WO2021077794A1 - 可溶性Tim-3重组蛋白及其突变型蛋白的制备和应用 - Google Patents

可溶性Tim-3重组蛋白及其突变型蛋白的制备和应用 Download PDF

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WO2021077794A1
WO2021077794A1 PCT/CN2020/099600 CN2020099600W WO2021077794A1 WO 2021077794 A1 WO2021077794 A1 WO 2021077794A1 CN 2020099600 W CN2020099600 W CN 2020099600W WO 2021077794 A1 WO2021077794 A1 WO 2021077794A1
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soluble tim
recombinant
protein
tim
function
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French (fr)
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杨英
陈智
朱海红
吴炜
刘艳宁
楼国华
施毓
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浙江大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • the present invention belongs to the field of biotechnology, and its specific content relates to the preparation and application of soluble Tim-3 recombinant protein and its mutant protein.
  • Biotechnology drugs mainly include proteins, peptides and nucleic acid molecules, covering almost hundreds of diseases including cancer, autoimmune diseases and infectious diseases.
  • Recombinant protein products manufactured by applying protein engineering technology play an indispensable role in people's production, life, and medical treatment.
  • Recombinant protein drugs are currently the most important type of biotechnology drugs. Compared with small molecule chemical drugs, they have a series of advantages such as good curative effect, small side effects and clear biological functions.
  • Liver failure is severe liver damage caused by a variety of factors, and a group of clinical symptoms mainly manifested by blood coagulation disorder, jaundice, hepatic encephalopathy, and ascites. The onset is sudden and the mortality rate is high, which is extremely harmful. It is a globally recognized and extremely challenging clinically critical illness that is life-threatening to patients, and there is still a lack of specific drugs and methods. At present, liver transplantation is considered to be the most effective method for the treatment of this disease, but due to the shortage of donor liver and the high cost of treatment, its clinical application is greatly restricted. The excessive activation of innate immune cells and monocytes plays an important role in the pathogenesis of liver failure.
  • Tim-3 T cell immunoglobulin-and mucin-domain-containing molecule-3) on the cell surface is expressed in monocytes, macrophages, dendritic cells and lymphocytes, etc. Tim-3 molecules on monocyte membranes The soluble Tim-3 (sTim-3) produced by shedding is negatively correlated with the expression of IL-12 and TNF- ⁇ in patients with sepsis. The expression level of TNF- ⁇ in human monocytes decreased, indicating that sTim-3 has the effect of inhibiting monocyte activation.
  • liver cancer patients account for 4% of the newly discovered malignant tumor patients worldwide each year. Liver cancer has become the second cause of tumor death in China, with extremely high morbidity and mortality.
  • surgical resection is the main method of treatment, but surgical treatment has a poor prognosis and is easy to relapse.
  • Tumor immunotherapy is a general term for many treatment methods, including immune checkpoint therapy, cytokine therapy, tumor vaccine and cell therapy.
  • Tim-3 The research on tumor immune checkpoint molecules mainly focuses on the three molecules of Tim-3, CTLA-4, and PD1. These molecules inhibit the activity of immune cells in the microenvironment.
  • Tumor immune checkpoint inhibitors are currently the most important aspect of tumor immunotherapy. By inhibiting the immune escape of tumor cells, mobilizing the function of the own immune system to eliminate tumors.
  • Tim-3 molecule stems from the search for surface markers that distinguish Th1 cells from Th2 cells.
  • a recent study found that by using a semi-quantitative RT-PCR method, the expression levels of soluble Tim-3 and CTLA-4 in tumor-bearing mice at different time periods were measured, and tumor growth was measured at the same time.
  • the present invention provides the application of a novel soluble Tim-3 recombinant protein and its purification method.
  • Soluble Tim-3 recombinant protein is used to prepare drugs with the function of regulating monocytes or enhancing tumor immune response.
  • the drugs that regulate the function of monocytes can be used to inhibit the excessive activation of monocytes in patients with inflammation, such as liver failure patients; the drugs that enhance the function of tumor immune response can be used to enhance the immune response of cancer patients, such as liver cancer patients, and avoid host immune escape.
  • the concentration of soluble Tim-3 recombinant protein is preferably 80 ng/ml.
  • the purification method of soluble Tim-3 recombinant protein includes the following steps: the eukaryotic CHO cell, prokaryotic E. coli and insect baculovirus expression system cell culture fluid containing recombinant human soluble Tim-3 is clarified by microfiltration, and concentrated by ultrafiltration. After the liquid, it passes through anion and cation chromatography column, molecular sieve chromatography, hydrophobic chromatography column and/or affinity chromatography column to obtain sufficient purity (purity determined by SDS-PAGE>96%) recombinant protein; the soluble Tim-3
  • the amino acid sequence of the recombinant protein is SEQ ID NO:1.
  • the present invention also provides the mutant protein of the soluble Tim-3 recombinant protein, and the preparation and application of the mutant protein and the mutant protein.
  • the mutant recombinant protein is obtained through directed evolution technology screening, has the function of regulating monocytes or enhancing the immune response of the body, and combines the host codon optimization method to improve its expression efficiency.
  • a soluble Tim-3 recombinant mutant protein is based on the gene sequence corresponding to the amino acid (SEQ ID NO: 1) of the extracellular domain of human Tim-3 (NP_116171.3), and the mutant is screened by directed evolution technology. Genes, whose mutation sites do not involve conserved sequences between species, occur in non-conserved sequence positions, and then express proteins.
  • the gene sequence corresponding to the amino acid sequence can be optimized according to the characteristics of the host, such as prokaryotic and eukaryotic codons.
  • the amino acid sequence of the soluble Tim-3 recombinant mutant protein is: one of SEQ ID NO: 2-7.
  • the preparation method of soluble Tim-3 recombinant mutant protein uses PCR kit to carry out mutation amplification and screening to obtain the gene sequence.
  • the mutant protein can retain the conservative amino acid sequence derived from different species; in human Tim-3 (NP_116171.3) Based on the gene sequence corresponding to the amino acid of the extracellular domain (SEQ ID NO: 1), the mutant gene is screened by directed evolution technology, and the amino acid is expressed as a soluble Tim-3 recombinant mutant protein.
  • the preferred amino acid sequence is: SEQ ID NO: 2-7 mutant proteins.
  • the amino acid expression refers to the expression of the pre-constructed recombinant protein gene expression vector in the eukaryotic expression system, the prokaryotic expression system and the insect baculovirus expression system, and the extraction and production of long-acting recombinant drugs.
  • the amino acid sequence of the soluble Tim-3 mutant protein is: the mutant protein of SEQ ID NO: 2-7.
  • the drug is achieved by regulating the function of monocytes or enhancing the function of tumor immune response.
  • the drugs for regulating the function of monocytes can be used to inhibit the excessive activation of monocytes in patients with inflammation such as liver failure patients, and the function of enhancing tumor immune response can be used to enhance cancer.
  • the immune response of patients such as liver cancer patients prevents the escape of host immunity.
  • the present invention constructs the soluble Tim-3 recombinant protein gene sequence into prokaryotic, eukaryotic and insect baculovirus expression vectors, and the recombinant protein expressed by the host adopts various purification methods such as ion exchange chromatography, affinity chromatography and hydrophobic chromatography. In combination, the soluble Tim-3 recombinant protein was purified.
  • mutant recombinant protein In order to obtain a mutant recombinant protein with stronger biological functions, a large number of mutations of the recombinant protein were screened by directed evolution technology, and PCR amplification was performed using GeneMorph II Random Mutant PCR Kit (Stratagene), and the target gene was constructed to eukaryotic expression Vector, prokaryotic expression vector or insect rod-shaped expression vector, and then the mutant recombinant expression vector is transformed into eukaryotic, prokaryotic or insect host for expression.
  • GeneMorph II Random Mutant PCR Kit GeneMorph II Random Mutant PCR Kit
  • codon optimization is performed through host codon optimization technology. Specifically, according to the characteristics of the host's eukaryotic, prokaryotic or insect codons, the recombinant PCR technology is used to carry out site-directed mutations to obtain the codon-optimized target sequence, and then the target sequence is digested and cloned into the corresponding host expression vector, and then the mutation Type recombinant expression vectors are transformed into eukaryotic, prokaryotic or insect hosts for expression.
  • soluble Tim-3 and mutant recombinant proteins adopt a combination of affinity chromatography, ion exchange chromatography and hydrophobic chromatography to purify soluble Tim-3 recombinant proteins and mutant recombinant proteins.
  • the principle of metal chelation affinity chromatography is to use some special amino acids on the protein surface to interact with metal ions, thereby affinity purification of the protein.
  • the fusion tag 6 ⁇ His-Tag is a combination of 6 histidine residues. Commonly used, it has the advantages of simple ligand, large adsorption capacity, mild separation conditions and strong versatility.
  • Ion chromatography can not only perform crude purification, effectively remove most of the charged host proteins and nucleic acids, but also achieve fine purification to remove trace impurities.
  • Hydrophobic chromatography can not only enrich protein but also remove most pigments.
  • the invention discloses a preparation method of soluble Tim-3 recombinant protein and its mutant protein in the technical field of bioengineering and its application in diseases such as liver diseases (liver failure and liver cancer).
  • Soluble Tim-3 recombinant protein is an extracellular secreted, soluble and small molecular weight recombinant protein.
  • the recombinant protein contains or does not contain the derivative protein of the soluble region of Tim-3 extracellular structure with 6 ⁇ His tag, and the mutant protein obtains the amino acid sequence containing the conserved structural region in evolution and optimized by the host codon through directed evolution technology.
  • This type of recombinant protein has the function of regulating monocyte function or enhancing tumor immune response.
  • Figure 1 is the SDS-PAGE identification diagram of the culture medium, natural protein, denatured protein and purified protein in Example 1;
  • Figure 2 is a graph showing the dose-dependent effect of soluble Tim-3 on monocyte TNF- ⁇ secretion in Example 2;
  • Figure 3 is a graph showing the inhibitory effect of soluble Tim-3 on monocyte HMGB1 secretion in Example 2;
  • Figure 4 is a graph showing the mTim-3 and sTim-3 expression levels of monocytes in patients with chronic-onset acute liver failure in Example 3;
  • Fig. 5 is a histological diagram of the liver in the improvement of D-GalN/LPS acute liver failure mice by sTim-3 of Example 3;
  • Figure 6 is a graph showing the effect of mutant and unmutated sTim-3 on monocyte TNF- ⁇ secretion.
  • Figure 7 contains the amino acid sequence of histidine and connecting peptide.
  • the present invention applies conventional techniques and methods used in the field of molecular biology, and those skilled in the art can use other conventional methods, experimental protocols and reagents in the field on the basis of the technical solutions described in the present invention, and are not limited to the specific embodiments of the present invention. The limit.
  • the amino acid sequence of the extracellular domain of human Tim-3 (NP_116171.3) is shown in SEQ ID NO:1.
  • the amino acid sequence of the fusion of human Tim-3 (NP_116171.3) extracellular domain, connecting peptide (G)nS and 6 histidines His ( Figure 7), n 1 to 4, preferably 4.
  • List the 6 mutant protein amino acid sequences SEQ ID NO: 2-7 based on SEQ ID NO:1.
  • the nucleotide sequence encoding the His amino acid or the His amino acid is constructed into a eukaryotic expression vector, a prokaryotic expression vector or an insect rod-shaped expression vector, and then the recombinant expression vector is transformed into a eukaryotic, prokaryotic or insect host for expression.
  • mutant recombinant protein In order to obtain a mutant recombinant protein with stronger biological functions, a large number of mutations of the recombinant protein were screened by directed evolution technology, and PCR amplification was performed using GeneMorph II Random Mutant PCR Kit (Stratagene), and the target gene was constructed to eukaryotic expression Vector, prokaryotic expression vector or insect rod-shaped expression vector, and then the mutant recombinant expression vector is transformed into eukaryotic, prokaryotic or insect host for expression.
  • GeneMorph II Random Mutant PCR Kit GeneMorph II Random Mutant PCR Kit
  • codon optimization is performed through host codon optimization technology. Specifically, according to the characteristics of the host's eukaryotic, prokaryotic or insect codons, the recombinant PCR technology is used to carry out site-directed mutations to obtain the codon-optimized target sequence, and then the target sequence is digested and cloned into the corresponding host expression vector, and then the mutation Type recombinant expression vectors are transformed into eukaryotic, prokaryotic or insect hosts for expression.
  • a combination of affinity chromatography, ion exchange chromatography, hydrophobic chromatography and other purification methods are used to purify soluble Tim-3 recombinant protein and mutant recombinant protein.
  • the principle of metal chelation affinity chromatography is to use some special amino acids on the protein surface to interact with metal ions, thereby affinity purification of the protein.
  • the fusion tag 6 ⁇ His-Tag is a combination of 6 histidine residues.
  • Figure 7 it has the advantages of simple ligand, large adsorption capacity, mild separation conditions and strong versatility.
  • Ion chromatography can not only perform crude purification, effectively remove most of the charged host proteins and nucleic acids, but also achieve fine purification to remove trace impurities.
  • Hydrophobic chromatography can not only enrich protein but also remove most pigments.
  • SEQ ID NO: 2 Amino acid sequence with histidine and connecting peptide (M1)
  • 9181P-TIM3-F AGTTTAAACGGATCTCTAGCgaattcGCCGCCACCATGTTCTCCCACCTGCC
  • 9181P-TIM3-R TCGAGGTCGGGGGATCCGCGGCCGCTCAGTGATGGTGGTGGTGGTGGGAGCCTC
  • 9181P-L140R-F1 GCCAAGGTGACCCCCGCCCCCACCAGACAAAGAGATTTCACAGCCGCC
  • 9181P-L140R-R1 GGCGGCTGTGAAATCTCTTTGTCTGGTGGGGGCGGGGGTCACCTTGGC
  • 9181P-TIM3-F AGTTTAAACGGATCTCTAGCgaattcGCCGCCACCATGTTCTCCCACCTGCC
  • 9181P-TIM3-R TCGAGGTCGGGGGATCCGCGGCCGCTCAGTGATGGTGGTGGTGGTGGGAGCCTC
  • 9181P-L140R-F1 GCCAAGGTGACCCCCGCCCCCACCAGACAAAGAGATTTCACAGCCGCC
  • 9181P-L140R-R1 GGCGGCTGTGAAATCTCTTTGTCTGGTGGGGGCGGGGGTCACCTTGGC
  • SEQ ID NO: 4 Amino acid sequence with histidine and connecting peptide (M3)
  • 9181P-TIM3-F AGTTTAAACGGATCTCTAGCgaattcGCCGCCACCATGTTCTCCCACCTGCC
  • 9181P-TIM3-R TCGAGGTCGGGGGATCCGCGGCCGCTCAGTGATGGTGGTGGTGGTGGGAGCCTC
  • 9181P-L140R-F1 GCCAAGGTGACCCCCGCCCCCACCAGACAAAGAGATTTCACAGCCGCC
  • 9181P-L140R-R1 GGCGGCTGTGAAATCTCTTTGTCTGGTGGGGGCGGGGGTCACCTTGGC
  • 9181P-TIM3-F AGTTTAAACGGATCTCTAGCgaattcGCCGCCACCATGTTCTCCCACCTGCC
  • 9181P-TIM3-R TCGAGGTCGGGGGATCCGCGGCCGCTCAGTGATGGTGGTGGTGGTGGGAGCCTC
  • 9181P-L140R-F1 GCCAAGGTGACCCCCGCCCCCACCAGACAAAGAGATTTCACAGCCGCC
  • 9181P-L140R-R1 GGCGGCTGTGAAATCTCTTTGTCTGGTGGGGGCGGGGGTCACCTTGGC
  • SEQ ID NO: 6 Amino acid sequence with histidine and connecting peptide (M5)
  • 9181P-TIM3-F AGTTTAAACGGATCTCTAGCgaattcGCCGCCACCATGTTCTCCCACCTGCC
  • 9181P-TIM3-R TCGAGGTCGGGGGATCCGCGGCCGCTCAGTGATGGTGGTGGTGGTGGGAGCCTC
  • 9181P-L140R-F1 GCCAAGGTGACCCCCGCCCCCACCAGACAAAGAGATTTCACAGCCGCC
  • 9181P-L140R-R1 GGCGGCTGTGAAATCTCTTTGTCTGGTGGGGGCGGGGGTCACCTTGGC
  • SEQ ID NO: 7 Amino acid sequence with histidine and connecting peptide (M6)
  • 9181P-TIM3-F AGTTTAAACGGATCTCTAGCgaattcGCCGCCACCATGTTCTCCCACCTGCC
  • 9181P-TIM3-R TCGAGGTCGGGGGATCCGCGGCCGCTCAGTGATGGTGGTGGTGGTGGGAGCCTC
  • 9181P-M6-F1 AGGATCCAGATCCCTAGAATCATGGCCGCCGAGAAGTTTAACCTGAAGCTGG
  • 9181P-M6-R1 CCAGCTTCAGGTTAAACTTCTCGGCGGCCATGATTCTAGGGATCTGGATCCT
  • the 10 ⁇ l PCR reaction system is as follows:
  • the expressed target recombinant protein was purified, and the culture medium, natural protein, denatured protein and purified protein were identified by SDS-PAGE and WB.
  • the results are shown in Figure 1.
  • the concentrations of purified recombinant protein M1, M2, M3, M4, M5 and M6 were 0.1mg/ml, 0.05mg/ml, 0.05mg/ml, 0.1mg/ml, 0.22mg/ml, 0.11mg/ml, 0.17mg, respectively /ml, freeze-dried and preserved.
  • Magnetic beads are used to sort the primary monocytes from the peripheral blood of healthy humans.
  • the soluble Tim-3 is simultaneously treated with the primary monocytes at the concentration of 10, 20, 40 and 80ng/ml, 1 ⁇ g/ml LPS was stimulated for 24 hours; 1 ⁇ g/ml LPS stimulated monocytes for 30 minutes, and then soluble Tim-3 was used to interfere with primary monocytes at concentrations of 10, 20, 40 and 80ng/ml, and 1 ⁇ g/ml LPS was stimulated for a total of 24 hours.
  • the instrument used is the American Bio-Rad Microplate Reader iMark Absorption Cursor Microplate Reader, and the reagents used are TNF- ⁇ ELISA kits. The specific steps are in accordance with the instructions.
  • soluble Tim-3 significantly inhibited the cytokine TNF- ⁇ secretion ability of LPS stimulated monocytes, with significant differences and a dose-dependent effect. Moreover, the simultaneous intervention of soluble Tim-3 and LPS has a better inhibitory effect than the cytokine TNF- ⁇ secretion stimulated by LPS for 30 minutes, and the highest dose of 80ng/ml soluble Tim-3 has the best TNF- ⁇ inhibitory effect.
  • Magnetic beads are used to sort the primary monocytes of healthy human peripheral blood.
  • the soluble Tim-3 will be treated with the concentration of 20ng/ml at the same time.
  • the primary monocytes are stimulated with 1 ⁇ g/ml LPS for 24 hours, and the cells are collected.
  • the supernatant was used to detect the level of HMGB1 by ELISA.
  • the instrument used was the Bio-Rad microplate reader iMark absorption cursor microplate reader, and the reagents used were the HMGB1 ELISA kit. The specific steps were operated in accordance with the instructions.
  • Example 3 The liver injury effect of soluble Tim-3 on liver failure
  • ACLF chronic acute liver failure
  • CHB chronic hepatitis B
  • HC healthy controls
  • use anti-human CD14-APC flow cytometry antibody and anti-human Tim-3-PE flow cytometry antibody to mix with 100 ⁇ l of blood samples, incubate at room temperature for 15 minutes in the dark, add 1 ml of red blood cell lysate to mix, and incubate at room temperature in the dark After 10 minutes, centrifuge, add washing solution to wash, and resuspend and prepare for testing on the machine. Open the flow analysis software, collect 100,000 cells, and analyze the Tim-3 positive percentage of CD14-positive monocytes.
  • D-galactosamine D-GalN
  • LPS endotoxin
  • the mutant sTim-3 protein sequence of the example is SEQ ID NO: 2-7 (amino acid sequence with histidine and connecting peptide, M1, M2, M3, M4, M5, M6). Both M0 and sTim-3 are unmutated sTim-3 protein sequences, where M0 is recombinantly expressed with mutant sTim-3, and sTim-3 is a commercial recombinant protein.
  • Magnetic beads are used to sort the primary monocytes from the peripheral blood of healthy people.
  • 1 ⁇ g/ml LPS stimulates the cells
  • the instrument used is the American Bio-Rad microplate reader iMark absorption cursor microplate reader, and the reagents used are domestic TNF- ⁇ ELISA kits. The specific steps are in accordance with the instructions. .
  • mutant sTim-3 proteins (M1, M2, M3 and M4), M0 and sTim-3 all significantly inhibited the ability of LPS to stimulate the secretion of the cytokine TNF- ⁇ of monocytes.
  • M5 nor M6 significantly inhibit the secretion ability of the cytokine TNF- ⁇ of LPS stimulated monocytes.
  • recombinant proteins (M0, M1, M2, M3, M4, M5, M6) have no significant difference in their ability to inhibit TNF- ⁇ secretion.

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Abstract

本发明提供了可溶性Tim-3重组蛋白用于制备具有调控单核细胞功能的药物或增强肿瘤免疫应答的功能的药物中的应用，所述可溶性Tim-3重组蛋白的氨基酸序列为SEQ ID NO:1。另外，本发明还提供了可溶性Tim-3重组蛋白的突变型蛋白及其制备和应用。所述突变型重组蛋白是通过定向进化技术筛选获得，具有调控单核细胞功能或增强机体免疫应答功能。

Description

可溶性Tim-3重组蛋白及其突变型蛋白的制备和应用 技术领域
本发明属于生物技术领域，具体内容涉及可溶性Tim-3重组蛋白及其突变型蛋白的制备和应用。
技术背景
生物技术药物主要包括蛋白质、多肽和核酸分子，几乎覆盖了包括癌症、自身免疫性疾病以及传染病等的上百种疾病。应用蛋白质工程技术制造的重组蛋白类产品在人们的生产、生活、医疗中发挥着不可或缺的作用。重组蛋白质药物是目前最主要的一类生物技术药物，与小分子化学药物相比，具有疗效好、副作用小和生物功能明确等一系列优点。
肝衰竭是多种因素引起的严重肝脏损害，出现以凝血机制障碍和黄疸、肝性脑病、腹水等为主要表现的一组临床症候群。发病急骤，病死率高，危害极大。它是全球公认的危及患者生命的、极具挑战性的临床危重症，目前尚缺乏特效药物和手段。目前认为肝移植是治疗本病的最有效手段，但由于供肝短缺及治疗费用高昂，其临床运用受到极大限制。固有免疫细胞单核细胞过度活化在肝衰竭发病机制中起到重要作用。细胞表面的Tim-3(T cell immunoglobulin-and mucin-domain-containing molecule-3)在单核细胞、巨噬细胞、树突状细胞和淋巴细胞等均有表达，单核细胞膜上Tim-3分子脱落产生的可溶性Tim-3(soluble Tim-3,sTim-3)，与败血症患者的IL-12和TNF-α表达成负相关，课题组前期体外研究发现，sTim-3可使LPS刺激的原代人单核细胞TNF-α表达水平下降，表明sTim-3具有抑制单核细胞活化作用。
目前，每年肝癌患者占到全世界新发现恶性肿瘤病人的4％，肝癌已成为国内肿瘤死亡病因的第二位，有极高的发病率和致死率。现以形成以手术切除为主的治疗方式，然而手术治疗预后差，易复发。研究表明，长期的免疫力低下也是癌症产生的重要原因之一，提高机体的免疫水平是肿瘤治疗的关键手段。因此寻找具有提高免疫力水平作用的抗癌药物是非常必要的。肿瘤免疫治疗是许多治疗方法的总称，有免疫检查点(check point)疗法、细胞因子疗法、肿瘤疫苗和细胞治疗。肿瘤免疫检查点分子的研究主要集中在Tim-3、CTLA-4、PD1三个分子上，这些分子抑制微环境中免疫细胞活性，肿瘤免疫检查点抑制剂是目前肿瘤免疫治疗的最主要方面，通过抑制肿瘤细胞的免疫逃逸，调动自身免疫系统功能消除肿瘤。Tim-3分子的发现是源于寻找区分Thl细胞和Th2细胞的表面标志物。最近研究发现，通过使用半定量RT-PCR方法，测定荷瘤小鼠不同时间段的可溶性Tim-3和CTLA-4等指标的表达水平，同时测定肿瘤生长。结果发现，肿瘤生长与CTLA-4表达成正相关，与可溶性Tim-3负相关，提示可溶 性Tim-3可能具有抑制肿瘤生长作用，目前未有研究报道可溶性Tim-3在肝癌中的作用。最近对HIV疫苗的细胞介导免疫应答的研究表明，可溶性PD1和可溶性Tim-3增强腺病毒载体SIV疫苗(rAd5-SIV)对小鼠T细胞增殖能力，产生更多的抗原特异IFN-γ(+)CD4(+)和CD8(+)T细胞。提示sTim-3可能具有免疫佐剂作用，增强机体的T细胞免疫应答。
发明内容
本发明提供了一种新型的可溶性Tim-3重组蛋白的应用及其纯化方法。
可溶性Tim-3重组蛋白用于制备具有调控单核细胞功能的药物或增强肿瘤免疫应答的功能的药物中的应用，所述可溶性Tim-3重组蛋白的氨基酸序列为SEQ ID NO:1(所述可溶性Tim-3重组蛋白包含或不包含组氨酸标签序列如HHHHHH；如果包含组氨酸标签序列时如HHHHHH，在标签序列前加上连接肽如(G)nS，n＝1到4，优选的n为4)。
所述调控单核细胞功能的药物可用于抑制炎症患者如肝衰竭患者的单核细胞过度活化；增强肿瘤免疫应答的功能的药物可用于增强癌症患者如肝癌患者的免疫应答，避免宿主免疫逃逸。
可溶性Tim-3重组蛋白的浓度优选为80ng/ml。
可溶性Tim-3重组蛋白的纯化方法，包括如下步骤：将含有重组人可溶性Tim-3的真核CHO细胞、原核大肠杆菌和昆虫杆状病毒表达系统细胞培养液通过微滤澄清、超滤浓缩换液后，再经过阴阳离子层析柱、分子筛层析、疏水层析柱和/或亲和层析柱，获得足够纯度(SDS-PAGE测定纯度>96％)重组蛋白；所述可溶性Tim-3重组蛋白的氨基酸序列为SEQ ID NO:1。
另外，本发明还提供了可溶性Tim-3重组蛋白的突变型蛋白，及其突变型蛋白和突变型蛋白的制备和应用。所述突变型重组蛋白是通过定向进化技术筛选获得，具有调控单核细胞功能或增强机体免疫应答功能，并结合宿主密码子优化方式，提高其表达效率。
一种可溶性Tim-3重组突变型蛋白，突变型蛋白在人Tim-3(NP_116171.3)胞外结构域氨基酸(SEQ ID NO:1)对应的基因序列基础上，通过定向进化技术筛选得到突变基因，其突变位点不涉及物种间的保守序列，发生在非保守序列位置，进而表达的蛋白。
进一步的，所述可溶性Tim-3重组突变型蛋白包含或不包含组氨酸标签序列如HHHHHH；如果包含组氨酸标签序列时如HHHHHH，在标签序列前加上连接肽如(G)nS，n＝1-4，优选的n为4。
更进一步的，所述氨基酸序列对应的基因序列可根据宿主如原核和真核等密码子的特点进行优化。
优选的，所述可溶性Tim-3重组突变型蛋白的氨基酸序列为：SEQ ID NO:2-7之一。
可溶性Tim-3重组突变型蛋白的制备方法，采用PCR试剂盒进行突变扩增筛选得到基因序列，该突变型蛋白可保留不同种属来源的保守氨基酸序列；在人Tim-3(NP_116171.3)胞外结 构域氨基酸(SEQ ID NO:1)对应的基因序列基础上，通过定向进化技术筛选得到突变基因，进而氨基酸表达成可溶性Tim-3重组突变型蛋白，优选氨基酸序列为：SEQ ID NO:2-7的突变型蛋白。
所述氨基酸表达指预构建的重组蛋白质基因表达载体于真核表达系统、原核表达系统及昆虫杆状病毒表达系统表达，提取生产长效重组药物。
可溶性Tim-3突变型蛋白在制备具有调控单核细胞功能药物或增强肿瘤免疫应答的功能的药物中的应用。优选的，可溶性Tim-3突变型蛋白的氨基酸序列为：SEQ ID NO:2-7的突变型蛋白。
药物通过调控单核细胞功能或增强肿瘤免疫应答功能来实现，所述调控单核细胞功能药物可用于抑制炎症患者如肝衰竭患者的单核细胞过度活化，增强肿瘤免疫应答的功能可用于增强癌症患者如肝癌患者的免疫应答，避免宿主免疫逃逸。
本发明把可溶性Tim-3重组蛋白基因序列构建到原核、真核和昆虫杆状病毒表达载体，经宿主表达的重组蛋白采用离子交换层析、亲和层析和疏水层析等多种纯化方法相结合，纯化出可溶性Tim-3重组蛋白。
为了得到更强生物学功能的突变型重组蛋白，通过定向进化技术对该重组蛋白进行大量突变筛选，通过使用GeneMorph II随机突变PCR试剂盒(Stratagene)进行PCR扩增，目的基因构建到真核表达载体、原核表达载体或昆虫杆状表达载体，然后把突变型重组表达载体转化到真核、原核或昆虫宿主进行表达。
为了得到更高表达效率的突变型重组表达质粒，通过宿主密码子优化技术进行密码子优化。具体根据宿主真核、原核或昆虫密码子特点，采用重组PCR技术进行定点突变，得到密码子优化了的目的序列，再把这段目的序列经过酶切克隆到相应宿主表达载体上，然后把突变型重组表达载体转化到真核、原核或昆虫宿主进行表达。
可溶性Tim-3及突变型重组蛋白的表达纯化采用亲和层析、离子交换层析和疏水层析等多种纯化方法相结合，纯化出可溶性Tim-3重组蛋白及突变型重组蛋白。金属螯合亲和层析原理是通过蛋白质表面的一些特殊氨基酸与金属离子发生相互作用，从而对蛋白质进行亲和纯化，其中以6个组氨酸残基组合的融合标签6×His-Tag较为常用，具有配体简单、吸附量大、分离条件温和和通用性强等优点。离子层析既能进行粗纯，有效去除绝大多数带有电荷的宿主蛋白质和核酸，又能做到精细纯化，去除痕量杂蛋白。疏水层析既能富集蛋白又能去除绝大多数色素类物质。
本发明公开一种生物工程技术领域的可溶性Tim-3重组蛋白及其突变型蛋白的制备方法及其在疾病如肝脏疾病(肝衰竭和肝癌)中的应用。可溶性Tim-3重组蛋白是一种胞外分泌、可 溶性和分子量小的重组蛋白。该重组蛋白含有或不含有6×His标签的Tim-3胞外结构可溶性区域的衍生蛋白，突变型蛋白通过定向进化技术得到含有进化中的保守结构区，又经宿主密码子优化的氨基酸序列。该类重组蛋白具有调控单核细胞功能或增强肿瘤免疫应答的功能。
附图说明
图1为实施例1对培养基、天然蛋白、变性蛋白和纯化蛋白进行SDS-PAGE鉴定图；
图2为实施例2的可溶性Tim-3对单核细胞TNF-α分泌的剂量依赖效应图；
图3为实施例2可溶性Tim-3对单核细胞HMGB1分泌的抑制作用图；
图4为实施例3慢加急性肝衰竭患者单核细胞的mTim-3和sTim-3表达水平图；
图5为实施例3的sTim-3改善D-GalN/LPS急性肝衰竭小鼠肝脏组织学图；
图6为突变型和未突变sTim-3对单核细胞TNF-α分泌的效应图。
图7包含有组氨酸和连接肽的氨基酸序列。
具体实施方式
为了更清楚地说明本发明，下面结合优选实施例和附图对本发明做进一步的说明。本领域技术人员应当理解，下面所具体描述的内容是说明性的而非限制性的，不应以此限制本发明的保护范围，还包括各具体实施方式间的任意组合，本领域普通技术人员通过阅读本发明说明书而对本发明技术方案采取的任何等效的变换，均为本发明的权利要求所涵盖。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法；所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
本发明应用分子生物学领域使用的常规技术和方法，本领域技术人员可以在本发明所记载的技术方案基础上，采用本领域其它常规方法、实验方案和试剂，而不限于本发明具体实施例的限定。
实施例1可溶性Tim-3重组蛋白及其突变型重组蛋白的获得
人Tim-3(NP_116171.3)胞外结构域的氨基酸序列见SEQ ID NO:1。人Tim-3(NP_116171.3)胞外结构域、连接肽(G)nS和6个组氨酸His融合的氨基酸序列(附图7)，n＝1到4，优选为4。列举以SEQ ID NO:1为基础的6条突变蛋白氨基酸序列SEQ ID NO:2-7。编码含His或不含His氨基酸的核苷酸序列构建到真核表达载体、原核表达载体或昆虫杆状表达载体，然后把重组表达载体转化到真核、原核或昆虫宿主进行表达。
为了得到更强生物学功能的突变型重组蛋白，通过定向进化技术对该重组蛋白进行大量突变筛选，通过使用GeneMorph II随机突变PCR试剂盒(Stratagene)进行PCR扩增，目的基因构 建到真核表达载体、原核表达载体或昆虫杆状表达载体，然后把突变型重组表达载体转化到真核、原核或昆虫宿主进行表达。
为了得到更高表达效率的突变型重组表达质粒，通过宿主密码子优化技术进行密码子优化。具体根据宿主真核、原核或昆虫密码子特点，采用重组PCR技术进行定点突变，得到密码子优化了的目的序列，再把这段目的序列经过酶切克隆到相应宿主表达载体上，然后把突变型重组表达载体转化到真核、原核或昆虫宿主进行表达。
可溶性Tim-3及突变型重组蛋白的表达纯化
采用亲和层析、离子交换层析和疏水层析等多种纯化方法相结合，纯化出可溶性Tim-3重组蛋白及突变型重组蛋白。金属螯合亲和层析原理是通过蛋白质表面的一些特殊氨基酸与金属离子发生相互作用，从而对蛋白质进行亲和纯化，其中以6个组氨酸残基组合的融合标签6×His-Tag较为常用(图7)，具有配体简单、吸附量大、分离条件温和和通用性强等优点。离子层析既能进行粗纯，有效去除绝大多数带有电荷的宿主蛋白质和核酸，又能做到精细纯化，去除痕量杂蛋白。疏水层析既能富集蛋白又能去除绝大多数色素类物质。
1.构建含有6条突变核苷酸序列(SEQ ID NO:2-7)连接有组氨酸和连接肽的氨基酸序列(如图7)的重组表达载体所用引物
如下：
SEQ ID NO:2连接有组氨酸和连接肽的氨基酸序列(M1)
9181P-TIM3-F：AGTTTAAACGGATCTCTAGCgaattcGCCGCCACCATGTTCTCCCACCTGCC
9181P-TIM3-R：TCGAGGTCGGGGGATCCGCGGCCGCTCAGTGATGGTGGTGGTGGTGGGAGCCTC
9181P-L140R-F1：GCCAAGGTGACCCCCGCCCCCACCAGACAAAGAGATTTCACAGCCGCC
9181P-L140R-R1：GGCGGCTGTGAAATCTCTTTGTCTGGTGGGGGCGGGGGTCACCTTGGC
SEQ ID NO:3连接有组氨酸和连接肽的氨基酸序列(M2)
9181P-TIM3-F：AGTTTAAACGGATCTCTAGCgaattcGCCGCCACCATGTTCTCCCACCTGCC
9181P-TIM3-R：TCGAGGTCGGGGGATCCGCGGCCGCTCAGTGATGGTGGTGGTGGTGGGAGCCTC
9181P-L140R-F1：GCCAAGGTGACCCCCGCCCCCACCAGACAAAGAGATTTCACAGCCGCC
9181P-L140R-R1：GGCGGCTGTGAAATCTCTTTGTCTGGTGGGGGCGGGGGTCACCTTGGC
SEQ ID NO:4连接有组氨酸和连接肽的氨基酸序列(M3)
9181P-TIM3-F：AGTTTAAACGGATCTCTAGCgaattcGCCGCCACCATGTTCTCCCACCTGCC
9181P-TIM3-R：TCGAGGTCGGGGGATCCGCGGCCGCTCAGTGATGGTGGTGGTGGTGGGAGCCTC
9181P-L140R-F1：GCCAAGGTGACCCCCGCCCCCACCAGACAAAGAGATTTCACAGCCGCC
9181P-L140R-R1：GGCGGCTGTGAAATCTCTTTGTCTGGTGGGGGCGGGGGTCACCTTGGC
SEQ ID NO:5连接有组氨酸和连接肽的氨基酸序列(M4)
9181P-TIM3-F：AGTTTAAACGGATCTCTAGCgaattcGCCGCCACCATGTTCTCCCACCTGCC
9181P-TIM3-R：TCGAGGTCGGGGGATCCGCGGCCGCTCAGTGATGGTGGTGGTGGTGGGAGCCTC
9181P-L140R-F1：GCCAAGGTGACCCCCGCCCCCACCAGACAAAGAGATTTCACAGCCGCC
9181P-L140R-R1：GGCGGCTGTGAAATCTCTTTGTCTGGTGGGGGCGGGGGTCACCTTGGC
SEQ ID NO:6连接有组氨酸和连接肽的氨基酸序列(M5)
9181P-TIM3-F：AGTTTAAACGGATCTCTAGCgaattcGCCGCCACCATGTTCTCCCACCTGCC
9181P-TIM3-R：TCGAGGTCGGGGGATCCGCGGCCGCTCAGTGATGGTGGTGGTGGTGGGAGCCTC
9181P-L140R-F1：GCCAAGGTGACCCCCGCCCCCACCAGACAAAGAGATTTCACAGCCGCC
9181P-L140R-R1：GGCGGCTGTGAAATCTCTTTGTCTGGTGGGGGCGGGGGTCACCTTGGC
SEQ ID NO:7连接有组氨酸和连接肽的氨基酸序列(M6)
9181P-TIM3-F：AGTTTAAACGGATCTCTAGCgaattcGCCGCCACCATGTTCTCCCACCTGCC
9181P-TIM3-R：TCGAGGTCGGGGGATCCGCGGCCGCTCAGTGATGGTGGTGGTGGTGGGAGCCTC
9181P-M6-F1：AGGATCCAGATCCCTAGAATCATGGCCGCCGAGAAGTTTAACCTGAAGCTGG
9181P-M6-R1：CCAGCTTCAGGTTAAACTTCTCGGCGGCCATGATTCTAGGGATCTGGATCCT
2.构建重组重组表达载体所用的PCR反应体系和程序
10μl PCR反应体系如下：
2×Taq酶：         5μl
正向引物F(10μM):0.4μl
反向引物F(10μM):0.4μl
模板：             1μl
双蒸水加至:        10μl
PCR反应程序：
初始变性：95℃，5min
变性：    95℃，30s
退火：    55℃，30s
延伸：    72℃，2min
循环数：30个
最后一步延伸：72℃，5min
储存：         保持16℃
3.构建重组表达载体，瞬时转染和表达纯化
构建pATX2表达载体，瞬时转染至80ml HEK293细胞，转染后第6天收集1.5ml细胞培养基和细胞。对表达的目的重组蛋白进行纯化，对培养基、天然蛋白、变性蛋白和纯化蛋白进行SDS-PAGE和WB进行鉴定，结果如图1。纯化的重组蛋白M1，M2，M3，M4，M5和M6浓度分别为0.1mg/ml，0.05mg/ml，0.05mg/ml，0.1mg/ml，0.22mg/ml，0.11mg/ml，0.17mg/ml，进行冻干保存。
实施例2可溶性Tim-3抑制单核细胞活化释放炎症因子
1.可溶性Tim-3对单核细胞的TNF-α细胞因子分泌能力的剂量依赖效应
磁珠分选健康人外周血原代单核细胞，在1μg/ml LPS刺激细胞时，将可溶性Tim-3以10、20、40和80ng/ml浓度同时处理原代单核细胞，1μg/ml LPS刺激24h；1μg/ml LPS先刺激单核细胞30min，再将可溶性Tim-3以10、20、40和80ng/ml浓度干预原代单核细胞，1μg/ml LPS总共刺激24h。收集细胞上清液，并采用ELISA检测其中TNF-α水平，所用的仪器是美国Bio-Rad伯乐酶标仪iMark吸收光标酶标仪，所用试剂是TNF-αELISA试剂盒，具体步骤按照说明书操作。
如图2所示，与LPS组相比，可溶性Tim-3显著抑制LPS刺激单核细胞的细胞因子TNF-α分泌能力，具有显著性差异，而且具有剂量依赖效应。而且可溶性Tim-3与LPS同时干预比LPS先刺激30min的细胞因子TNF-α分泌的抑制效果更好，其中最高剂量80ng/ml可溶性Tim-3的TNF-α抑制效果最好。
2.可溶性Tim-3对单核细胞的HMGB1分泌能力效应
磁珠分选健康人外周血原代单核细胞，在1μg/ml LPS刺激细胞时，将可溶性Tim-3以20ng/ml浓度同时处理原代单核细胞，1μg/ml LPS刺激24h，收集细胞上清液，并采用ELISA检测其中HMGB1水平，所用的仪器是美国Bio-Rad伯乐酶标仪iMark吸收光标酶标仪，所用试剂是HMGB1ELISA试剂盒，具体步骤按照说明书操作。
如图3所示，与LPS组相比，可溶性Tim-3显著抑制LPS刺激单核细胞的炎症介质HMGB1分泌能力，具有显著性差异。
实施例3可溶性Tim-3对肝衰竭的肝损伤作用
1.慢加急性肝衰竭患者单核细胞的可溶性Tim-3和膜式Tim-3表达水平
收集慢加急性肝衰竭患者(ACLF,n＝8)、慢性乙型肝炎患者(CHB,n＝8)和健康对照(HC,n＝8)外周血。对于外周血样本，用抗人CD14-APC流式抗体和抗人Tim-3-PE流式抗体与血液标本100μl混匀，室温避光孵育15min，加入红细胞裂解液1ml混匀，室温避光孵育10min 后，离心，加洗涤液洗涤，重悬后准备上机测定。打开流式分析软件，收集100000个细胞，分析CD14阳性单核细胞的Tim-3阳性百分数。图4的结果表明，慢加急性肝衰竭患者(ACLF,n＝8)、慢性乙型肝炎患者(CHB,n＝8)和健康对照(HC,n＝8)的血浆可溶性Tim-3(sTim-3)水平随疾病进展表达水平显著上升，而单核细胞膜上Tim-3(mTim-3)水平随疾病进展而显著降低。2.可溶性Tim-3对急性肝衰竭小鼠的肝损伤作用
模型组采用D-氨基半乳糖胺(D-GalN)联合内毒素(LPS)诱导小鼠急性肝衰竭模型，可溶性Tim-3治疗组是急性肝衰竭模型在造模后30min，再经尾静脉注射可溶性Tim-3，对照组是小鼠经尾静脉注射生理盐水。所用的C57BL/6小鼠为：雄性，6-8周龄，18-20g。HE染色检测的肝组织学表现，发现sTim-3显著改善肝细胞坏死，对该模型具有保护作用(图5)。
实施例4突变型和未突变sTim-3蛋白对单核细胞TNF-α分泌的抑制效应
实施例的突变型sTim-3蛋白序列是SEQ ID NO:2-7(连接有组氨酸和连接肽的氨基酸序列，M1,M2,M3,M4,M5,M6)。M0和sTim-3都是未突变的sTim-3蛋白序列，其中M0是与突变型sTim-3一起重组表达，sTim-3是商用重组蛋白。
磁珠分选健康人外周血原代单核细胞，在1μg/ml LPS刺激细胞时，将可溶性Tim-3以20ng/ml浓度提前30min处理原代单核细胞，再用1μg/ml LPS刺激24h。收集细胞上清液，并采用ELISA检测其中TNF-α水平，所用的仪器是美国Bio-Rad伯乐酶标仪iMark吸收光标酶标仪，所用试剂是国产TNF-αELISA试剂盒，具体步骤按照说明书操作。
如图6所示，与LPS组相比，突变型sTim-3蛋白(M1，M2,M3和M4)，M0和sTim-3都显著抑制LPS刺激单核细胞的细胞因子TNF-α分泌能力，具有显著性差异，而M5和M6都未显著抑制LPS刺激单核细胞的细胞因子TNF-α分泌能力。重组蛋白(M0，M1，M2，M3，M4，M5，M6)与商业sTim-3相比，抑制TNF-α分泌能力无显著差异。

Claims (10)

  1. 可溶性Tim-3重组蛋白用于制备具有调控单核细胞功能的药物或增强肿瘤免疫应答的功能的药物中的应用，所述可溶性Tim-3重组蛋白的氨基酸序列为SEQ ID NO:1。
  2. 根据权利要求1所述的应用，其特征在于：所述调控单核细胞功能的药物可用于抑制炎症患者的单核细胞过度活化；所述增强肿瘤免疫应答的功能的药物可用于增强癌症患者的免疫应答，避免宿主免疫逃逸。
  3. 可溶性Tim-3重组蛋白的纯化方法，其特征在于：包括如下步骤：将含有重组人可溶性Tim-3的真核CHO细胞、原核大肠杆菌和昆虫杆状病毒表达系统细胞培养液通过微滤澄清、超滤浓缩换液后，再经过阴阳离子层析柱、分子筛层析、疏水层析柱和/或亲和层析柱，获得足够纯度可溶性Tim-3重组蛋白；所述可溶性Tim-3重组蛋白的氨基酸序列为SEQ ID NO:1。
  4. 一种可溶性Tim-3重组突变型蛋白，其特征在于:该突变型蛋白为：在人Tim-3(NP_116171.3)胞外结构域氨基酸(SEQ ID NO:1)对应的基因序列基础上，通过定向进化技术筛选得到突变基因，其突变位点不涉及物种间的保守序列，发生在非保守序列位置，进而表达的可溶性Tim-3重组突变型蛋白。
  5. 可溶性Tim-3重组突变型蛋白，其氨基酸序列为：SEQ ID NO:2-7之一。
  6. 根据权利要求4或5所述的可溶性Tim-3重组突变型蛋白，其特征在于:所述可溶性Tim-3重组突变型蛋白还包含组氨酸标签序列；组氨酸标签序列前加上连接肽。
  7. 根据权利要求6所述的可溶性Tim-3重组突变型蛋白，其特征在于:所述组氨酸标签序列为HHHHHH，所述连接肽为(G)nS，n＝1-4。
  8. 权利要求4-7任一所述可溶性Tim-3重组突变型蛋白的制备方法，其特征在于：在人Tim-3(NP_116171.3)胞外结构域氨基酸(SEQ ID NO:1)对应的基因序列基础上，通过定向进化技术筛选得到突变基因，进而氨基酸表达成可溶性Tim-3重组突变型蛋白。
  9. 权利要求4或5所述的可溶性Tim-3突变型蛋白用于制备具有调控单核细胞功能的药物或增强肿瘤免疫应答的功能的药物中的应用。
  10. 根据权利要求9所述的应用，其特征在于：所述调控单核细胞功能的药物可用于抑制炎症患者的单核细胞过度活化；所述增强肿瘤免疫应答的功能的药物可用于增强癌症患者的免疫应答，避免宿主免疫逃逸。
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