A recombinant human interleukin-11 mutant and its conjugate with a chemical molecule, and a preparation method and application thereof

Abstract

The application provides a recombinant human interleukin-11 mutant and a conjugate of the mutant and a chemical molecule, and a preparation method and application thereof. The recombinant human interleukin-11 mutant is obtained by using a method of tandem fusion of Sumo sequences, and is realized in soluble expression in an E. coli expression system. The conjugate is obtained by site-specific coupling of the mutant and the chemical molecule on a mutant amino acid residue at a W147 site through a covalent bond. The chemical molecule is at least one of polyethylene glycol, an alkanoic fatty chain and a hydrophilic polymer containing the alkanoic fatty chain. The conjugate retains the binding ability to human interleukin-11 receptor A, and the binding ability to human IL-6 signal transducer receptor is greatly reduced. The conjugate can block the signal transduction between human IL-11 and IL-11RA and GP130 in vivo or in vitro, and can be used for treating diseases related to tissue fibrosis as a clinical symptom caused by primary or secondary IL-11 elevation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, and particularly relates to a recombinant human interleukin-11 mutant, a conjugate of the mutant and a chemical molecule, and a preparation method and application thereof. BACKGROUND

[0002] Human interleukin-11 (hIL-11) is one of many important cytokines in the human body, belonging to the human IL-6 family of cytokines. In addition, the IL-6 family of cytokines also includes IL-6, IL-27, IL-31, LIF, CNTF, CT-1 and OSM, etc. The physiological function of human IL-11 in the body is mainly through the formation of a complex with IL-11RA and IL-6ST / GP130 receptors to conduct physiological signal transduction, and its mechanism of action includes three pathways: classic signaling, trans-signaling and cluster signaling (Emerging roles for Interleukin-11 in disease, 2019). In these three signaling pathways, IL-11 needs to first bind to IL-11RA with relatively low affinity (Kd = ~ 10 nM) to form an IL-11-IL-11RA dimer complex, and then the IL-11-IL-11RA dimer complex binds to the IL-6ST / GP130 receptor with higher affinity (Kd = 300-800 pM) to form an IL-11-IL-11RA-IL-6ST / GP130 trimer complex, and finally two trimer complexes are assembled into an IL-11-IL-11RA-IL-6ST / GP130 hexamer complex to successfully achieve IL-11 signaling. The IL-6ST / GP130 receptor involved in signal transduction is a commonly used receptor for IL-6 family cytokine signaling, and IL11-RA is a specific receptor for IL-11.

[0003] The natural mature human interleukin-11 (UniProtKB-P20809) is composed of 178 amino acids, which contains a high proportion of proline and leucine, does not contain cysteine, and has no glycosylation site. Its theoretical molecular weight is about 19 kDa, and the theoretical isoelectric point is about 11.16. IL-11 can synergize with various cytokines in the human body to exert physiological effects, stimulate the growth of human erythrocyte, megakaryocyte precursor cells, and induce megakaryocyte maturation, and promote platelet production. rhIL-11 (Oprelvekin, Neulasta) produced by Genetic Institute Co., Ltd. is a recombinant human IL-11, which is used for the prevention of chemotherapy-induced neutropenia and thrombocytopenia, and is also used for the treatment of thrombocytopenia caused by acute myeloid leukemia and myelodysplastic syndrome. )Early in 1997, it was approved by the FDA for the treatment of cancer patients during the treatment of radiation therapy, chemotherapy-induced thrombocytopenia. The rhIL-11 produced by the Genetic Institute contains 177 amino acids, and compared with natural human IL-11, it lacks the N-terminal proline. Although one amino acid is missing, its biological activity in vivo and in vitro is not significantly different from that of IL-11 with natural structure.

[0004] In recent years, studies have found that IL-11 levels in patients with idiopathic pulmonary fibrosis (IPF) are significantly elevated, suggesting that IL-11 may be involved in the development of pulmonary fibrosis disease process, and play a certain physiological and pathological role. After neutralizing mouse IL-11 with anti-IL-11 antibody, it was found that it could alleviate and delay pulmonary fibrosis caused by bleomycin (Interleukin-11 is a therapeutic target in idiopathic pulmonary fibrosis, 2019), indicating that inhibiting the activity of endogenous IL-11 is expected to control or reverse the progression of pulmonary fibrosis disease. In addition, studies have shown that increasing the expression level of endogenous IL-11 in mice through transgenic means or directly by continuously injecting exogenous IL-11 into healthy mice can cause fibrosis in multiple organs of mice, including liver fibrosis, kidney fibrosis, etc., and even eventually lead to multiple organ failure in mice (IL-11 is a crucial determinant of cardiovascular fibrosis, 2017), indicating that IL-11 may be involved in the pathological changes of tissue and organ fibrosis in the body, suggesting that IL-11 may be a potential therapeutic target molecule for inhibiting tissue and organ fibrosis. In addition, studies have found that IL-11 is involved in the occurrence and progression of gastric, intestinal and other tumors and vascular diseases (IL-11 signaling as a therapeutic target for cancer, 2015; A neutralizing IL-11 antibody reduces vessel hyperplasia in a mouse carotid artery wire injury model, 2021).

[0005] For the development of inhibitors interfering with the IL-11 signaling pathway, currently mainly antibody drugs are used, mainly targeting IL-11 or IL-11RA to screen antibodies, and the antibodies are combined with IL-11 or IL-11RA to block the IL-11 signaling pathway. For example, Boehringer-Ingelheim developed BI765423 antibody targeting IL-11 as a target, its main mechanism of action is to inhibit the formation of IL-11-IL-11RA dimer complex between IL-11 and IL-11RA, and then block IL-11 signaling. Another way is to inhibit the formation of IL-11-IL-11RA-IL-6ST / GP130 trimer complex by inhibiting the formation of IL-11-IL-11RA dimer complex and IL-6ST / GP130 receptor, and then block the IL-11 signaling pathway, for example, based on the structure of IL-11, site-directed mutagenesis is used to obtain IL-11 mutants with reduced affinity to IL-6ST / GP130, such as IL-11(W147A) mutant, but the IL-11(W147A) mutant still has the problems of insufficient affinity to IL-6ST / GP130 receptor and easy to be quickly cleared in vivo. SUMMARY

[0006] The present application is based on the structural characteristics of human IL-11 and its mechanism of participating in the signal pathway in vivo, and aims to solve the problems in the prior art and the above-mentioned difficulties. A mutant IL-11 (hereinafter referred to as IL-11M) is designed and screened, which retains the binding affinity to the specific receptor IL-11RA of human IL-11 and the ability to form an IL-11M-IL-11RA dimer complex, but greatly reduces the binding affinity of the IL-11M-IL-11RA dimer complex to the common receptor IL-6ST / GP130 of the IL-11 family of cytokines and the ability to form an IL-11M-IL-11RA-IL-6ST / GP130 trimer complex. In addition, the IL-11M mutant is coupled with a chemical molecule through site-directed modification at the mutation site (e.g., W147C) to obtain an IL-11M conjugate. Compared with IL-11M, the IL-11M conjugate retains the binding affinity to the specific receptor IL-11RA of human IL-11 and the ability to form an IL-11M conjugate-IL-11RA dimer complex, but the binding affinity to the common receptor IL-6ST / GP130 of the IL-11 family of cytokines is greatly reduced, making it difficult to form an IL-11M conjugate-IL-11RA-IL-6ST / GP130 trimer. In addition, the IL-11M conjugate exhibits a longer in vivo circulation half-life than IL-11M, and significantly delays the progression of fibrosis in a bleomycin-induced idiopathic pulmonary fibrosis (IPF) model in mice, and even improves or reverses the effect.

[0007] To achieve the above object, the technical scheme adopted by the present application is as follows:

[0008] A recombinant human interleukin-11 mutant, which is obtained by adding, deleting, truncating or substituting one or more amino acid sequences at the end or middle of the amino acid sequence of human interleukin-11, specifically, the tryptophan at position 147 is mutated to cysteine or a non-natural amino acid carrying acetylene or / and azide.

[0009] The preparation method of the recombinant human interleukin-11 mutant as described above comprises the following steps:

[0010] Step 1: converting the gene sequence of human interleukin-11 and optimizing the host preferred codon of Escherichia coli, and replacing the codon at position W147 to obtain a human interleukin-11M mutant;

[0011] Step 2, splice the gene sequence of human interleukin-11M mutant with the 3' end of the gene sequence of small ubiquitin-related modifier tandem gene sequence to obtain the gene sequence of His-Sumo-IL-11M;

[0012] Step 3, insert the gene sequence of His-Sumo-IL-11M obtained in step 2 into a prokaryotic expression plasmid vector, and then transform the plasmid vector into a prokaryotic competent cell;

[0013] Step 4, select the dominant growth colony by resistance coating, and perform expansion culture, and induce expression of the target gene by IPTG or other lactose structural analogs; lyse the bacterial body, centrifuge to obtain the supernatant, and obtain the recombinant His-Sumo-IL-11M fusion protein with high purity through purification, and obtain the recombinant human interleukin-11 mutant after removing the His-Sumo fusion sequence by enzyme digestion.

[0014] Further, the recombinant human interleukin-11 mutant is covalently coupled with a chemical molecule at the mutant amino acid residue of W147 to obtain a site-directed coupling product.

[0015] Further, the molecular weight of the chemical molecule is 0.1-100 kDa.

[0016] The application provides a recombinant human interleukin-11 mutant, a conjugate of the mutant and a chemical molecule, and a preparation method and application thereof. The recombinant human interleukin-11 mutant is obtained by using a Sumo sequence tandem fusion method to realize soluble expression in an E. coli expression system. The conjugate is obtained by covalently coupling the mutant with a chemical molecule at the mutant amino acid residue of W147. The chemical molecule is at least one of polyethylene glycol, an alkanoic fatty chain, and a hydrophilic polymer containing an alkanoic fatty chain. The conjugate retains the binding ability with human interleukin-11 receptor A, and the binding ability with human IL-6 signal transduction receptor is greatly reduced. The conjugate can block the signal transduction between human IL-11 and IL-11RA and GP130 in vivo or in vitro, and can be used for treating diseases related to tissue fibrosis caused by primary or secondary IL-11 increase.

[0017] A preparation method of the conjugate of the recombinant human interleukin-11 mutant and the chemical molecule, comprising the following steps:

[0018] S1, the recombinant human interleukin-11 mutant is replaced into phosphate or Tris-HCl buffer system; the pH value of the solution is adjusted to 6.0-9.0; the ionic strength is adjusted to 5-50 mS / cm; the IL-11M protein concentration is adjusted to 0.2-10 mg / ml;

[0019] S2, the chemical molecules are mixed with the recombinant human interleukin-11M protein solution obtained in S1 at a molar ratio of 0.2-50:1, stirring, the reaction temperature is 2-40℃, and the reaction time is 0.5-48h;

[0020] S3, the sample after the coupling reaction obtained in step S2 is subjected to hydrophobic chromatography or ion exchange chromatography purification to obtain a recombinant human interleukin-11 mutant and chemical molecule conjugate.

[0021] Further, in S1, the pH value of the solution is adjusted to 6.5-7.5; the ionic strength is adjusted to 5-10 mS / cm; the IL-11M protein concentration is adjusted to 0.5-3.0 mg / ml.

[0022] Further, in S2, the molar ratio of the chemical molecules to the recombinant human interleukin-11M protein is 1-5:1; in S2, the reaction temperature is 4-25℃, and the reaction time is 2-8h.

[0023] Further, in S3, the hydrophobic chromatography filler is a medium with hydrophobic ligands butyl sulfide, butyl, octyl or phenyl; the ion exchange chromatography filler is a medium with sulfonic acid groups or quaternary ammonium salt.

[0024] The recombinant human interleukin-11 mutant and chemical molecule conjugate as described above is used in the preparation of drugs for diseases related to tissue fibrosis as a clinical manifestation of primary or secondary IL-11 elevation.

[0025] Further, the tissue fibrosis is at least one of pulmonary fibrosis, myocardial fibrosis, liver fibrosis, bladder fibrosis and renal fibrosis.

[0026] Further, based on the human-derived IL-11 amino acid sequence (UniProtKB-P20809, with or without the N-terminal proline (Pro) reserved), the gene sequence of IL-11 is obtained by codon E. coli preference optimization, and on this basis, a specific amino acid (W147) site mutation is carried out, and the mutation site nucleotide codon is TGC, TGT or TAA, TAG, TGA, to derive the IL-11M mutant. Through PCR technology, under the action of specific restriction enzymes and specific primers, the gene sequence of IL-11M can be inserted into the E. coli expression vector, and the obtained cloning vector containing the IL-11M target gene is introduced into the E. coli competent cells, and the positive cloning vector is screened by plating culture on solid medium to obtain the positive cloning colonies. The selected dominant cloning bacteria are cultured and induced for expression, and the IL-11M protein is obtained by multi-step chromatography purification. Then the IL-11M protein is covalently coupled with the chemical molecule at the residue of the mutated amino acid at the W147 site to obtain the IL-11M conjugate.

[0027] Preferably, the recombinant human interleukin-11 protein expressed in E. coli by the method has an amino acid sequence comprising the entire human IL-11 amino acid sequence or truncated by one or more amino acid residues at the N-terminus, and on the basis of this amino acid sequence, the tryptophan residue (Trp or W) at position 147 (numbered from the N-terminus of the complete human interleukin-11 amino acid sequence) in the sequence is mutated to a cysteine residue (Cys, C) or other types of unnatural amino acid residues that can be used for click chemistry coupling (such as unnatural amino acids carrying ethynyl (—C≡CH) and azido (—N≡N) and their derivative structures), to obtain an interleukin-11 mutant (IL-11M), and the nucleotide codon sequence of the W147 mutant is TGC, TGT or TAA, TAG, TGA.

[0028] Further, in the IL-11M conjugate molecule, the covalent bond of IL-11M and the chemical molecule occurs at the amino acid residue of the mutation site, in particular, for example, the free thiol group on the cysteine residue of the W147C mutant.

[0029] Further, the covalently coupled chemical molecule is characterized by a molecular weight generally distributed in the range of 0.1-100 kDa, and representative backbone structures include polyethylene glycol (PEG), alkyl fatty chains (C8-C20), and other hydrophilic polymer structures, but are not limited to this.

[0030] Further, the structure of the coupling molecule can be linear or branched, and one of its ends can be derived by chemical synthesis to have a functional group that can be efficiently coupled with a group on a specific amino acid residue. These activation derivation methods or functional group structures include maleimide (-Maleimide), vinyl sulfone (-Vinyl sulfone), iodoacetamide (Iodoacetamide), acetylene (—C≡CH), and azide (—N≡N) and their derivatives, but are not limited to these.

[0031] Further, the IL-11M and IL-11M conjugate molecules retain the ability to bind to human IL-11RA with high affinity, and the ability of both to form a dimeric complex with human IL-11RA and then bind to human IL-11ST / GP130 receptors to form a trimeric complex is greatly reduced or even completely lost. That is, the ability of IL-11M and IL-11M conjugate molecules to form a complete trimeric complex or hexameric complex with IL-11RA and IL-11ST / GP130 receptors is greatly reduced or even completely lost, which can potentially be used as an antagonist of the IL-11 signaling pathway in vitro or in vivo, to interfere with or block the physiological signal transduction of natural human IL-11 in vitro or in vivo.

[0032] The technical key points of the present application are:

[0033] IL-11M is based on the human-derived IL-11 amino acid sequence (UniProtKB-P20809, with or without the N-terminal proline (Pro) or substitution), with a specific mutation at position 147 of the tryptophan residue (W147). The mutation site nucleotide codon is TGC, TGT, or TAA, TAG, TGA, preferably IL-11M (W147C) mutant.

[0034] In the IL-11M mutant, one of the purposes of selecting and mutating the mutation site is to reduce the affinity of the IL-11M-IL-11RA dimeric complex with the IL-11ST / GP130 receptor, reduce the ability of the IL-11M-IL-11RA dimeric complex to form an IL-11M-IL-11RA-IL-11ST / GP130 trimeric complex with the IL-11ST / GP130 receptor, and ultimately form an IL-11M-IL-11RA-IL-11ST / GP130 hexameric complex. Another more important and key purpose is to facilitate the site-specific coupling of chemical molecules.

[0035] The key to site-directed modification of IL-11M mutant with chemical molecules is to site-direct the coupling of chemical molecules to the amino acid residue mutated at the 147th tryptophan position, such as the free thiol group on the cysteine residue in the IL-M(W147C) mutant. Alternatively, it can be coupled to other types of unnatural amino acid mutants, which have the structural feature of having a reactive click chemistry functional group on their amino acid residues, such as acetylene (—C≡CH) and azide (—N≡N) and their structural derivatives, but not limited to this. One of the purposes of introducing unnatural amino acids at the 147th tryptophan site is also to achieve site-directed coupling of chemical molecules.

[0036] One of the purposes of site-directed modification of IL-11M mutant is to further reduce or even completely lose the ability of IL-11M conjugate-IL-11RA dimer complex to form IL-11M-IL-11RA-IL-11ST / GP130 trimer complex with IL-11ST / GP130 receptor; another purpose of site-directed coupling of IL-11M with chemical molecules is to prolong the circulating retention half-life of IL-11M in vivo, as well as other improvements in physicochemical properties, such as water solubility, stability and immunogenicity.

[0037] IL-11M and IL-11M conjugate chemical molecule are characterized by being able to bind to IL-11RA with relatively high affinity, but after forming a dimer complex with IL-11RA, it is difficult or completely unable to bind to IL-11ST / GP130 receptor, with a significantly reduced affinity between them, making it difficult to form a trimer complex, and further unable to assemble into a complete hexamer complex capable of transmitting IL-11 physiological activity, especially IL-11M conjugate chemical molecules, even making IL-11M conjugate molecule-IL-11RA dimer complex completely lose the ability to form a trimer complex with IL-11ST / GP130 receptor, playing a role in antagonizing, interfering or blocking the natural IL-11 protein signaling in vivo or in vitro.

[0038] The application field of IL-11M and IL-11M conjugate molecules in the present application is mainly in the prevention and treatment of diseases related to tissue and organ fibrosis as a clinical symptom of primary or secondary endogenous human IL-11 elevation, such as lung, heart, liver, kidney and bladder organ fibrosis diseases.

[0039] The sequences involved in the specification are as follows:

[0040] SEQ ID NO: 1 Amino acid sequence of IL-11M(W147C) mutant

[0041] GPPPGPPRVSPDPRAELDSTVLLTRSLLADTRQLAAQLRDKFPADGDHNLDSLPTLAMS AGALGALQLPGVLTRLRADLLSYLRHVQWLRRAGGSSLKTLEPELGTLQARLDRLLRRLQ LLMSRLALPQPPPDPPAPPLAPPSSACGGIRAAHAILGGLHLTLDWAVRGLLLLKTRL.

[0042] SEQ ID NO: 2 Gene sequence of IL-11M (W147C) mutant optimized by E. coli preferred codon

[0043] GGTCCTCCTCCTGGTCCGCCTCGTGTTAGTCCGGATCCGCGTGCAGAACTGGATAGCACCGTTCTGCTGACCCGTAGCCTGCTGGCAGATACCCGTCAGCTGGCAGCACAGCTGCGTGATAAATTTCCGGCAGATGGTGATCATAATCTGGATAGCCTGCCGACACTGGCAATGAGCGCAGGCGCACTGGGTGCACTGCAGCTGCCTGGTGTTCTGACCCGTCTGCGTGCCGATCTGCTGAGCTATCTGCGTCATGTTCAGTGGCTGCGTCGTGCCGGTGGTAGCAGCCTGAAAACCCTGGAACCGGAACTGGGCACCCTGCAGGCACGTCTGGATCGTCTGCTGCGTCGCCTGCAACTGCTGATGAGCCGTCTGGCACTGCCGCAGCCTCCGCCTGATCCTCCGGCACCGCCTCTGGCACCTCCGAGCAGTGCATGCGGTGGTATTCGTGCAGCACATGCAATTTTAGGTGGTCTGCATCTGACCCTGGATTGGGCAGTTCGTGGTCTGCTGCTGCTGAAAACACGTCT.

[0044] SEQ ID NO: 3 Amino acid sequence of His-Sumo-IL-11M (W147C) mutant fusion protein

[0045] HHHHHHSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGGPPPGPPRVSPDPRAELDSTVLLTRSLLADTRQLAAQLRDKFPADGDHNLDSLPTLAMSAGALGALQLPGVLTRLRADLLSYLRHVQWLRRAGGSSLKTLEPELGTLQARLDRLLRRLQLLMSRLALPQPPPDPPAPPLAPPSSACGGIRAAHAILGGLHLTLDWAVRGLLLLKTRL.

[0046] SEQ ID NO: 4 Gene sequence of His-Sumo-IL-11M (W147C) mutant fusion protein

[0047] CATCATCACCATCATCATAGCGATAGCGAAGTTAATCAAGAAGCCAAACCGGAAGTTAAGCCGGAAGTGAAACCTGAAACACATATTAACCTGAAAGTGAGTGATGGTAGCAGCGAGATCTTTTTCAAAATCAAAAAGACCACACCGCTGCGTCGTCTGATGGAAGCATTTGCAAAACGTCAGGGTAAAGAAATGGATAGCCTGCGTTTTCTGTATGATGGTATTCGTATTCAGGCAGATCAGACACCGGAAGATCTGGATATGGAAGATAACGATATTATCGAAGCACATCGTGAGCAGATTGGTGGTGGTCCTCCTCCTGGTCCGCCTCGTGTTAGTCCGGATCCGCGTGCAGAACTGGATAGCACCGTTCTGCTGACCCGTAGCCTGCTGGCAGATACCCGTCAGCTGGCAGCACAGCTGCGTGATAAATTTCCGGCAGATGGTGATCATAATCTGGATAGCCTGCCGACACTGGCAATGAGCGCAGGCGCACTGGGTGCACTGCAGCTGCCTGGTGTTCTGACCCGTCTGCGTGCCGATCTGCTGAGCTATCTGCGTCATGTTCAGTGGCTGCGTCGTGCCGGTGGTAGCAGCCTGAAAACCCTGGAACCGGAACTGGGCACCCTGCAGGCACGTCTGGATCGTCTGCTGCGTCGCCTGCAACTGCTGATGAGCCGTCTGGCACTGCCGCAGCCTCCGCCTGATCCTCCGGCACCGCCTCTGGCACCTCCGAGCAGTGCATGCGGTGGTATTCGTGCAGCACATGCAATTTTAGGTGGTCTGCATCTGACCCTGGATTGGGCAGTTCGTGGTCTGCTGCTGCTGAAAACACGTCTG.

[0048] SEQ ID NO: 5 Gene sequence of His-Sumo-IL-11M (W147C)-pET30a expression vector plasmid

[0049]

[0050] Therefore, by means of the above technical scheme, the present application has the following beneficial effects:

[0051] The present application provides a recombinant human interleukin-11 mutant, a conjugate of the mutant and a chemical molecule, and a preparation method and application thereof. The recombinant human interleukin-11 mutant is obtained by using a method of tandem fusion of Sumo sequences to realize soluble expression in an Escherichia coli expression system. The conjugate is obtained by site-specific coupling of the mutant and the chemical molecule at a mutant amino acid residue at a W147 site through a covalent bond. The chemical molecule is at least one of polyethylene glycol, an alkanoic fatty chain, and a hydrophilic polymer containing an alkanoic fatty chain. The conjugate retains the binding ability to human interleukin-11 receptor A, but the binding ability to human IL-6 signal transducer receptor is greatly reduced. The conjugate can block the signal transduction between human IL-11 and IL-11RA and GP130 in vivo or in vitro, and can be used for the treatment of diseases related to tissue fibrosis as a clinical symptom caused by primary or secondary IL-11 elevation. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 is a schematic diagram of the characteristic structure of the construction plasmid of the His-Sumo-IL-11M(W147C) fusion protein in Example 1 of the present application (top) and a nucleic acid electrophoresis identification chart (bottom).

[0053] Figure 2 is an SDS-PAGE identification chart of the soluble expression of IL-11M(W147C) in Example 1 of the present application.

[0054] Figure 3 is an electrophoresis identification result chart of the PEG-modified IL-11M(W147C) conjugate in Example 2 of the present application.

[0055] Figure 4 is a circular dichroism spectrum identification result of IL-11M(W147C) and its PEG coupling modification conjugate in Example 3 of the present application.

[0056] Figure 5 is an intrinsic fluorescence emission spectrum identification result of IL-11M(W147C) and its PEG coupling modification conjugate in Example 3 of the present application.

[0057] Figure 6 is a high-efficiency gel filtration chromatography identification result of IL-11M(W147C) and its PEG coupling modification conjugate in Example 3 of the present application.

[0058] Figure 7Binding affinity detection results of rhIL-11, IL-11M(W147C) and PEG modified conjugates thereof to IL-11RA and IL-6ST / GP130 in Example 4 of the present application.

[0059] Figure 8 Stimulation of TF-1 cell survival and proliferation activity determination results of IL-11M(W147C) and PEG modified IL-11M(W147C) conjugates in Example 5 of the present application.

[0060] Figure 9 Intervention determination results of IL-11M(W147C) and PEG modified IL-11M(W147C) conjugates on the fibrotic differentiation response of human lung fibroblasts (HLFs) after TGF-β stimulation in Example 6 of the present application.

[0061] Figure 10 Pharmacokinetic experiment results of IL-11M(W147C) and PEG modified IL-11M(W147C) conjugates in SD rats in Example 7 of the present application.

[0062] Figure 11 In vivo pharmacodynamic evaluation results of IL-11M(W147C) and PEG modified IL-11M(W147C) conjugates on bleomycin-induced pulmonary fibrosis model in mice in Example 8 of the present application. DETAILED DESCRIPTION

[0063] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0064] Example 1

[0065] Construction, expression and preparation of expression vector of IL-11M(W147C) mutant

[0066] As Figure 1 and Figure 2As shown, first, the truncated human IL-11 amino acid sequence (177AA, without N-terminal Pro) was subjected to genetic sequence conversion and E. coli host-preferred codon optimization, and codon substitution at W147 site to obtain IL-11M mutant, with the amino acid sequence SEQ ID NO: 1 and the corresponding gene sequence SEQ ID NO: 2. The IL-11M gene sequence was commissioned to be synthesized, and the IL-11M gene sequence was spliced with the 3'-end of the His-SUMO fusion tag gene, with the amino acid sequence of the His-SUMO-IL-11M fusion protein being SEQ ID NO: 3. Then, the His-SUMO-IL-11M gene (SEQ ID NO: 4) was inserted into the pET-30a plasmid vector to obtain the His-SUMO-IL-11M-pET-30a expression plasmid (gene sequence of the plasmid: SEQ ID NO: 5). The His-SUMO-IL-11M-pET-30a recombinant plasmid was transformed into E. coli competent cells, and cultured in Amp+ resistant solid LB medium, and single colonies were screened and expanded for culture, and the bacteria were broken and purified to obtain the His-SUMO-IL-11M (W147C) protein. After removal of the His-SUMO fusion tag by ULP1 enzyme, the protein was purified by IMAC-Ni chromatography (flow-through mode), and finally the IL-11M (W147C) mutant protein was obtained, and 12% SDS-PAGE was performed to identify the purification results. The results showed that the His-SUMO-IL-11M was mainly expressed in a soluble form in E. coli, and the purity after purification was higher than 95%.

[0067] Example 2

[0068] PEGylation of IL-11M (W147C) mutant protein

[0069] As Figure 3As shown, the buffer of the purified IL-11M (W147C) protein was replaced with 50 mM Tris-HCl, pH 7.0 buffer, and the concentration of IL-11M (W147C) protein was adjusted to approximately 2.0 mg / ml. Three 5 ml portions of the protein solution were measured. 5 mg mPEG10k-MAL, 10 mg mPEG10k-MAL, 20 mg mPEG20k-MAL, and 40 mg mPEG40k-MAL were weighed and added to the IL-11M (W147C) protein solution, respectively. After reacting for 6 h, the protein was purified using a Hitrap Butyl-sepharose 1.0 ml chromatography column. Hydrophobic chromatography procedure (adsorption-elution mode): Before sample loading, the column was pre-washed with buffer B (20 mmol / L Tris-HCl, pH 8.0) for 3–5 column volumes, then equilibrated with buffer A (20 mmol / L Tris-HCl, 0.8 M (NH4)2SO4, pH 8.0) for 3–5 column volumes. After sample loading, the column was equilibrated again with buffer A for 3–5 column volumes, followed by elution with 60% and 100% buffer B sequentially. The elution products were collected, and the purification results were identified using 12% SDS-PAGE. The results showed that the purity of PEG-modified IL-11M was higher than 95%.

[0070] Example 3

[0071] Structural characterization of IL-11M(W147C) and mPEG-modified IL-11M(W147) conjugates

[0072] like Figures 4-6 As shown, the secondary structures of rhIL-11, IL-11M(W147C), and their PEG-modified conjugates were identified by circular dichroism analysis. First, the protein sample buffer was replaced with 5.0 mM PB at pH 7.0, and the protein concentration was concentrated and adjusted to 0.2-0.3 mg / ml. A 0.1 cm thick sample cell was used, with a scanning wavelength interval of 1.0 nm, 5 scans, a scanning wavelength range of 190-260 nm, and a sample scanning speed of 1200 nm / min. The results show that the secondary structures of rhIL-11, IL-11M(W147C), and their PEG-modified conjugates are consistent with the theoretical structures, and the PEG modification has almost no effect on the structure.

[0073] Fluorescence spectroscopy identification:

[0074] The endogenous fluorescence properties of rhIL-11, IL-11M(W147C) and PEG modified conjugates thereof were identified by fluorescence spectrophotometer. First, the protein sample buffer was replaced into 5.0 mM PB, pH 7.0, concentrated and adjusted the protein concentration to about 0.1 mg / ml, the sample was placed in a 1.0 cm light path quartz cuvette sample cell, the excitation wavelength was set to 280 nm, the emission wavelength was set to 280-450 nm, the scanning wavelength interval was 1.0 nm, and the sample scanning speed was 1000 nm / min. The results can be seen that the structure of rhIL-11, IL-11M(W147C) and PEG modified conjugates thereof is compact.

[0075] High performance gel filtration analysis:

[0076] Gel filtration analysis was performed using a HPLC system, the gel filtration column model was TKS3000-GSW, the mobile phase was 50 mM Na2HPO4 / NaH2PO4, 0.15 M Na2SO4, pH 7.2. The flow rate was 0.5 ml / min, the detection wavelength was 280 nm, the results showed that the apparent molecular exclusion volume of IL-11M(W147) modified by PEG was greatly increased compared with rhIL-11 and IL-11M(W147C), and was positively correlated with the size of the modified PEG molecule.

[0077] Example 4

[0078] Detection of the binding affinity of IL-11M(W147C) and mPEG modified IL-11M(W147) conjugates to IL-11RA and IL-11ST / GP130 (surface plasmon resonance, SPR)

[0079] As shown in Figure 7 IL-11RA receptor or IL-6ST / GP30 receptor was covalently coupled on a CM5 chip, then rhIL-11, IL-11M(W147C) and PEG modified conjugates thereof sample or rhIL-11, IL-11M(W147C) and PEG modified conjugates thereof sample pre-mixed with IL-11RA receptor at a molar ratio of 1:1 were reacted with the receptor-coupled CM5 chip at a concentration of 100 nM, and the detection results were analyzed by kinetics fitting according to the 1:1 model. The results showed that compared with rhIL-11, the affinity of IL-11M(W147C) and PEG modified conjugates thereof to IL-11RA did not decrease significantly, but the affinity to IL-11ST / GP30 receptor decreased significantly, especially the PEG modified conjugates, which showed a greater degree of decrease compared with the IL-11M(W147C) mutant, and the degree of decrease was closely related to the size of the PEG molecule.

[0080] Example 5

[0081] Assay of TF-1 cell survival and proliferation activity stimulated by IL-11M(W147C) and mPEG-modified IL-11M(W147) conjugate

[0082] like Figure 8 As shown, cell viability was measured using TF-1 cells. TF-1 cells were cultured in RPMI-1640 medium containing 2.0 ng / ml GM-CSF and fetal bovine serum. After three passages and stable cell growth, the cells were harvested, and the cell concentration was adjusted to 2 x 10⁻⁶ cells / ml using RPMI-1640 medium containing fetal bovine serum. 5 Cells were cultured at a concentration of 1 / ml in 96-well plates for 24 hours, followed by co-culture in RPMI + fetal bovine serum containing different concentrations of IL-11 for 72 hours. Finally, cell proliferation was detected using CCK-8 working solution. The results showed that, compared with rhIL-11, IL-11M (W147C) and its PEG-modified conjugates could not stimulate the survival or proliferation of TF-1 cells.

[0083] Example 6

[0084] Interventional assay of the fibrotic differentiation response of human lung fibroblasts (HLFs) after TGF-β stimulation by IL-11M(W147C) and mPEG-modified IL-11M(W147) conjugate.

[0085] like Figure 9 As shown, human lung fibroblasts were used to assess their TGF-β-stimulated fibrotic differentiation response. HLF cells were cultured in RPMI-1640 medium containing 5.0 ng / ml TGF-β and fetal bovine serum. After three passages and stable cell growth, cells were harvested, and the cell concentration was adjusted to 2 x 10⁻⁶ cells / mL using RPMI-1640 medium containing fetal bovine serum. 5 Cells were cultured at a concentration of 100 ng / ml in 96-well plates for 24 hours, followed by co-culturing in RPMI + fetal bovine serum medium containing 100 ng / ml of IL-11M and its PEG-modified conjugate for 72 hours. After cell immobilization, the cells were incubated with fluorescently labeled anti-collagen antibody working solution, and fluorescence confocal microscopy was used to identify fibrosis marker proteins. The results showed that, compared with the untreated control group, IL-11M (W147C) and its PEG-modified conjugate could reduce the level of collagen expression in HLF cells and delay the fibrotic differentiation of HLF cells.

[0086] Example 7

[0087] Pharmacokinetics of IL-11M(W147C) and mPEG-modified IL-11M(W147) conjugate in SD rats

[0088] like Figure 10 As shown, 6-week-old healthy SD rats were administered a single dose of 1.0 mg / kg IL-11M (W147C) and an mPEG-modified IL-11M (W147) conjugate via tail vein. Blood samples were then collected via orbital vein at specific time points and centrifuged to obtain serum samples. Serum drug concentrations were then measured using a human IL-11 ELISA kit, and drug-time curves were plotted and pharmacokinetic parameters were analyzed. The results showed that compared to unmodified IL-11M (W147C), the PEG-conjugated conjugate had a significantly increased circulating half-life in rats, and the magnitude of this increase was positively correlated with the molecular weight of the PEG conjugated.

[0089] Example 8

[0090] In vivo evaluation of IL-11M(W147C) and mPEG-modified IL-11M(W147) conjugates in a bleomycin-induced mouse pulmonary fibrosis model.

[0091] like Figure 11 As shown, a mouse model of idiopathic pulmonary fibrosis was established in 5-week-old C57 mice by instilling bleomycin (2.5 mg / kg) via endotracheal intubation. Following bleomycin administration via endotracheal intubation, IL-11M (W147C) and an mPEG-modified IL-11M (W147) conjugate were subcutaneously administered twice weekly at a dose of 0.1 mg / kg for 3 weeks. Mice were then euthanized by cervical dislocation, and lung tissue was dissected for histological sections. Hematoxylin-eosin (HE) staining and Masson's trichrome staining were performed. Stain staining analysis of lung tissue fibrosis showed that, compared with normal PBS mice and untreated IPF model mice, mice treated with PEG-modified IL-11M conjugates showed varying degrees of improvement in lung fibrosis. In particular, the IPF model mice treated with mPEG20k and mPEG40k modified IL-11M showed a very significant improvement in lung fibrosis.

[0092] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A recombinant human interleukin-11 mutant, characterized in that, The amino acid sequence of the recombinant human interleukin-11 mutant is shown in SEQ ID NO:

1.

2. The method for preparing the recombinant human interleukin-11 mutant as described in claim 1, characterized in that, The method comprises the following steps: Step 1, converting the human interleukin-11 amino acid sequence into a gene sequence and optimizing the E. coli host preferred codon, and replacing the codon at the W147 site to obtain a human interleukin-11M mutant; Step 2, splicing the gene sequence of the human interleukin-11M mutant with the 3' end of the small ubiquitin-related modifier tandem gene sequence to obtain the gene sequence of His-Sumo-IL-11M; Step 3, inserting the gene sequence of His-Sumo-IL-11M obtained in step 2 into a prokaryotic system expression plasmid vector, and then transforming the plasmid vector into a prokaryotic competent cell; Step 4, selecting dominant growth colonies by resistance coating, expanding culture, and inducing expression of the target gene by IPTG; lysing the bacterial body, centrifuging to obtain supernatant, and obtaining a higher purity recombinant His-Sumo-IL-11M fusion protein by purification; and obtaining the recombinant human interleukin-11 mutant by removing the His-Sumo fusion sequence through enzyme digestion.

3. A conjugate of a recombinant human interleukin-11 mutant and a chemical molecule, wherein the recombinant human interleukin-11 mutant is a mutant of SEQ ID NO: 1, and the chemical molecule is a molecule of SEQ ID NO:

2. The recombinant human interleukin-11 mutant is covalently coupled to the chemical molecule at the mutated amino acid residue of the W147 site by site-directed coupling, wherein the chemical molecule is polyethylene glycol.

4. The recombinant human interleukin-11 mutant and chemical molecule conjugate according to claim 3, wherein the chemical molecule is a drug. The molecular weight of the chemical molecule is 0.1-100 kDa.

5. A method for preparing a conjugate of a recombinant human interleukin-11 mutant of claim 3 or 4 with a chemical molecule, characterized in that, The method comprises the following steps: S1, replacing the recombinant human interleukin-11 mutant into a phosphate or Tris-HCl buffer system; adjusting the pH value of the solution to 6.0-9.0; adjusting the ionic strength to 5-50 mS / cm; and adjusting the concentration of the recombinant human interleukin-11 mutant protein to 0.2-10 mg / ml; S2, mixing the chemical molecule with the recombinant human interleukin-11 mutant protein solution obtained in S1 at a molar ratio of 0.2-50:1, stirring, and reacting at a temperature of 2-40℃ for 0.5-48 h; S3, purifying the sample after the coupling reaction in step S2 by hydrophobic chromatography or ion exchange chromatography to obtain a recombinant human interleukin-11 mutant and chemical molecule conjugate.

6. The method for preparing the recombinant human interleukin-11 mutant and chemical conjugate according to claim 5, characterized in that, In S1, the pH value of the solution is adjusted to 6.5-7.5; the ionic strength is adjusted to 5-10 mS / cm; and the concentration of the recombinant human interleukin-11 mutant protein is adjusted to 0.5-3.0 mg / ml.

7. The method for preparing the recombinant human interleukin-11 mutant and chemical conjugate according to claim 5, characterized in that, In S2, the molar ratio of the chemical molecule to the recombinant human interleukin-11 mutant protein is 1-5:1; in S2, the reaction temperature is 4-25℃, and the reaction time is 2-8 h.

8. The method for preparing the recombinant human interleukin-11 mutant and chemical conjugate according to claim 5, characterized in that, In S3, the hydrophobic chromatography filler is a medium with hydrophobic ligands such as butyl sulfide, butyl, octyl or phenyl; and the ion exchange chromatography filler is a medium with sulfonic acid groups or quaternary ammonium salt.

9. Use of the recombinant human interleukin-11 mutant and chemical molecule conjugate as claimed in claim 3 or 4 in the manufacture of a medicament for the treatment of a tissue fibrosis disease caused by an increase in primary or secondary recombinant human interleukin-11; the tissue fibrosis being at least one of pulmonary fibrosis, myocardial fibrosis, liver fibrosis, bladder fibrosis and renal fibrosis.

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