and targeting the interaction surface between IL6Ra and IL6, have designed a class of small proteins with ultra-high affinity for IL6Ra. The binding site of this small protein almost completely covers the binding site of the wild-type IL6Ra / IL6. Experiments show that the high-affinity small protein of this invention has a much higher affinity than the wild-type IL6 protein and exhibits better ability to inhibit cytokine storms. This invention was completed based on these findings.

[0102] This invention relates to high-affinity small proteins and fusion proteins targeting IL6Ra.

[0103] In this invention, a class of high-affinity small proteins targeting IL6Ra and a fusion protein or conjugate thereof comprising said small protein are provided.

[0104] As used herein, the terms "small protein of the present invention" and "high-affinity small protein of the present invention targeting IL6Ra" are used interchangeably and both refer to the small protein having the ultra-high affinity for IL6Ra described in the first aspect of the present invention.

[0105] Preferably, the small protein of the present invention has an amino acid sequence as shown in SEQ ID NO:1, 3, 9 or 11.

[0106] As used herein, the term "fusion protein of the present invention" refers to a fusion protein formed by the high-affinity small protein targeting IL6Ra described in this invention and other fusion elements. For example, the small protein of the present invention can form a fusion protein with elements such as hinge regions, Fc regions, and self-assembling proteins. The fusion protein of the present invention can block the binding of IL6 to IL6Ra.

[0107] As used herein, the term "self-assembling protein" refers to a protein capable of self-assembling to form a multimer. In some embodiments, the high-affinity small protein targeting IL6Ra of the present invention can form a fusion protein with the self-assembling protein, thereby self-assembling to form a multimer (e.g., a trimer) comprising multiple small proteins of the present invention. The multimers of the present invention can be homopolymers or heteropolymers, that is, the multimers can contain the same or different high-affinity small proteins targeting IL6Ra.

[0108] Preferably, the ultra-high affinity fusion protein of the present invention can be any high-affinity small protein that targets IL6Ra or a partial amino acid fragment thereof (typically at least 70% of the amino acid length).

[0109] Typically, the fusion protein of the present invention may have the following structure:

[0110] High-affinity small proteins or fragments targeting IL6Ra in a Y-shaped structure of Hinge-CH2-CH3;

[0111] High-affinity small proteins or fragments targeting IL6Ra in a Y-shaped structure of Hinge-CH3;

[0112] High-affinity small proteins or fragments targeting IL6Ra - tracer markers;

[0113] High-affinity small proteins or fragments targeting IL6Ra—multimeric structures of self-assembled proteins;

[0114] High-affinity small proteins or fragments targeting IL6Ra.

[0115] It should be understood that the above structural types are merely illustrative and do not limit the present invention. High-affinity small proteins or fragments thereof targeting IL6Ra can be single or multiple (e.g., 2, 3 or 4 ultra-high affinity small proteins or fragments thereof in tandem).

[0116] As used herein, the terms "high-affinity small protein targeting IL6Ra" or "fusion protein" also include variants having IL6Ra binding activity and IL6 / IL6Ra blocking activity. These variants include (but are not limited to): deletions, insertions, and / or substitutions of 1-3 amino acids (typically 1-2, more preferably 1); additions or deletions of one or more amino acids (typically up to 3, preferably up to 2, more preferably up to 1) at the C-terminus and / or N-terminus; or additions of smaller amino acid side chains as linkers (such as glycine, serine, etc.) at the N-terminus or C-terminus of the small protein. For example, in the art, substitution with amino acids of similar or comparable properties typically does not alter the function of the protein. Similarly, additions or deletions of one or more amino acids at the C-terminus and / or N-terminus typically do not alter the structure and function of the protein. Furthermore, the term also includes the polypeptides of the invention in monomeric and multimeric forms. The term also includes linear and non-linear polypeptides (such as cyclic peptides).

[0117] The present invention also includes active fragments, derivatives, and analogs of the aforementioned small proteins or fusion proteins targeting IL6Ra (especially fusion proteins formed with the Fc fragment). As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the function or activity of the high-affinity small proteins or fusion proteins targeting IL6Ra of the present invention.

[0118] The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, or (ii) polypeptides having substituent groups in one or more amino acid residues, or (iii) polypeptides formed by fusing a polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, for example, polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (fusion proteins formed by fusing with a leader sequence, secretion sequence, or tag sequence such as 6His). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0119] A preferred class of active derivatives refers to polypeptides formed by replacing up to five, more preferably up to three, and even more preferably up to one amino acid with an amino acid of similar or analogous properties, compared to the amino acid sequence of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0120] Table A

[0121]

[0122]

[0123] This invention also provides analogs of the fusion protein of this invention. These analogs may differ from the peptides of this invention in terms of amino acid sequence, or in the form of modifications that do not affect the sequence, or both. Analogs also include those having residues different from naturally occurring L-amino acids (such as D-amino acids), and those having non-naturally occurring or synthetic amino acids (such as β- or γ-amino acids). It should be understood that the peptides of this invention are not limited to the representative peptides exemplified above.

[0124] Furthermore, the high-affinity small proteins or fusion proteins targeting IL6Ra of this invention can also be modified. Modifications (generally without altering the primary structure) include chemically derived forms of the peptide, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during peptide synthesis and processing or further processing steps. This modification can be accomplished by exposing the peptide to glycosylation enzymes (such as mammalian glycosylation or deglycosylation enzymes). Modifications also include sequences containing phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). Modifications also include peptides modified to improve their resistance to proteolytic hydrolysis or optimize their solubility.

[0125] The term "polynucleotide of the present invention" can be a polynucleotide that encodes a high-affinity small protein or fusion protein targeting IL6Ra of the present invention, or it can also include polynucleotides that include additional coding and / or non-coding sequences.

[0126] This invention also relates to variants of the aforementioned polynucleotides that encode fragments, analogs, and derivatives of polypeptides or fusion proteins having the same amino acid sequence as those of this invention. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the high-affinity small protein or fusion protein it encodes that targets IL6R.

[0127] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions (or strict conditions). In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%.

[0128] The high-affinity small protein or fusion protein and polynucleotide targeting IL6Ra of the present invention are preferably provided in isolated form, more preferably purified to homogenization.

[0129] The full-length polynucleotide sequences of this invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in this invention, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequences are long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0130] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0131] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0132] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0133] The application of PCR technology to amplify DNA / RNA is preferred for obtaining the polynucleotides of the present invention. Especially when it is difficult to obtain full-length cDNA from a library, the RACE (RACE-cDNA end amplification) method is preferred. Primers used for PCR can be appropriately selected based on the sequence information disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0134] As used herein, “C3-50-70-IL6Ra-5”, “C3-50-70-IL6Ra-rd-5”, “C3-70-11-IL6Ra-rd-5”, and “C3-50-70-11-IL6Ra-rd-5” have the same meaning and can be used interchangeably. They all refer to the multivalent high-affinity protein shown in SEQ ID NO:7.

[0135] As used in this article, “IL6Ra-5-C3-50-70”, “IL6Ra-rd-5-C3-50-70”, “IL6Ra-rd-5-C3-70-11”, and “IL6Ra-rd-5-C3-50-70-11” have the same meaning and can be used interchangeably. They all refer to the multivalent high-affinity protein shown in SEQ ID NO:5.

[0136] expression carrier

[0137] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells genetically engineered using the vectors of the present invention or the coding sequences of the high-affinity small protein or fusion protein of the present invention targeting IL6Ra, and methods for generating the polypeptides of the present invention via recombinant technology.

[0138] Using conventional recombinant DNA techniques, the polynucleotide sequences of this invention can be used to express or produce recombinant fusion proteins. Generally, the following steps are involved:

[0139] (1) Transform or transduce suitable host cells using the polynucleotide (or variant) encoding the fusion protein of the present invention, or using a recombinant expression vector containing the polynucleotide;

[0140] (2) Host cells cultured in a suitable culture medium;

[0141] (3) Isolate and purify proteins from culture media or cells.

[0142] In this invention, the polynucleotide sequence encoding the fusion protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well-known in the art. Any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0143] In the method for preparing a high-affinity small protein or its fusion protein targeting IL6Ra of the present invention, any suitable vector can be used, selected from pET, pDR1, pcDNA3.1(+), pcDNA3.1 / ZEO(+), pDHFR, and the expression vector includes a fusion DNA sequence linked with suitable transcription and translation regulatory sequences.

[0144] Both eukaryotic and prokaryotic host cells can be used for the expression of the high-affinity small protein or its fusion protein targeting IL6Ra in this invention. The eukaryotic host cells are preferably mammalian or insect host cell culture systems, preferably COS, CHO, NSO, sf9 and sf21 cells; the prokaryotic host cells are preferably one of lemo21, DH5a, BL21(DE3) and TG1.

[0145] Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence encoding the fusion protein of this invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, LTRs of retroviruses, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0146] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0147] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express proteins.

[0148] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces, Salmonella typhimurium bacterial cells, fungal cells such as yeast, and plant cells (such as ginseng cells).

[0149] When the polynucleotides of this invention are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0150] Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0151] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0152] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0153] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0154] The high-affinity small protein or its fusion protein targeting IL6Ra disclosed in this invention can be separated and purified using affinity chromatography. Depending on the characteristics of the affinity column used, conventional methods such as high-salt buffer or pH adjustment can be used to elute the high-affinity small protein or its fusion protein targeting IL6Ra bound to the affinity column.

[0155] Using the above method, high-affinity small proteins or their fusion proteins targeting IL6Ra can be purified into essentially homogeneous substances, such as a single band on SDS-PAGE electrophoresis.

[0156] Pharmaceutical Composition

[0157] In this invention, a pharmaceutical composition containing a small protein or fusion protein or an immunoconjugate thereof targeting IL6Ra is also provided.

[0158] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the small protein or fusion protein (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or aqueous solutions containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 mg / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents. The small protein or fusion protein targeting IL6Ra, or its immunoconjugate, can be formulated with pharmaceutically acceptable excipients to achieve more stable therapeutic effects. These formulations ensure the structural integrity of the amino acid core sequence of the small protein or fusion protein targeting IL6Ra of the present invention, while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination, or oxidation). The formulation can be in various forms. Generally, for liquid formulations, it can be stably stored for at least one year at 2°C-8°C, and for lyophilized formulations, it remains stable for at least six months at 30°C. The formulation can be a suspension, injection, or lyophilized formulation commonly used in the pharmaceutical industry, preferably an injection or lyophilized formulation.

[0159] For the pharmaceutical compositions (such as aqueous injections or lyophilized formulations) targeting IL6Ra of the present invention, pharmaceutically acceptable excipients include one or a combination of surfactants, solution stabilizers, isotonic modifiers, and buffers. Surfactants include nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (Tween 20 or 80); poloxamer (such as poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; tetradecyl, linoleic, or octadecyl sarcosine; Pluronics; MONAQUAT™, etc., added in an amount that minimizes the tendency of protein granulation. Solution stabilizers can be sugars, including reducing and non-reducing sugars; amino acids including monosodium glutamate or histidine; alcohols including one or a combination of triols, higher sugar alcohols, propylene glycol, and polyethylene glycol. The amount of solution stabilizer added should be such that the final formulation is considered by a person skilled in the art to remain stable for a stable period of time. Isotonic modifiers can be one of sodium chloride and mannitol. Buffers can be one of TRIS, histidine buffer, and phosphate buffer.

[0160] When using the pharmaceutical composition, a safe and effective amount of the small protein or fusion protein of the present invention, or its immunoconjugate, is administered to a mammal. This safe and effective amount is typically at least about 50 micrograms per kilogram of body weight, and in most cases does not exceed about 100 milligrams per kilogram of body weight; preferably, the dose is between about 100 micrograms per kilogram of body weight and about 50 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise. Typically, the total dose should not exceed a certain range, for example, an intravenous dose of 10 to 3000 mg / day / 50 kg, preferably 100 to 1000 mg / day / 50 kg.

[0161] The main advantages of this invention include:

[0162] 1) The small protein targeting IL6Ra provided by this invention has a binding site that can cover the binding of wild-type IL6Ra to IL6.

[0163] 2) The small protein of the present invention has an extremely high affinity for human IL6Ra, which is much higher than the affinity of wild-type IL6 for IL6Ra.

[0164] 3) The small protein of the present invention has ultra-high structural stability, with a Tm value greater than 95°C.

[0165] 4) The protein of this invention exhibits a strong ability to suppress inflammatory storms and can significantly improve the survival rate of animal models.

[0166] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0167] The sequence of the present invention is shown in Table 1 below.

[0168] Table 1. Sequence of the present invention

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175] Example 1: Construction, expression, and purification of a small protein with high affinity for IL6R

[0176] A nucleotide sequence targeting the IL6R protein was synthesized using gene synthesis methods. Auxiliary sequences MGS and GSENLYFQSLEHHHHHH (SEQ ID NO:19) were added to both ends of this sequence. The sequence was then inserted into the pET29b vector in the form of MGS-IL6R protein nucleotide sequence-GSENLYFQSLEHHHHHH. After transforming this vector into *E. coli*, it was cultured in LB medium at 37°C and 270 rpm until OD290. 600 =0.6. Then, 1 mM IPTG was used to induce bacterial protein expression overnight. After centrifugation, the bacteria were collected and resuspended, followed by lysis using a low-temperature mechanical homogenizer and high-speed centrifugation to collect the supernatant. After purification via Ni column, the protein sample was desalted and concentrated, and the concentration of the purified protein was determined using a micro-spectrophotometer. The purified protein was then filtered through a 0.22 μm filter membrane for sterilization and stored at 4°C for subsequent experiments.

[0177] Example 2: Detection of the thermal stability of a high-affinity small protein structure targeting IL6R

[0178] The thermal stability of the protein structure was assessed using the Unchained Labs Uncle multi-functional protein stability analyzer. The sample temperature range was 25℃-95℃ at a rate of 0.5℃ / min. Static light scattering was measured at an excitation wavelength of 266nm. As the protein unfolded, fluorescence typically decreased and shifted to longer wavelengths. Data analysis software determined the Tm value based on the centroid average (BCM) of the fluorescence intensity curves from 300-430nm, and the aggregation (Tagg) was observed based on the intensity of scattered light at 266nm. The changes in the secondary structure conformation of the protein at different temperatures were obtained, thereby assessing the structural stability of the bound protein.

[0179] like Figure 2 As shown, the denaturation curves indicate that neither IL6Ra_rd-5 nor IL6Ra_rd-7 exhibits a significant Tm, with IL6Ra_rd-7 showing a denaturation variation of 5 nm, indicating a larger conformational change. The aggregation curves show that IL6Ra_rd-7 has an initial aggregation temperature (Tagg) of 44 °C, indicating a low degree of aggregation; IL6Ra_rd-5 shows no significant aggregation. These experimental results demonstrate the proteins exhibiting exceptional thermal stability.

[0180] Example 3: Detection of the thermal recovery of high-affinity small protein structures targeting IL6R

[0181] The thermal resilience of protein structure was assessed using the Unchained Labs Uncle multi-functional protein stability analyzer. Samples were heated from 20°C to 95°C and then cooled back to 20°C in 15°C increments. Changes in protein conformation and aggregation during these temperature fluctuations were observed. As the temperature increased, the conformation of the sample opened up; as the temperature decreased, the conformation folded back up, indicating good thermal resilience.

[0182] like Figure 3 As shown, the conformation of IL6Ra_rd-7 opens up with increasing temperature and folds back down with decreasing temperature, indicating good conformational thermal recovery. The aggregation signal increases with increasing temperature and decreases slightly with decreasing temperature, with a small number of samples undergoing deaggregation, but overall, the aggregation is irreversible. The conformation of IL6Ra_rd-5 changes slightly with temperature, but the overall change is small, indicating strong conformational stability. Furthermore, the aggregation signal changes slightly with temperature, but the overall change is small, indicating that this sample is not prone to aggregation. Compared to IL6Ra_rd-7, IL6Ra_rd-5 is more stable.

[0183] Example 4: Determination of Affinity to High-Affinity Small Proteins Targeting IL6Ra

[0184] In this embodiment, the affinity of high-affinity proteins was detected using the ForteBio Octet. First, 3 μg / ml of IL6Ra-Fc protein was loaded onto the ProA detection probe (1200 s), and unbound IL6Ra-Fc protein was eluted in PBST solution. Then, the detection probe carrying human IL6Ra protein was simultaneously immersed in a 2-fold serially diluted solution of IL6 or a high-affinity protein targeting IL6Ra, and the binding signal was detected (180 s). Next, the probe was immersed in PBST (300 s), and the dissociation signal of the bound protein was detected. Finally, the affinity of the high-affinity blocking protein was calculated.

[0185] like Figure 4 As shown, IL6Ra_rd-5 and IL6Ra_rd-7 exhibited extremely strong binding activity, with affinities of 4.951 × 10⁻⁶ and 4.951 × 10⁻⁶, respectively. -9 M and 5.805×10 -9 M. The affinity of IL6 for IL6Ra is 1.18 × 10⁻⁶. -6 M. Compared with wild-type IL6, IL6Ra_rd-5 and IL6Ra_rd-7 have nearly 500 times greater affinity.

[0186] like Figure 7 As shown, the affinities of IL6Ra-rd-15 and L6Ra-rd-30 are 6.126 × 10⁻⁶ and 6.126 × 10⁻⁶, respectively. -10 M and 1.056×10 -8 M.

[0187] Example 5: Construction, expression, and purification of a multivalent high-affinity protein targeting IL6Ra

[0188] Gene sequences of IL6Ra_rd-5, IL6Ra_rd-15, or IL6Ra_rd-30 were synthesized using gene synthesis methods and ligated to the N-terminus or C-terminus of a trimeric protein nucleotide sequence (see patent application number: CN202411374093.4 for trimeric protein). This sequence was then inserted into the pET29b vector in the format MGS-protein nucleotide sequence-GSENLYFQSLEHHHHHH, where ENLYFQSLE (SEQ ID NO: 20) was used for restriction enzyme digestion and HHHHHH (SEQ ID NO: 21) was used for protein purification. After transforming the vector into *E. coli*, it was cultured in LB medium at 37°C and 270 rpm until OD200. 600=0.6. Then, 1 mM IPTG was used to induce bacterial protein expression overnight. After centrifugation, the bacteria were collected and resuspended, followed by lysis using a low-temperature mechanical homogenizer and high-speed centrifugation to collect the supernatant. After purification via Ni column, the protein sample was desalted and concentrated, and the concentration of the purified protein was determined using a micro-spectrophotometer. The purified protein was then filtered through a 0.22 μm filter membrane for sterilization and stored at 4°C for subsequent experiments.

[0189] Example 6: Affinity determination of multivalent high-affinity protein targeting IL6Ra

[0190] In this embodiment, the affinity of high-affinity proteins was detected using the ForteBio Octet. First, 3 μg / ml of IL6Ra-Fc protein was loaded onto the ProA detection probe (1200 s), and unbound IL6Ra-Fc protein was eluted in PBST solution. Then, the detection probe containing human IL6Ra protein was simultaneously immersed in a 2-fold serially diluted solution of a multivalent high-affinity protein targeting IL6R, and the binding signal was detected (180 s). Next, the probe was immersed in PBST (300 s), and the dissociation signal of the bound protein was detected. Finally, the affinity of the high-affinity blocking protein was calculated.

[0191] like Figure 5 As shown, both IL6Ra-5-C3-50-70 and C3-50-70-IL6Ra-5 exhibited extremely strong binding activity, with affinities of 2.192 × 10⁻⁶ and 2.192 × 10⁻⁶, respectively. -9 M and 4.987×10 -9 M.

[0192] like Figure 8 As shown, the affinities of C3-70-11-GS16-IL6Ra-rd-30 and C3-70-11-GS18-IL6Ra-rd-30 are 2.02 × 10⁻⁶ and 2.02 × 10⁻⁶, respectively. -12 M and 1.876×10 -12 M.

[0193] Example 7: Protective effect of targeting IL6Ra multivalent high-affinity protein on an animal model of intraperitoneal infection

[0194] In this embodiment, the protective effect of high-affinity blocking proteins against cytokine storm was verified using two animal models of intraperitoneal injection of lipopolysaccharide (LPS). First, 6-8 week old C57BL / 6 mice were selected and an intraperitoneal infection model was established using 10 mg / kg LPS. Then, the mice were intraperitoneally injected with 0.5 mg / kg tocilizumab (TCZ) or a high-affinity multivalent protein targeting IL6Ra. Figure 6As shown, the survival of mice in each group over 7 days was observed. Compared with the model animals given the multivalent high-affinity protein C3-70-11-IL6Ra-rd-5 targeting IL6Ra, the mortality rate of the TCZ group decreased by 43.8%, showing better protective activity.

[0195] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A small protein targeting IL6Ra, characterized in that, The amino acid sequence of the small protein is selected from the following group: (1) An amino acid sequence as shown in SEQ ID NO: 1, 3, 9 or 11; (2) An amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO:1, 3, 9 or 11 (i.e., homology ≥90%, preferably ≥95%, more preferably ≥98%) and retains binding activity with IL6Ra (preferably, retains ≥70%, more preferably ≥80%).

2. The small protein targeting IL6Ra as described in claim 1, characterized in that, The amino acid sequence of the small protein is shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 9 or SEQ ID NO:

11.

3. A recombinant protein, characterized in that, The recombinant protein comprises two or more small proteins targeting IL6Ra as described in claim 1 or 2, connected in tandem, and the amino acid sequence of the recombinant protein is shown in SEQ ID NO: 5, 7, 13, 15 or 17.

4. A fusion protein, characterized in that, The fusion protein comprises one or more polypeptides with structures as shown in Formula I or Formula II. P-Mx-HF (Formula I) PFH-Mx (Formula II) in, P represents the absence of a signal peptide sequence; M is the IL6Ra binding region, which includes the small protein targeting IL6Ra as described in claim 1, or the recombinant protein as described in claim 3; H represents the area with no hinge or no connection. F represents the constant region of an immunoglobulin, or a self-assembled protein, or a fragment thereof; "-" indicates a peptide bond or linking peptide that connects the above elements; x is a positive integer between 1 and 4.

5. A polynucleotide, characterized in that, The polynucleotide encodes the small protein targeting IL6Ra as described in claim 1 or 2, the recombinant protein as described in claim 3, or the fusion protein as described in claim 4.

6. A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 5.

7. A host cell, characterized in that, The host cell contains the vector as described in claim 6, or the genome is integrated with the polynucleotide as described in claim 5.

8. An immunoconjugate, characterized in that, This immunoconjugate contains: (a) the small protein targeting IL6Ra as described in claim 1 or 2, the recombinant protein as described in claim 3, or the fusion protein as described in claim 4; and (b) The coupling part selected from the following group: detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.

9. A pharmaceutical composition, characterized in that, include: (a) the small protein targeting IL6Ra as described in claim 1 or 2, or the recombinant protein as described in claim 3, or the fusion protein as described in claim 4, or its encoding gene; or the immunoconjugate as described in claim 8; and (b) Pharmaceutically acceptable carriers.

10. A method for preparing a small protein targeting IL6Ra as described in claim 1 or 2, or a recombinant protein as described in claim 3, or a fusion protein as described in claim 4, characterized in that, Including the following steps: (a) Culturing the host cells as described in claim 7 under suitable conditions to obtain a culture containing the small protein, recombinant protein, or fusion protein; and (b) The culture obtained in step (a) is purified and / or separated to obtain the small protein, recombinant protein or fusion protein targeting IL6Ra.

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