Recombinant human interleukin-11 mutant, and conjugate of mutant conjugated to chemical molecule and use thereof

Abstract

Provided are a recombinant human interleukin-11 mutant, and a conjugate of the mutant conjugated to a chemical molecule and a use thereof. The recombinant human interleukin-11 mutant achieves soluble expression in an Escherichia coli expression system by means of an SUMO sequence tandem fusion method. The conjugate is obtained by the site-specific conjugation of the mutant to the chemical molecule at a mutant amino acid residue in position W147 by means of a covalent bond. The chemical molecule is at least one of polyethylene glycol, an alkanoic acid aliphatic chain, and a hydrophilic high-molecular polymer containing an alkanoic acid aliphatic chain. The conjugate retains the binding capacity to human interleukin-11 receptor A while demonstrating significantly reduced binding capacity to human IL-6 signal transducer receptor. The conjugate shows the ability to block the signaling between human IL-11 and IL-11 RA and between human IL-11 and GP130 either in vivo or in vitro and can be used for the treatment of associated diseases with tissue fibrosis as a clinical manifestation caused by elevated primary or secondary IL-11.

Description

A recombinant human interleukin-11 mutant and its conjugate with chemical molecules and its application Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a recombinant human interleukin-11 mutant and a conjugate thereof coupled with a chemical molecule and an application thereof. Background Art

[0002] Human interleukin-11 (hIL-11) is one of the many important cytokines in the human body and belongs to the human IL-6 family of cytokines. The IL-6 family of cytokines also includes IL-6, IL-27, IL-31, LIF, CNTF, CT-1, and OSM. Human IL-11 exerts its physiological functions in the body primarily by forming a complex with the IL-11RA and IL-6ST / GP130 receptors, which transmit physiological signals. Its mechanisms of action include three pathways: classical signaling, trans-signaling, and cluster signaling (Emerging roles for interleukin-11 in disease, 2019). In all three signaling pathways, IL-11 must first bind to IL-11RA with relatively low affinity (Kd = ~10 nM) to form an IL-11-IL-11RA dimer complex. The IL-11-IL-11RA dimer complex then 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. Finally, the two trimer complexes assemble into an IL-11-IL-11RA-IL-6ST / GP130 hexamer complex to successfully achieve IL-11 signaling. The IL-6ST / GP130 receptor involved in this signaling is a common receptor in IL-6 family cytokine signaling, while IL-11RA is a specific receptor for IL-11.

[0003] Natural mature human interleukin-11 (UniProtKB-P20809) is composed of 178 amino acids, which contain a high proportion of proline and leucine, no cysteine, and no glycosylation sites. Its theoretical molecular weight is approximately 19kDa and its theoretical isoelectric point is approximately 11.16. IL-11 can synergize with various cytokines in the human body to exert physiological effects, jointly stimulating the growth of human precursor cells such as red blood cells and megakaryocytes, inducing megakaryocyte maturation, and promoting platelet production. rhIL-11 (Oprelvekin, As early as 1997, it was approved by the US FDA for the treatment of regenerative thrombocytopenia caused by radiation and chemotherapy in cancer patients. The rhIL-11 produced by Genetic Institute contains 177 amino acids. Compared to native human IL-11, it lacks the N-terminal proline. Despite this missing amino acid, its biological activity in vivo and in vitro is not significantly different from that of native IL-11.

[0004] Recent studies have found significantly elevated levels of IL-11 in patients with idiopathic pulmonary fibrosis (IPF), suggesting that IL-11 may potentially be involved in the progression of pulmonary fibrosis and play a role in its pathophysiology. Neutralization of IL-11 in mice using anti-IL-11 antibodies has been shown to alleviate and delay bleomycin-induced pulmonary fibrosis (Interleukin-11 is a therapeutic target in idiopathic pulmonary fibrosis, 2019), suggesting that inhibiting endogenous IL-11 activity may hold promise for controlling or reversing the progression of pulmonary fibrosis. Furthermore, studies have demonstrated that increasing endogenous IL-11 expression in mice through transgenic means or by continuously injecting healthy mice with exogenous IL-11 can lead to fibrosis in multiple organs, including liver and kidney fibrosis, and even to multiple organ failure (IL-11 is a crucial determinant of cardiovascular fibrosis, 2017). This suggests that IL-11 may be involved in the pathological changes of tissue and organ fibrosis in vivo and may be a potential therapeutic target for inhibiting fibrotic lesions. Furthermore, studies have found that IL-11 in humans is involved in the development and progression of gastric and intestinal 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] The development of inhibitors that interfere with the IL-11 signaling pathway currently focuses on antibody drugs, primarily targeting IL-11 or IL-11RA. These antibodies bind to IL-11 or IL-11RA, blocking the IL-11 signaling pathway. For example, the BI 765423 antibody developed by Boehringer-Ingelheim targets IL-11. Its primary mechanism of action is to inhibit the formation of the IL-11-IL-11RA dimer complex between IL-11 and IL-11RA, thereby blocking IL-11 signaling. Another approach is to inhibit the formation of an IL-11-IL-11RA-IL-6ST / GP130 trimer complex by inhibiting the IL-11-IL-11RA dimer complex and the IL-6ST / GP130 receptor, thereby blocking the IL-11 signaling pathway. For example, based on the IL-11 structure, site-directed mutagenesis is performed to obtain IL-11 mutants with reduced affinity for IL-6ST / GP130, such as the IL-11(W147A) mutant. However, the IL-11(W147A) mutant still has shortcomings such as insufficient affinity for the IL-6ST / GP130 receptor and easy rapid clearance in the body.

[0006] Summary of the Invention

[0007] The present invention aims to design and screen an IL-11 mutant (hereinafter referred to as IL-11M) based on the structural characteristics of human IL-11 and its mechanism of involvement in signaling pathways in vivo, addressing the deficiencies of the prior art and the aforementioned difficulties. The mutant retains its binding affinity to the IL-11RA receptor, a specific human IL-11 receptor, and its ability to form an IL-11M-IL-11RA dimer complex, while significantly reducing its binding affinity to the IL-6ST / GP130 receptor, a common receptor for IL-11 family cytokines, and its ability to form an IL-11M-IL-11RA-IL-6ST / GP130 trimer complex with the IL-6ST / GP130 receptor. Furthermore, the IL-11M mutant is conjugated to a chemical molecule through site-directed modification at the selected mutation site (e.g., W147C) to produce an IL-11M conjugate. Compared to IL-11M, the IL-11M conjugate is characterized by retaining its binding affinity to the IL-11RA receptor, a specific human IL-11 receptor, and the ability to form an IL-11M conjugate-IL-11RA dimer complex with it. However, its binding affinity to the IL-6ST / GP130 receptor, a common receptor for IL-11 family cytokines, is significantly reduced, making it difficult for the IL-11M conjugate-IL-11RA-IL-6ST / GP130 trimer to form. Furthermore, the IL-11M conjugate exhibits a longer in vivo circulation half-life than IL-11M and significantly delays, inhibits, and even improves or reverses the progression of fibrosis in mice modeled with bleomycin-induced idiopathic non-fibrotic (IPF).

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] A recombinant human interleukin-11 mutant is obtained by adding, deleting, truncating or replacing one or more amino acid sequences at the end or in the 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 and / or azide.

[0010] The method for preparing the above-mentioned recombinant human interleukin-11 mutant comprises the following steps:

[0011] Step 1: performing gene sequence conversion and codon optimization for the Escherichia coli host on the human interleukin-11 amino acid sequence, and performing codon substitution at the W147 site to obtain a human interleukin-11M mutant;

[0012] Step 2: splicing the gene sequence of the human interleukin-11M mutant with the 3' end of the tandem gene sequence of the small ubiquitin-related modifier to obtain the gene sequence of His-Sumo-IL-11M;

[0013] Step 3: insert the His-Sumo-IL-11M gene sequence obtained in step 2 into a prokaryotic expression plasmid vector, and then transform the plasmid vector into prokaryotic competent cells;

[0014] Step 4: Select dominant growing colonies through resistance plating, expand culture, and induce expression of the target gene using IPTG or other lactose structural analogs; lyse the bacteria, obtain the supernatant by centrifugation, and obtain a high-purity recombinant His-Sumo-IL-11M fusion protein through purification. After enzymatic excision of the His-Sumo fusion sequence, a recombinant human interleukin-11 mutant is obtained.

[0015] Furthermore, the above-mentioned recombinant human interleukin-11 mutant is obtained by site-specific coupling with a chemical molecule on the residue of the mutant amino acid at the W147 position through a covalent bond; wherein the chemical molecule is at least one of polyethylene glycol, an alkanoic acid fatty chain, and a hydrophilic high molecular polymer containing an alkanoic acid fatty chain.

[0016] Furthermore, the molecular weight of the chemical molecule is 0.1 to 100 kDa.

[0017] The present invention provides a recombinant human interleukin-11 mutant, a conjugate coupled with a chemical molecule, and a preparation method and application thereof. The recombinant human interleukin-11 mutant utilizes a Sumo sequence tandem fusion method to achieve soluble expression in an Escherichia coli expression system. The conjugate is obtained by site-specific coupling of the mutant and the chemical molecule via a covalent bond at the residue of the mutant amino acid at position W147; the chemical molecule is at least one of polyethylene glycol, an alkanoic acid fatty chain, and a hydrophilic polymer containing an alkanoic acid fatty chain. The conjugate retains binding ability to human interleukin-11 receptor A, but significantly reduces binding ability to the human IL-6 signal transducer receptor. It has been shown in vitro and in vivo to block signal transduction between human IL-11 and IL-11RA and GP130, and can be used to treat diseases associated with tissue fibrosis as a clinical symptom due to primary or secondary IL-11 elevation.

[0018] A method for preparing the above-mentioned recombinant human interleukin-11 mutant and chemical molecule coupling conjugate comprises the following steps:

[0019] S1. Replace the recombinant human interleukin-11 mutant into a phosphate or Tris-HCl buffer system; adjust the solution pH to 6.0-9.0; adjust the ionic strength to 5-50 mS / cm; and adjust the IL-11M protein concentration to 0.2-10 mg / ml;

[0020] S2. Mix the chemical molecule with the recombinant human interleukin-11M protein solution obtained in S1 at a molar ratio of 0.2 to 50:1, stir, and perform the reaction at a temperature of 2 to 40° C. for 0.5 to 48 hours.

[0021] S3. The sample obtained after the coupling reaction in step S2 is purified by hydrophobic chromatography or ion exchange chromatography to obtain a conjugate of the recombinant human interleukin-11 mutant and the chemical molecule.

[0022] Furthermore, 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 IL-11M protein concentration is adjusted to 0.5-3.0 mg / ml.

[0023] Furthermore, in S2, the molar ratio of the chemical molecule to the recombinant human interleukin-11M protein is 1 to 5:1; in S2, the reaction temperature is 4 to 25° C., and the reaction time is 2 to 8 hours.

[0024] Furthermore, in S3, the hydrophobic chromatography filler is a medium with a hydrophobic ligand butyl sulfide, butyl, octyl or phenyl; and the ion exchange chromatography filler is a medium with a sulfonic acid group or a quaternary ammonium salt.

[0025] The use of the above-mentioned recombinant human interleukin-11 mutant and chemical molecule coupling conjugate in the preparation of drugs for related diseases with tissue fibrosis as a clinical symptom due to primary or secondary IL-11 elevation.

[0026] Furthermore, the tissue fibrosis is at least one of pulmonary fibrosis, myocardial fibrosis, liver fibrosis, bladder fibrosis and kidney fibrosis.

[0027] Furthermore, based on the human IL-11 amino acid sequence (UniProtKB-P20809, retaining or not retaining the N-terminal proline (Pro)), the IL-11 gene sequence was obtained by codon preference optimization of Escherichia coli, and on this basis, a specific amino acid (W147) site mutation was performed, and the nucleotide codons of the mutation site were TGC, TGT or TAA, TAG, TGA to derive IL-11M mutants. Through PCR technology, under the action of specific restriction endonucleases and specific primers, the IL-11M gene sequence can be inserted into an Escherichia coli expression vector, and the resulting cloning vector containing the IL-11M target gene is introduced into the competent Escherichia coli. Clonal colonies positive for the cloning vector are obtained by streaking and screening on solid culture medium. The screened dominant clones are cultured and induced for expression, and the IL-11M protein is purified by multi-step chromatography. Then, the IL-11M protein is site-specifically coupled to the chemical molecule at the mutated amino acid residue at the W147 site through a covalent bond to obtain an IL-11M coupled molecule conjugate.

[0028] Preferably, the recombinant human interleukin-11 protein expressed in Escherichia coli by the present method has an amino acid sequence comprising the entire human IL-11 amino acid sequence or one or several amino acid residues truncated at its N-terminus, and based on this amino acid sequence, the tryptophan residue (Trp or W) at position 147 (numbering starting from the N-terminus of the complete human interleukin-11 amino acid sequence) in the sequence is site-directedly mutated to a cysteine ​​residue (Cys, C) or other types of non-natural amino acid residues that can be used for click chemistry coupling (such as non-natural amino acids carrying acetylene (—C≡CH) and azide (—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.

[0029] Furthermore, in the IL-11M-coupled molecule conjugate, the covalent bond between IL-11M and the chemical molecule occurs on the amino acid residue at the mutation site, in particular, for example, the free thiol group on the cysteine ​​residue of the W147C mutant.

[0030] Furthermore, the covalently coupled chemical molecules are characterized by having a molecular weight generally ranging from 0.1 to 100 kDa, and representative backbone structures include hydrophilic polymers composed of polyethylene glycol (PEG), alkanoic acid fatty chains (C8-C20) and other structural units, but are not limited thereto.

[0031] Furthermore, the structure of the coupled molecule can be linear or branched, and one of its ends can be chemically derivatized to form a functional group that can efficiently couple with a group on a specific amino acid residue. These activated derivatization methods or functional group structures include, but are not limited to, maleimide, vinyl sulfone, iodoacetamide, acetylene (—C≡CH), azide (—N≡N), and their derivatives. The remaining ends of the coupled chemical molecule are generally chemically inert functional groups, including, but are not limited to, alkyl (—CH3), methoxy (—OCH3), hydroxyl (—OH), and carboxyl (—COOH).

[0032] Furthermore, the IL-11M and IL-11M-coupled molecule conjugates retain their high-affinity binding ability to human IL-11RA. After each of them binds to human IL-11RA to form a dimeric complex, their ability to bind to the human IL-11ST / GP130 receptor to form a trimeric complex is significantly reduced, or even completely lost. Specifically, the ability of the IL-11M and IL-11M-coupled molecule conjugates to form complete trimeric or hexameric complexes with IL-11RA and IL-11ST / GP130 receptors is significantly reduced, or even completely lost. These conjugates can potentially be used as antagonists of the IL-11 signaling pathway in vitro or in vivo, interfering with or blocking the physiological signaling of natural human IL-11 in vitro or in vivo.

[0033] The key technical points of the present invention are:

[0034] IL-11M is based on the human IL-11 amino acid sequence (UniProtKB-P20809, retaining or not retaining the N-terminal proline (Pro) or replacing it), and undergoes a specific mutation at the tryptophan residue at position 147 (W147), and the nucleotide codon at the mutation site is TGC, TGT or TAA, TAG, TGA, preferably, the IL-11M (W147C) mutant.

[0035] In the IL-11M mutant, one of the selection and mutation purposes of the mutation site is to reduce the affinity of the dimer complex formed by IL-11M and IL-11RA receptor to the IL-11ST / GP130 receptor, and reduce the ability of the IL-11M-IL-11RA dimer complex to form an IL-11M-IL-11RA-IL-11ST / GP130 trimer complex with the IL-11ST / GP130 receptor, and finally to form an IL-11M-IL-11RA-IL-11ST / GP130 hexameric complex; another more important and critical purpose is to facilitate the site-specific coupling of chemical molecules.

[0036] The key to site-specific modification of IL-11M mutants with chemical molecules is to site-specifically conjugate the chemical molecule to the amino acid residue mutated at tryptophan position 147, such as the free sulfhydryl group on the cysteine ​​residue in the IL-11M(W147C) mutant. Alternatively, conjugation can be performed with other types of unnatural amino acid mutants characterized by the presence of active click chemistry functional groups on their amino acid residues, such as, but not limited to, acetylene (—C≡CH) and azide (—N≡N) and their derivatives. One purpose of introducing an unnatural amino acid at tryptophan position 147 is to enable site-specific conjugation of the chemical molecule.

[0037] One of the purposes of site-specific modification of IL-11M mutants is to further reduce or even completely lose the ability of the IL-11M conjugate-IL-11RA dimer complex to form an IL-11M-IL-11RA-IL-11ST / GP130 trimer complex with the IL-11ST / GP130 receptor; another purpose of site-specific conjugation of IL-11M with chemical molecules is to prolong the circulation half-life of IL-11M in the body and improve other physical and chemical properties, such as water solubility, stability and immunogenicity.

[0038] IL-11M and IL-11M-coupled chemical molecular conjugates are characterized by being able to bind to IL-11RA with relatively high affinity. However, after forming a dimeric complex with IL-11RA, it is difficult or completely impossible to bind to the IL-11ST / GP130 receptor. The affinity between them is greatly reduced, making it difficult to form a trimeric complex, and further, it is impossible to assemble into a complete hexameric complex capable of transmitting the physiological activity of IL-11. In particular, the IL-11M-coupled chemical molecular conjugate may even cause the IL-11M-coupled molecule conjugate-IL-11RA dimer complex to completely lose the ability to form a trimeric complex with the IL-11ST / GP130 receptor, thereby antagonizing, interfering with or blocking the signal transduction of the natural IL-11 protein in vivo or in vitro.

[0039] The application areas of IL-11M and IL-11M-coupled molecule conjugates in the present invention are mainly in the prevention and treatment of related diseases with tissue and organ fibrosis as clinical symptoms due to primary or secondary increase in endogenous human IL-11, such as organ fibrosis diseases of the lungs, heart, liver, kidneys and bladder.

[0040] The sequences mentioned in the specification are as follows:

[0041] SEQ ID NO: 1 Amino acid sequence of IL-11M (W147C) mutant:

[0042] SEQ ID NO: 2 Gene sequence of IL-11M (W147C) mutant after codon optimization based on E. coli preference:

[0043] SEQ ID NO: 3 Amino acid sequence of His-Sumo-IL-11M (W147C) mutant fusion protein:

[0044] SEQ ID NO: 4 Gene sequence of His-Sumo-IL-11M (W147C) mutant fusion protein:

[0045] SEQ ID NO: 5 Gene sequence of His-Sumo-IL-11M (W147C)-pET30a expression vector plasmid:

[0046] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0047] The present invention provides a recombinant human interleukin-11 mutant and its conjugate with a chemical molecule and its application. The recombinant human interleukin-11 mutant utilizes a Sumo sequence tandem fusion method to achieve soluble expression in an Escherichia coli expression system. The conjugate is obtained by site-specific coupling of the mutant and the chemical molecule via a covalent bond at the residue of the mutant amino acid at position W147; the chemical molecule is at least one of polyethylene glycol, an alkanoic acid fatty chain, and a hydrophilic polymer containing an alkanoic acid fatty chain. The conjugate retains its binding ability to human interleukin-11 receptor A, but significantly reduces its binding ability to the human IL-6 signal transducer receptor. It has been shown in vitro and in vivo to block signal transduction between human IL-11 and IL-11RA and GP130, and can be used to treat diseases caused by primary or secondary IL-11 elevation, characterized by tissue fibrosis as a clinical symptom. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG1 is a schematic diagram of the characteristic structure of the plasmid for constructing the His-Sumo-IL-11M (W147C) fusion protein in Example 1 of the present invention (top) and a nucleic acid electrophoresis identification diagram (bottom).

[0049] FIG2 is an SDS-PAGE identification diagram of the soluble expression of IL-11M (W147C) in Example 1 of the present invention.

[0050] FIG3 is a diagram showing the electrophoresis identification results of the PEG-modified IL-11M (W147C) conjugate in Example 2 of the present invention.

[0051] FIG4 is the circular dichroism spectrum identification result of IL-11M (W147C) and its PEG-coupled modified conjugate in Example 3 of the present invention.

[0052] FIG5 is the result of identifying IL-11M (W147C) and its PEG-coupled modified conjugate by endogenous fluorescence emission spectrum in Example 3 of the present invention.

[0053] FIG6 is the result of high performance gel filtration chromatography identification of IL-11M (W147C) and its PEG-coupled modified conjugate in Example 3 of the present invention.

[0054] FIG7 shows the binding affinity test results of rhIL-11, IL-11M(W147C) and their PEG-modified conjugates with IL-11RA in Example 4 of the present invention.

[0055] FIG8 is the result of the binding affinity test between rhIL-11, IL-11M(W147C) and their PEG-modified conjugates and IL-6ST / GP130 in Example 4 of the present invention.

[0056] FIG9 shows the results of the assay of the survival and proliferation activities of TF-1 cells stimulated by IL-11M (W147C) and PEG-modified IL-11M (W147) conjugates in Example 5 of the present invention.

[0057] 10 is the result of the intervention assay of the fibrotic differentiation response of IL-11M(W147C) and PEG-modified IL-11M(W147) conjugates in Example 6 of the present invention after TGF-β stimulation of human lung fibroblasts (HLFs).

[0058] FIG11 shows the pharmacokinetic experimental results of IL-11M(W147C) and PEG-modified IL-11M(W147) conjugates in Example 7 of the present invention in SD rats.

[0059] FIG12 shows the in vivo efficacy evaluation results of IL-11M(W147C) and PEG-modified IL-11M(W147) conjugates in Example 8 of the present invention on the bleomycin-induced mouse pulmonary fibrosis model (hematoxylin-eosin staining).

[0060] FIG13 shows the in vivo efficacy evaluation results of IL-11M(W147C) and PEG-modified IL-11M(W147) conjugates in Example 8 of the present invention on the bleomycin-induced mouse pulmonary fibrosis model (Masson's trichrome staining). DETAILED DESCRIPTION

[0061] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0062] Example 1

[0063] Construction, expression and preparation of expression vector of IL-11M(W147C) mutant

[0064] As shown in Figures 1 and 2, the truncated human IL-11 amino acid sequence (177AA, without an N-terminal Pro) was first subjected to gene sequence conversion and codon optimization for the E. coli host, and codon substitution was performed at the W147 site to obtain an IL-11M mutant, whose amino acid sequence is SEQ ID NO: 1 and the corresponding gene sequence is SEQ ID NO: 2. The IL-11M gene sequence was commissioned for synthesis and spliced ​​to the 3'-end of the His-SUMO fusion tag gene. The amino acid sequence of the His-SUMO-IL-11M fusion protein is SEQ ID NO: 3. The His-SUMO-IL-11M gene (SEQ ID NO: 4) was then inserted into the pET-30a plasmid vector to obtain the His-SUMO-IL-11M-pET-30a expression plasmid (plasmid gene sequence: SEQ ID NO: 5). The His-SUMO-IL-11M-pET-30a recombinant plasmid was transformed into competent Escherichia coli cells and cultured in Amp+-resistant solid LB medium. Single colonies were screened, expanded, disrupted, and purified to obtain the His-SUMO-IL-11M(W147C) protein. The His-SUMO fusion tag was removed by ULP1 enzyme, and the IL-11M(W147C) mutant protein was purified by IMAC-Ni chromatography (flow-through mode). The purification results were characterized by 12% SDS-PAGE, which showed that His-SUMO-IL-11M was primarily expressed in a soluble form in E. coli with a purity exceeding 95%.

[0065] Example 2

[0066] Site-directed polyethylene glycol modification of IL-11M(W147C) mutant protein

[0067] As shown in Figure 3, the buffer of the purified IL-11M (W147C) protein was replaced with 50mM Tris-HCl, pH 7.0 buffer, and the IL-11M (W147C) protein concentration was adjusted to approximately 2.0mg / ml. Three 5ml protein solutions were measured; and 5mg mPEG10k-MAL, 10mg mPEG10k-MAL, 20mg mPEG20k-MAL, and 40mg mPEG40k-MAL were weighed respectively and added to the IL-11M (W147C) protein solution. After the reaction was completed for 6h, purification was carried out using a Hitrap Butyl-sepharose 1.0ml chromatography column. Hydrophobic chromatography (adsorption elution mode): Before loading the sample, the column was prewashed with buffer B (20 mmol / L Tris-HCl, pH 8.0) for 3-5 column volumes and then equilibrated with buffer A (20 mmol / L Tris-HCl, 0.8 M (NH₄)₂SO₄, pH 8.0) for 3-5 column volumes. After loading the sample, the column was equilibrated again with buffer A for 3-5 column volumes, followed by elution with 60% and then 100% buffer B. The eluted product was collected and purified using 12% SDS-PAGE, which showed that the purity of the PEG-modified IL-11M was greater than 95%.

[0068] Example 3

[0069] Structural characterization of IL-11M(W147C) and mPEG-modified IL-11M(W147) conjugates

[0070] As shown in Figures 4-6, 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 5.0mM PB, pH 7.0, concentrated, and the protein concentration was adjusted to 0.2-0.3mg / ml. A 0.1cm thick sample cell was used, with a scanning wavelength interval of 1.0nm, 5 scan repetitions, a scanning wavelength range of 190-260nm, and a sample scanning speed of 1200nm / min. The results show that the secondary structures of rhIL-11, IL-11M (W147C), and their PEG-modified conjugates are consistent with the theoretical structure, and PEG modification has little effect on the structure.

[0071] Fluorescence spectroscopy identification:

[0072] The intrinsic fluorescence properties of rhIL-11, IL-11M(W147C), and their PEG-modified conjugates were characterized by spectrofluorometry. The protein sample was first buffered into 5.0 mM PB, pH 7.0, concentrated, and adjusted to a protein concentration of approximately 0.1 mg / ml. The sample was placed in a 1.0 cm quartz cuvette. The excitation wavelength was set to 280 nm, the emission wavelength was set to 280–450 nm, the scanning interval was 1.0 nm, and the sample scan rate was 1000 nm / min. The results show that rhIL-11, IL-11M(W147C), and their PEG-modified conjugates have compact structures.

[0073] High performance gel filtration analysis:

[0074] Gel filtration analysis was performed using a TKS3000-GSW HPLC system with a mobile phase consisting of 50 mM Na₂HPO₄ / NaH₂PO₄, 0.15 M Na₂SO₄, pH 7.2. The flow rate was 0.5 ml / min, and the detection wavelength was 280 nm. The results showed that the apparent molecular exclusion volume of PEG-modified IL-11M(W147) was significantly increased compared to rhIL-11 and IL-11M(W147C), and was positively correlated with the molecular size of the modified PEG.

[0075] Example 4

[0076] Binding affinity detection of IL-11M(W147C) and mPEG-modified IL-11M(W147) conjugates to IL-11RA and IL-11ST / GP130 (surface plasmon resonance, SPR)

[0077] As shown in Figures 7 and 8, the IL-11RA receptor or IL-6ST / GP30 receptor was covalently coupled to a CM5 chip. Then, rhIL-11, IL-11M (W147C), and their PEG-modified conjugate samples, or samples 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 subjected to kinetic fitting analysis according to the 1:1 model. The results showed that compared with rhIL-11, the affinity of IL-11M(W147C) and its PEG-modified conjugates to IL-11RA was not significantly reduced, but the affinity to IL-11ST / GP30 receptor was greatly reduced, especially the PEG-modified conjugate, which showed a greater degree of reduction compared with the IL-11M(W147C) mutant, and the degree of reduction was closely related to the molecular weight of PEG.

[0078] Example 5

[0079] IL-11M(W147C) and mPEG-modified IL-11M(W147) conjugates stimulate TF-1 cell survival and proliferation activity assay

[0080] As shown in Figure 9, TF-1 cells were used for cell viability assay. TF-1 cells were cultured in RPMI-1640 medium containing 2.0 ng / ml GM-CSF and fetal bovine serum. After the cells were passaged for three generations and their growth was stable, the cells were harvested and the cell concentration was adjusted to 2 x 10 5 / ml, and then distributed in a 96-well plate and cultured for 24 hours. Then, RPMI + fetal bovine serum culture medium containing different concentrations of IL-11 was added for co-culture for 72 hours, and 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 conjugate could not stimulate the survival or proliferation of TF-1 cells.

[0081] Example 6

[0082] Interventional assay of IL-11M(W147C) and mPEG-modified IL-11M(W147) conjugates on the fibrotic differentiation response of human lung fibroblasts (HLFs) stimulated by TGF-β

[0083] As shown in Figure 10, human lung fibroblasts were used to perform a TGF-β-stimulated fibrotic differentiation response assay. HLFs cells were cultured in RPMI-1640 medium containing 5.0 ng / ml TGF-β and fetal bovine serum. After the cells were passaged for three generations and their growth was stable, the cells were harvested and the cell concentration was adjusted to 2 x 10 5 The cells were then plated in 96-well plates and cultured for 24 hours. After 72 hours of co-culture, 100 ng / ml of IL-11M and its PEG-modified conjugates in RPMI + fetal bovine serum were added. After solidification, the cells were incubated with a fluorescently labeled anti-collagen antibody working solution and fluorescent 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 conjugates were able to reduce the level of collagen expression in HLFs cells and delay the degree of fibrotic differentiation of HLFs.

[0084] Example 7

[0085] Pharmacokinetics of IL-11M(W147C) and mPEG-modified IL-11M(W147) conjugates in SD rats

[0086] As shown in Figure 11, 6-week-old SD healthy rats were given a single dose of 1.0 mg / kg of IL-11M(W147C) and mPEG-modified IL-11M(W147) conjugates via the tail vein. Blood was then collected from the orbital vein at specific time points and centrifuged to obtain serum samples. Blood drug concentrations were then measured using a human IL-11 ELISA kit, and drug-time curves were plotted and pharmacokinetic parameters analyzed. The results showed that compared to unmodified IL-11M(W147C), the PEG-modified conjugate had a significantly improved circulation half-life in rats, and the magnitude of the improvement was positively correlated with the molecular weight of the PEG-modified conjugate.

[0087] Example 8

[0088] In vivo evaluation of IL-11M(W147C) and mPEG-modified IL-11M(W147) conjugates in bleomycin-induced mouse pulmonary fibrosis model

[0089] As shown in Figures 12 and 13, 5-week-old C57 mice were instilled with bleomycin (dose 2.5 mg / kg) via tracheal intubation to establish an idiopathic pulmonary fibrosis mouse model. After bleomycin modeling drug was administered via tracheal intubation, IL-11M (W147C) and mPEG-modified IL-11M (W147) conjugates were subcutaneously administered to the mice at a dose of 0.1 mg / kg twice a week for 3 weeks. The mice were then killed by cervical dislocation, and lung tissues were dissected for tissue sections and stained with hematoxylin-eosin (HE) and Masson's Trichrome. Stain staining was used to identify the degree of lung fibrosis. The results showed that compared with the normal PBS group and the IPF model mouse group that did not receive the drug, the degree of lung fibrosis in the mouse groups that received the PEG-modified IL-11M conjugate drug was improved to varying degrees. In particular, the IPF model mouse groups that received the mPEG20k and mPEG40k-modified IL-11M drugs showed very significant improvement in lung fibrosis.

[0090] 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 in the scope of protection of the present invention.

Claims

1. A recombinant human interleukin-11 mutant, characterized in that: The recombinant human interleukin-11 mutant is obtained by adding, deleting, truncating or replacing one or more amino acid sequences at the end or in the 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 and / or azide.

2. A conjugate of a recombinant human interleukin-11 mutant and a chemical molecule, characterized in that: The invention is obtained by site-specific coupling of the recombinant human interleukin-11 mutant as claimed in claim 1 with a chemical molecule on the residue of the mutant amino acid at the W147 position through a covalent bond; wherein the chemical molecule is at least one of polyethylene glycol, an alkanoic acid fatty chain, and a hydrophilic high molecular polymer containing an alkanoic acid fatty chain.

3. The recombinant human interleukin-11 mutant and chemical molecule coupling conjugate according to claim 2, characterized in that: The molecular weight of the chemical molecule is 0.1 to 100 kDa.

4. Use of the recombinant human interleukin-11 mutant and chemical molecule coupling conjugate according to claim 3 in the preparation of drugs for related diseases characterized by tissue fibrosis as a clinical symptom due to primary or secondary IL-11 elevation.

5. The use according to claim 4, characterized in that The tissue fibrosis is at least one of pulmonary fibrosis, myocardial fibrosis, liver fibrosis, bladder fibrosis and kidney fibrosis.

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