Ultrahigh-affinity small protein targeting IL6R and application

Abstract

The invention provides an ultra-high affinity small protein targeting IL6R (Interleukin-6 Receptor) and application thereof. Specifically, the invention provides a binding protein which targets IL6R and has ultrahigh affinity, the protein provided by the invention can be competitively bound with wild type IL6, and the affinity of the protein is far higher than that of the wild type IL6 to the IL6R. The invention also provides a fusion protein comprising the ultra-high affinity targeting IL6R (Interleukin-6 Receptor).

Description

Technical Field

[0001] The present invention belongs to the fields of biotechnology and medicine, and particularly relates to a class of small proteins with ultra-high affinity targeting IL6R and their uses. Background Art

[0002] Interleukin 6 (IL-6) is the most typical cytokine related to inflammation and plays an important role in host defense by regulating immune and inflammatory responses. After IL6R (IL6 receptor) binds to IL6, a complex is formed through IL6RA (the α subunit of IL6R) and other signal transduction factors to conduct signals, activate regeneration and anti-inflammatory signals, and promote the immunomodulatory properties of IL6. Therefore, the IL6R / IL6 signaling pathway is one of the important signaling pathways for the body to regulate immunity and exert immunosuppressive effects. At the same time, the abnormal activation of the IL6 signaling pathway is closely related to the occurrence and development of various diseases. Blocking the IL6RA / IL6 immunosuppressive signal has become one of the important strategies for current anti-tumor treatment.

[0003] In recent years, targeted therapies against the IL6RA / IL6 signaling pathway have shown good prospects in autoimmune diseases, inflammatory diseases, tumor treatment, and anti-bacterial / viral infections. How to more effectively block the binding of IL6RA / IL6 and thus more effectively inhibit the cytokine storm is an urgent problem to be solved currently.

[0004] In summary, there is an urgent need in this field to develop a candidate drug that can more efficiently block the binding of IL6RA / IL6, thereby more effectively blocking the binding of IL6RA / IL6 and more effectively inhibiting the cytokine storm. Summary of the Invention

[0005] The object of the present invention is to provide a class of small proteins with ultra-high affinity targeting IL6R, which can block the binding of IL6RA / IL6 and more effectively inhibit the cytokine storm.

[0006] Another object of the present invention is to provide a class of fusion proteins based on small proteins with ultra-high affinity targeting IL6 and their preparation methods.

[0007] In the first aspect of the present invention, a small protein targeting IL6R is provided, and the amino acid sequence of the small protein is selected from the following group:

[0008] (1) The amino acid sequence shown in SEQ ID NO: 1 or 3;

[0009] (2) An amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO: 1 or 3 (i.e., having a homology of ≥ 90%, preferably ≥ 95%, more preferably ≥ 98%), and retains the binding activity to IL6R (preferably, retains a binding activity of ≥ 70%, more preferably ≥ 80%).

[0010] In another preferred embodiment, the small protein blocks the binding of IL6R to IL6.

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

[0012] The present invention also provides a recombinant protein, which comprises two or more small proteins targeting IL6R of the present invention linked together.

[0013] In the second aspect of the present invention, there is provided a fusion protein, which comprises one or more polypeptides having a structure as shown in Formula I or Formula II.

[0014] P-Mx-H-F (Formula I)

[0015] P-F-H-Mx (Formula II)

[0016] Wherein,

[0017] P is an optional signal peptide sequence;

[0018] M is an IL6R binding region, and the IL6R binding region comprises a small protein targeting IL6R as described in the first aspect of the present invention, or the recombinant protein of the present invention;

[0019] H is an optional hinge region;

[0020] F is a constant region of an immunoglobulin, or a self-assembling protein, or a fragment thereof;

[0021] "-" represents a peptide bond or a linker peptide connecting the above elements;

[0022] x is a positive integer from 1 to 4.

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

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

[0025] In another preferred embodiment, the fusion protein is a homomultimer or a heteromultimer.

[0026] In another preferred embodiment, the fusion protein comprises 3 of the polypeptides and forms a trimer through the self-assembling protein in the polypeptides.

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

[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) the sequence as shown in SEQ ID NO: 5 or 7;

[0031] (ii) based on SEQ ID NO: 5 or 7, one or more amino acid residues are substituted, 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 the amino acid sequence.

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

[0033] In the third aspect of the present invention, there is provided a polynucleotide encoding the small protein targeting IL6R of the first aspect of the present invention, or its recombinant protein or the fusion protein of the second aspect of the present invention.

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

[0035] In the fourth aspect of the present invention, there is provided a vector containing the polynucleotide of the third aspect of the present invention.

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

[0037] In the fifth aspect of the present invention, there is provided a host cell containing the vector of the fourth aspect, or the polynucleotide of the third aspect is integrated into the genome.

[0038] In the sixth aspect of the present invention, there is provided an immunoconjugate, which contains:

[0039] (a) the small protein targeting IL6R of the first aspect of the present invention or its recombinant protein or the fusion protein of the second aspect; and

[0040] (b) a conjugate moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

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

[0042] In another preferred embodiment, the coupling moiety is a detectable label.

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

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

[0045] (a) a small protein targeting IL6R or its recombinant protein as described in the first aspect of the present invention, or the fusion protein as described in the second aspect of the present invention, or its encoding gene; or the immunoconjugate as described in the sixth aspect of the present invention; and

[0046] (b) a pharmaceutically acceptable carrier.

[0047] In another preferred embodiment, the pharmaceutical composition is used for diagnosing or treating IL6R-related diseases.

[0048] In another preferred embodiment, the IL6R-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%, preferably 10-99.9 wt%, 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 pharmaceutical 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 preparation is selected from the group consisting of: suspension preparations, liquid preparations, or lyophilized preparations.

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

[0056] In another preferred embodiment, the shelf life of the liquid preparation is from one to three years, preferably from one to two years, more preferably one year.

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

[0058] In another preferred embodiment, the shelf life of the freeze-dried preparation is half a year to two years, preferably half a year to one year, more preferably half a year.

[0059] In another preferred embodiment, the storage temperature of the freeze-dried preparation is ≤42°C, preferably ≤37°C, more preferably ≤30°C.

[0060] In another preferred embodiment, the pharmaceutically acceptable carrier includes: surfactants, solution stabilizers, isotonicity regulators, buffers, 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; 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 saccharide solution stabilizers, amino acid solution stabilizers, alcohol solution stabilizers, or combinations thereof.

[0063] In another preferred embodiment, the saccharide solution stabilizer is selected from the group consisting of reducing saccharide solution stabilizers or non-reducing saccharide 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 trihydric alcohols, higher polyhydric alcohols, propylene glycol, polyethylene glycol, or combinations thereof.

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

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

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

[0069] In another preferred embodiment, the non-human animals include: rodents (such as rats, mice), primates (such as monkeys).

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

[0071] In another preferred embodiment, the pharmaceutical composition or preparation is used for treating IL6R-related diseases.

[0072] In another preferred embodiment, the IL6R-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 preparation can be administered in combination with other antiviral or anti-inflammatory drugs.

[0076] In another preferred embodiment, the other antiviral or anti-inflammatory drugs for combined administration 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 drugs include anti-estrogens, aromatase inhibitors, or anti-androgens; preferably, the anti-estrogens are selected from the group consisting of: tamoxifen, droloxifene, exemestane, or combinations thereof; the aromatase inhibitors are selected from the group consisting of: aminoglutethimide, lulutron, letrozole, arimidex, or combinations thereof; the anti-androgens are selected from the group consisting of: flutamide, RH-LH agonists / antagonists: zoladex, enantone, or combinations thereof.

[0078] In another preferred embodiment, the biological response modifiers include: interferons, interleukin-2, thymopeptides, or combinations thereof.

[0079] In an eighth aspect of the present invention, there is provided a method for preparing the small protein targeting IL6R of the first aspect of the present invention, or its recombinant protein, or the fusion protein of the third aspect of the present invention, comprising the steps of:

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

[0081] (b) Purifying and / or separating the culture obtained in step (a) to obtain the small protein targeting IL6R, its recombinant protein, or the fusion protein.

[0082] In a ninth aspect of the present invention, there is provided the use of the small protein targeting IL6R described in the first aspect of the present invention, or its fusion protein, or its immunoconjugate, for preparing a medicament, a reagent, a test plate or a kit; wherein, the reagent, the test plate or the kit is used for: detecting IL6R in a sample; wherein, the medicament is used for treating and / or preventing IL6R-related diseases.

[0083] In another preferred embodiment, the IL6R-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 detection reagents, cellular immunofluorescence detection reagents, magnetic nanoparticles and imaging agents.

[0087] In another preferred embodiment, the reagent for detecting IL6R in the sample is a contrast agent for detecting IL6R (in vivo).

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

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

[0090] In another preferred embodiment, the medicament is used to block the interaction between IL6R and IL6.

[0091] In a tenth aspect of the present invention, there is provided a method for treating and / or preventing IL6R-related diseases, comprising the step of: administering a safe and effective amount of the small protein targeting IL6R described in the first aspect of the present invention or its recombinant protein, 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 to a subject in need thereof.

[0092] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described hereinafter (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 Shows a structural simulation diagram of the complex of the ultra-high affinity binding small protein targeting IL6R and human IL6R.

[0094] Figure 2 Shows the detection results of the thermal stability of the small protein structure targeting IL6R with high affinity.

[0095] Figure 3 Shows the detection results of the thermal recoverability of the small protein structure targeting IL6R with high affinity.

[0096] Figure 4 Shows the affinity determination results of the small protein targeting IL6R with high affinity.

[0097] Figure 5 Shows the affinity determination results of the multivalent high-affinity protein targeting IL6R.

[0098] Figure 6 Shows the protective effect of the multivalent high-affinity protein targeting IL6R on the animal model of abdominal infection. Detailed implementation manners

[0099] Through extensive and in-depth research, based on the wild-type IL6RA / IL6 protein structure, targeting the interaction surface between IL6R and IL6, through protein design, the inventors obtained a class of ultra-high-affinity small proteins targeting IL6R. The binding site of this small protein can almost completely cover the wild-type IL6R / IL6 binding site. Experiments show that the high-affinity small protein of the present invention has a much higher affinity than the wild-type IL6 protein and has a better ability to inhibit the cytokine storm. Based on this, the present invention was completed.

[0100] The high-affinity small protein and fusion protein targeting IL6R of the present invention

[0101] In the present invention, a class of high-affinity small proteins targeting IL6R and a fusion protein or conjugate containing the small protein are provided.

[0102] As used herein, the terms "the small protein of the present invention" and "the high-affinity small protein targeting IL6R of the present invention" can be used interchangeably, and both refer to the small protein having ultra-high affinity for IL6R as described in the first aspect of the present invention.

[0103] Preferably, the small protein of the present invention has the amino acid sequence shown in SEQ ID NO: 1 or 3.

[0104] As used herein, the term "the fusion protein of the present invention" refers to a fusion protein formed by the high-affinity small protein targeting IL6R of the present invention and other fusion elements. For example, the small protein of the present invention can form a fusion protein with elements such as a hinge region, an Fc region, and a self-assembling protein. The fusion protein of the present invention can block the binding of IL6 to IL6R.

[0105] As used herein, the term "self-assembling protein" refers to a protein that can self-assemble to form a multimer. In some embodiments, the high-affinity small protein targeting IL6R of the present invention can form a fusion protein with a self-assembling protein, thereby self-assembling to form a multimer (e.g., trimer) containing multiple small proteins of the present invention. The multimer of the present invention can be a homomultimer or a heteromultimer, that is, the multimer can contain the same or different high-affinity small proteins targeting IL6R.

[0106] Preferably, the ultra-high-affinity fusion protein of the present invention can be any that contains at least a complete high-affinity small protein targeting IL6R or a partial amino acid fragment thereof (usually an amino acid fragment with at least 70% length).

[0107] Typically, the fusion protein of the present invention may have the following structures:

[0108] A Y-shaped structure of a high-affinity small protein or fragment targeting IL6R - Hinge - CH2 - CH3;

[0109] A Y-shaped structure of a high-affinity small protein or fragment targeting IL6R - Hinge - CH3;

[0110] A high-affinity small protein or fragment targeting IL6R - tracer label;

[0111] A high-affinity small protein or fragment targeting IL6R - a multimeric structure of a self-assembling protein;

[0112] A high-affinity small protein or fragment targeting IL6R.

[0113] It should be understood that the above structural types are only exemplary and do not limit the present invention. The high-affinity small protein targeting IL6R or a fragment thereof can be single or multiple (such as 2, 3, or 4 ultra-high-affinity small proteins or fragments thereof in tandem form).

[0114] As used herein, the term "small protein with high affinity targeting IL6R" or "fusion protein" also includes variant forms having IL6R binding activity and IL6 / IL6R blocking activity. These variant forms include (but are not limited to): deletion, insertion and / or substitution of 1-3 (usually 1-2, preferably 1) amino acids, addition or deletion of one or several (usually within 3, preferably within 2, more preferably within 1) amino acids at the C-terminus and / or N-terminus, or addition of amino acid fragments with 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, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding or deleting one or several amino acids at the C-terminus and / or N-terminus usually does not change the structure and function of the protein. In addition, the term also includes polypeptides of the present invention in monomeric and multimeric forms. The term also includes linear and non-linear polypeptides (such as cyclic peptides).

[0115] The present invention also includes active fragments, derivatives and analogs of the above-mentioned small protein or fusion protein targeting IL6R (especially the fusion protein formed with Fc fragment). As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the function or activity of the high-affinity small protein or fusion protein targeting IL6R of the present invention.

[0116] The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) polypeptides having substitution groups in one or more amino acid residues, or (iii) polypeptides formed by fusion of a polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to this polypeptide sequence (fusion proteins formed by fusion with a leader sequence, a secretory sequence or a tag sequence such as 6His). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0117] A preferred class of active derivatives refers to polypeptides formed by replacing at most 5, preferably at most 3, and more preferably at most 1 amino acid with similar or similar properties compared to the amino acid sequence of the present invention. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.

[0118] Table A

[0119] Initial residue Representative substitution Preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0120] The present invention also provides analogs of the fusion proteins of the present invention. These analogs may differ from the polypeptides of the present invention in terms of amino acid sequence, or in modified forms that do not affect the sequence, or both. Analogs also include those with residues different from natural L-amino acids (such as D-amino acids), and those with non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0121] In addition, the high-affinity small proteins or fusion proteins targeting IL6R of the present invention can also be modified. Modification forms (usually without changing the primary structure) include: chemically derivatized forms of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modification also includes glycosylation, such as polypeptides produced by glycosylation modification during the synthesis and processing of polypeptides or further processing steps. Such modification can be achieved by exposing the polypeptide to glycosylating enzymes (such as mammalian glycosylating enzymes or deglycosylating enzymes). Modification forms also include sequences having phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that are modified to improve their proteolytic resistance or optimize their solubility.

[0122] The term "polynucleotide of the present invention" can be a polynucleotide encoding the high-affinity small protein or fusion protein targeting IL6R of the present invention, or a polynucleotide that also includes additional coding and / or non-coding sequences.

[0123] The present invention also relates to variants of the above polynucleotides, which encode fragments, analogs, and derivatives of polypeptides or fusion proteins having the same amino acid sequence as the present invention. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, allelic variants are alternative forms of a polynucleotide that may be substitutions, deletions, or insertions of one or more nucleotides, but do not substantially change the function of the encoded high-affinity small protein or fusion protein targeting IL6R.

[0124] The present invention also relates to polynucleotides that hybridize with the above sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions (or tight conditions). In the present invention, "stringent conditions" refer to: (1) hybridization and washing at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) adding denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least above 90%, preferably above 95%.

[0125] The high-affinity small proteins or fusion proteins targeting IL6R of the present invention and the polynucleotides are preferably provided in isolated form, and more preferably, purified to homogeneity.

[0126] The full-length sequences of the polynucleotides of the present invention can generally be obtained by PCR amplification, recombination or artificial synthesis methods. For PCR amplification, primers can be designed according to the nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art can be used as a template for amplification to obtain the relevant sequences. When the sequence is relatively long, it is often necessary to perform PCR amplification two or more times, and then splice the fragments amplified each time together in the correct order.

[0127] Once the relevant sequences are obtained, the relevant sequences can be obtained in large quantities by recombination methods. This is usually to clone them into a vector, then transfer them into cells, and then isolate the relevant sequences from the proliferated host cells by conventional methods.

[0128] In addition, artificial synthesis methods can also be used to synthesize the relevant sequences, especially when the fragment length is relatively short. Usually, a very long fragment can be obtained by first synthesizing multiple small fragments and then ligating them.

[0129] Currently, it is already possible to completely obtain the DNA sequence encoding the protein (or its fragment, or its derivative) of the present invention by chemical synthesis. Then this DNA sequence can be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.

[0130] The method of using PCR technology to amplify DNA / RNA is preferably used to obtain the polynucleotides of the present invention. Especially when it is difficult to obtain full-length cDNA from a library, the RACE method (rapid amplification of cDNA ends) can be preferably used. The primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein and can be synthesized by conventional methods. The amplified DNA / RNA fragments can be separated and purified by conventional methods such as gel electrophoresis.

[0131] Expression vector

[0132] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells genetically engineered with the vectors of the present invention or the coding sequences of the high-affinity small proteins or fusion proteins targeting IL6R of the present invention, and methods for producing the polypeptides of the present invention by recombinant techniques.

[0133] By conventional recombinant DNA techniques, the polynucleotide sequences of the present invention can be used to express or produce recombinant fusion proteins. Generally, there are the following steps:

[0134] (1). Transform or transduce a suitable host cell with the polynucleotide (or variant) encoding the fusion protein of the present invention, or with a recombinant expression vector containing the polynucleotide.

[0135] (2). A host cell cultured in a suitable medium.

[0136] (3). Isolate and purify the protein from the medium or the cells.

[0137] In the present 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, phages, 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 be stable in the host. An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene and translation control elements.

[0138] In the method for preparing the high-affinity small protein targeting IL6R or its fusion protein of the present invention, any suitable vector can be used, which can be selected from one of pET, pDR1, pcDNA3.1(+), pcDNA3.1 / ZEO(+), pDHFR. The expression vector includes a fusion DNA sequence ligated with appropriate transcriptional and translational regulatory sequences.

[0139] Both eukaryotic / prokaryotic host cells can be used for the expression of the high-affinity small protein targeting IL6R or its fusion protein of the present invention. Eukaryotic host cells are preferably mammalian or insect host cell culture systems, preferably cells such as COS, CHO, NS0, sf9 and sf21; prokaryotic host cells are preferably one of lemo21, DH5a, BL21(DE3), TG1.

[0140] Methods well known to those skilled in the art can be used to construct an expression vector containing the DNA sequence encoding the fusion protein of the present invention and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, etc. The said DNA sequence can be effectively ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of Escherichia coli; the λ phage PL promoter; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the LTRs of retroviruses and some other known promoters that can 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.

[0141] In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0142] Vectors containing the appropriate DNA sequences described above, along with appropriate promoters or control sequences, can be used to transform appropriate host cells to enable them to express proteins.

[0143] The host cell can 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. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, plant cells (such as ginseng cells).

[0144] When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs in length, which act on promoters to enhance gene transcription. Examples include the 100 to 270 base pair SV40 enhancer on the late side of the replication origin, the polyomavirus enhancer on the late side of the replication origin, and the adenovirus enhancer, etc.

[0145] Those of ordinary skill in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0146] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as Escherichia coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, and the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0147] The obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional media. Culturing is carried out under conditions suitable for the growth of the host cell. When the host cell grows to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.

[0148] The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be separated and purified by various separation methods using its 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 renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0149] The high-affinity small protein targeting IL6R or its fusion protein disclosed in the present invention can be separated and purified by affinity chromatography. Depending on the characteristics of the affinity column used, conventional methods such as high-salt buffer, pH change, etc. can be used to elute the high-affinity small protein targeting IL6R or its fusion protein bound to the affinity column.

[0150] Using the above method, the high-affinity small protein targeting IL6R or its fusion protein can be purified into a substantially homogeneous substance, such as a single band on SDS-PAGE electrophoresis.

[0151] Pharmaceutical composition

[0152] In the present invention, there is also provided a pharmaceutical composition containing the small protein targeting IL6R or its fusion protein or its immunoconjugate of the present invention.

[0153] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001 - 99 wt%, preferably 0.01 - 90 wt%, more preferably 0.1 - 80 wt%) of the small protein or fusion protein (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and their combinations. The pharmaceutical preparation should be matched with the administration method. The pharmaceutical composition of the present invention can be made into an injection form, for example, prepared by a conventional method with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, such as about 10 micrograms per kilogram of body weight per day - about 50 milligrams per kilogram of body weight. In addition, the polypeptide of the present invention can also be used together with other therapeutic agents. The small protein or fusion protein targeting IL6R or its immunoconjugate can form a pharmaceutical preparation together with pharmaceutically acceptable excipients to exert its efficacy more stably. These preparations can ensure the structural integrity of the amino acid core sequence of the small protein or its fusion protein targeting IL6R of the present invention, and at the same time protect the multiple functional groups of the protein from degradation (including but not limited to aggregation, deamination or oxidation). The preparations can be in various forms. Generally, for liquid preparations, they can be stored stably at least for one year at 2°C - 8°C, and for freeze-dried preparations, they can remain stable at 30°C for at least six months. Here, the preparations can be suspensions, aqueous injections, freeze-dried preparations, etc. commonly used in the pharmaceutical field, preferably aqueous injections or freeze-dried preparations.

[0154] For the pharmaceutical composition targeting IL6R of the present invention (such as aqueous injection or freeze-dried preparation), the pharmaceutically acceptable excipients include one or a combination of surfactants, solution stabilizers, isotonicity regulators, and buffer solutions. The surfactants include non-ionic 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, linoleyl or octadecyl sarcosine; Pluronics; MONAQUATTM, etc. The added amount should minimize the tendency of the protein to granulate. The solution stabilizers can be sugars, including reducing sugars and non-reducing sugars, amino acids including monosodium glutamate or histidine, alcohols including trihydric alcohols, higher polyhydric alcohols, propylene glycol, polyethylene glycol, or a combination of them. The added amount of the solution stabilizer should make the finally formed preparation maintain a stable state within a stable time considered by those skilled in the art. The isotonicity regulator can be one of sodium chloride and mannitol, and the buffer solution can be one of TRIS, histidine buffer solution, and phosphate buffer solution.

[0155] When using the pharmaceutical composition, a safe and effective amount of the small protein, fusion protein or immunoconjugate of the present invention is administered to a mammal, wherein the safe and effective amount is usually 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 about 100 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight. Of course, the specific dose should also consider factors such as the administration route and the patient's health condition, which are all within the scope of the skills of a skilled physician. Typically, usually, the total administered amount cannot exceed a certain range. For example, the dose for intravenous injection is 10 to 3000 mg / day / 50 kg, preferably 100 to 1000 mg / day / 50 kg.

[0156] The main advantages of the present invention include:

[0157] 1) The small protein targeting IL6R provided by the present invention has a binding site that can cover the binding of wild-type IL6R to IL6.

[0158] 2) The small protein of the present invention has an extremely high affinity for human IL6, far higher than the affinity of wild-type IL6R for IL6.

[0159] 3) The small protein of the present invention has extremely high structural stability, and its Tm value is greater than 95 °C.

[0160] 4) The protein of the present invention exhibits extremely strong ability to inhibit cytokine storm and can significantly improve the survival rate of animal models.

[0161] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out 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 according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0162] The sequences of the present invention are shown in Table 1 below.

[0163] Table 1. Sequences of the present invention

[0164]

[0165]

[0166] Example 1: Construction, expression and purification of a small protein with high affinity for IL6R

[0167] The nucleotide sequence of the IL6R protein was synthesized by gene synthesis, and auxiliary sequences MGS and GSENLYFQSLEHHHHHH (SEQ ID NO:9) were added to both ends of the sequence. It was inserted into the pET29b vector in the form of MGS-IL6R protein nucleotide sequence-GSENLYFQSLEHHHHHH. After transforming this vector into Escherichia coli, it was cultured in LB medium at 37 °C and 270 rpm until OD 600 = 0.6. Then, 1 mM IPTG was used to induce protein expression in the bacterial liquid overnight. After centrifuging to collect the bacteria and resuspending them, the bacteria were lysed with a cryogenic mechanical disruptor and then centrifuged at high speed to obtain the supernatant. After purification by Ni column, the protein sample was desalted and concentrated, and the concentration of the purified protein was measured with a micro-spectrophotometer. The purified protein was filtered and sterilized with a 0.22 μm filter membrane and then placed in a 4 °C refrigerator for subsequent experiments.

[0168] Example 2: Detection of the thermal stability of the structure of the high-affinity small protein targeting IL6R

[0169] The thermal stability of the protein structure was detected with the Unchained Labs all-in-one protein stability analyzer Uncle. The temperature range for sample heating was 25 °C - 95 °C, and the heating rate was 0.5 °C / min. Static light scattering was measured at an excitation wavelength of 266 nm. As the protein unfolds, fluorescence usually decreases and the wavelength shifts towards a longer direction. The data analysis software determines the Tm value based on the average value of the center of mass (BCM) of the fluorescence intensity curve at 300 - 430 nm, and observes the aggregation situation Tagg based on the light intensity scattered at 266 nm. The changes in the secondary structure conformation of the protein at different temperatures were obtained, and then the structural stability of the binding protein was evaluated.

[0170] As Figure 2 shown, the denaturation curves indicate that neither IL6_8A_rd-5 nor IL6_8A_rd-7 has an obvious Tm. The change amplitude of the denaturation curve of IL6_8A_rd-7 is 5 nm, and the conformational change is greater. The aggregation curves show that the initial aggregation temperature Tagg of IL6_8A_rd-7 is 44 °C, and the degree of aggregation is not high; there is no obvious aggregation in IL6_8A_rd-5. The experimental results show that the protein exhibits extremely strong thermal stability.

[0171] Example 3: Detection of the thermal recoverability of the structure of the high-affinity small protein targeting IL6R

[0172] The thermal recovery of protein structure was tested with the help of Uncle, an all-purpose protein stability analyzer from Unchained Labs. The sample was heated from 20°C to 95°C and then cooled to 20°C, with a step temperature of 15°C, to detect changes in protein conformation and aggregation during the heating and cooling process. As the temperature of the sample increases, the conformation opens, and when the temperature decreases, the conformation folds back. The sample has a good ability to recover from thermal shock.

[0173] like Figure 3 As shown in the figure, the conformation of IL6_8A_rd-7 opens with the increase of temperature, and folds back with the decrease of temperature. The conformational thermal recovery ability of this sample is relatively good; the aggregation signal of this sample increases with the increase of temperature, and decreases slightly with the decrease of temperature. A small amount of samples disaggregate, but the overall aggregation is irreversible. The conformation of IL6_8A_rd-5 changes slightly with the change of temperature, but the overall change is not large, indicating that the conformational stability of this sample is relatively strong; in addition, the aggregation signal changes slightly with the change of temperature, but the overall change is not large, indicating that this sample is not easy to aggregate. Compared with IL6_8A_rd-7, IL6_8A_rd-5 is more stable.

[0174] Example 4: Affinity determination of a small protein with high affinity targeting IL6R

[0175] In this embodiment, affinity detection of high-affinity blocking proteins was performed with the aid of ForteBio Octet. First, 3 μg / ml of biotin-labeled human IL6 protein was loaded onto the detection probe coupled with avidin (600s), and the unbound biotin-labeled human IL6 protein was eluted in a PBST solution. Then the detection probe with human IL6 protein was simultaneously immersed in a two-fold diluted solution of the targeted IL6 high-affinity small protein to detect the binding signal (120s). The probe was then immersed in PBST to detect the dissociation signal of the binding protein. Finally, the affinity of the high-affinity blocking binding protein was calculated.

[0176] like Figure 4 As shown in Figure 2, IL6_8A_rd-5 and IL6_8A_rd-7 showed strong binding activity, with affinities of 4.951×10 -9 M and 5.805×10 -9 M.

[0177] Example 5: Construction, expression and purification of multivalent high-affinity proteins targeting IL6R

[0178] The gene sequence of IL6_8A_rd-5 was synthesized by gene synthesis and linked to the N-terminus or C-terminus of the trimer protein nucleotide sequence through GSlinker (for the trimer protein, see patent application number: CN202411374093.4). And this sequence was inserted into the pET29b vector in the form of MGS - protein nucleotide sequence - GSENLYFQSLEHHHHHH, where the ENLYFQSLE (SEQ ID NO:10) sequence was used for enzyme digestion and HHHHHH (SEQ ID NO:11) was used for protein purification. After transforming this vector into Escherichia coli, it was cultured in LB medium at 37 °C and 270 rpm until OD 600 = 0.6. Then, 1 mM IPTG was used to induce protein expression in the bacterial liquid overnight. After centrifuging to collect the bacteria and resuspending them, the bacteria were lysed with a cryogenic mechanical crusher and then centrifuged at high speed to collect the supernatant. After purification by Ni column, the protein sample was desalted and concentrated, and the concentration of the purified protein was measured with a micro - spectrophotometer. The purified protein was filtered and sterilized with a 0.22 - μm filter membrane and then placed in a 4 °C refrigerator for subsequent experiments.

[0179] Example 6: Affinity determination of the multivalent high - affinity protein targeting IL6R

[0180] In this example, the ForteBio Octet was used to detect the affinity of the high - affinity protein. First, 3 μg / ml of IL6Ra - Fc protein was loaded onto the detection probe of ProA (1200 s), and the unbound IL6Ra - Fc protein was eluted in PBST solution. Then, the detection probe with human IL6Ra protein was simultaneously immersed in the solution of the multivalent high - affinity protein targeting IL6R diluted two - fold serially, and the binding signal was detected (180 seconds). Then the probe was immersed in PBST (300 seconds) to detect the dissociation signal of the bound protein. Finally, the affinity of the high - affinity blocking binding protein was calculated.

[0181] As Figure 5 shown, both IL6Ra - 5 - C3 - 50 - 70 and C3 - 50 - 70 - IL6Ra - 5 showed extremely strong binding activity, with affinities of 2.192×10 -9 M and 4.987×10 -9 M respectively.

[0182] Example 7: Protective effect of the multivalent high - affinity protein targeting IL6R on the animal model of abdominal infection

[0183] In this embodiment, two animal models of cecal ligation and puncture (CLP) and lipopolysaccharide (LPS) intraperitoneal injection were used to verify the protective effect of the high-affinity blocking protein on the cytokine storm. First, C57BL / 6 mice aged 6-8 weeks were selected, and an intraperitoneal infection model was constructed by using the standard CLP surgery and the method of 10 mg / kg LPS intraperitoneal injection. Then, the mice were intraperitoneally injected with 0.5 mg / kg tocilizumab (TCZ) or the multivalent high-affinity protein targeting IL6R. As Figure 6 shown, by observing the survival of mice in each group for 7 days, it was found that the mortality rate of the model animals given the multivalent high-affinity protein targeting IL6R decreased by 43.8% compared with the TCZ group, showing better protective activity.

[0184] All the documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A small protein targeting IL6R, characterized in that: The amino acid sequence of the small protein is selected from the group consisting of: (1) the amino acid sequence shown in SEQ ID NO: 1 or 3; (2) An amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO: 1 or 3 (i.e., homology ≥90%, preferably ≥95%, and more preferably ≥98%), and that retains binding activity to IL6R (preferably, retains ≥70%, and more preferably ≥80% of the binding activity).

2. The small protein targeting IL6R according to claim 1, characterized in that The amino acid sequence of the small protein is shown in SEQ ID NO: 1 or SEQ ID NO:

3.

3. A recombinant protein, characterized in that The recombinant protein comprises two or more small proteins targeting IL6R according to claim 1 or 2 connected in series.

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 is none or a signal peptide sequence; M is an IL6R binding region, and the IL6R binding region comprises the small protein targeting IL6R as claimed in claim 1, or the recombinant protein as claimed in claim 3; H is none or hinge region; F is the constant region of an immunoglobulin, or a self-assembling protein, or a fragment thereof; "-" indicates a peptide bond or connecting peptide connecting the above elements; x is a positive integer from 1 to 4.

5. A polynucleotide, characterized in that The polynucleotide encodes the small protein targeting IL6R 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 vector contains the polynucleotide according to claim 5.

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

8. An immunoconjugate, characterized in that: The immunoconjugate contains: (a) the small protein targeting IL6R 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) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

9. A pharmaceutical composition, characterized in that include: (a) the small protein targeting IL6R as described in claim 1 or 2, or the recombinant protein as described in claim 3, or the fusion protein as described in claim 3, or the encoding gene thereof; or the immunoconjugate as described in claim 8; and (b) a pharmaceutically acceptable carrier.

10. A method for preparing the small protein targeting IL6R as claimed in claim 1 or 2, or the recombinant protein as claimed in claim 3, or the fusion protein as claimed in claim 4, characterized in that: Includes steps: (a) culturing the host cell according to claim 7 under suitable conditions to obtain a culture containing the small protein or recombinant protein or fusion protein; and (b) purifying and / or separating the culture obtained in step (a) to obtain the small protein or recombinant protein or fusion protein targeting IL6R.

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