WO2018223764A1 - 人sDR5-Fc重组融合蛋白及其作为制备治疗生殖系统炎症的药物中的应用 - Google Patents

人sDR5-Fc重组融合蛋白及其作为制备治疗生殖系统炎症的药物中的应用 Download PDF

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WO2018223764A1
WO2018223764A1 PCT/CN2018/082316 CN2018082316W WO2018223764A1 WO 2018223764 A1 WO2018223764 A1 WO 2018223764A1 CN 2018082316 W CN2018082316 W CN 2018082316W WO 2018223764 A1 WO2018223764 A1 WO 2018223764A1
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seq
fusion protein
human
recombinant fusion
sdr5
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French (fr)
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万晓春
陈倩
张青梅
夏蒙
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深圳市中科艾深医药有限公司
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype

Definitions

  • the present invention relates to a fusion protein, in particular to a human sDR5-Fc recombinant fusion protein and its use as a medicament for the preparation of a treatment for seminal vesiculitis.
  • the seminal vesicle is an organ that stores sperm. It has a rich vascular layer. Because the seminal vesicle and prostate are closely adjacent to the urinary tract and rectum, it is easy to cause infection. Stimulation of inflammatory substances after infection can lead to edema and congestion of the seminal vesicle wall. When the patient is discharged, the smooth muscles contract violently, causing the small blood vessels to rupture and bloody sperm. When the ejaculatory duct is spread by inflammation, it will lead to narrowing of the ampulla of the seminal vesicle and the opening of the ejaculatory duct, further aggravating the inflammatory reaction and contributing to the formation of stones.
  • Seminal vesiculitis often occurs simultaneously with prostatitis.
  • the pathogens are mostly hemolytic streptococcus, staphylococcus aureus and Escherichia coli. They are classified into acute and chronic seminal vesiculitis. If the acute phase inflammation is not completely controlled, it is easy to change to Chronic seminal vesiculitis.
  • the main symptoms include blood sperm, perineal bulge, ejaculation pain and even some patients with obstructive azoospermia, which has a longer course.
  • Seminal vesiculitis affects the male reproductive system and can cause complications such as infertility.
  • the basic treatment principle of seminal vesiculitis is: the acute phase is mainly antibiotic treatment.
  • Therapeutic drugs mainly include the following:
  • the cefuroxime axetil tablet is a second-generation cephalosporin antibiotic. After oral absorption through the gastrointestinal tract, it is rapidly hydrolyzed to cefuroxime by an esterase to exert an antibacterial effect.
  • the mechanism of action of cefuroxime axetil tablets is to inhibit the synthesis of bacterial cell walls. Common adverse reactions are gastrointestinal reactions such as nausea, vomiting, diarrhea, and loose stools. Occasionally pseudomembranous colitis.
  • Erythromycin is a macrolide antibiotic, and its antibacterial spectrum is similar to penicillin, and it has a strong inhibitory effect on Gram-positive bacteria.
  • the mechanism of action is mainly combined with the 50S subunit of ribonucleoprotein, inhibiting peptide acyltransferase, affecting the translocation process of ribonucleoprotein, impeding the growth of peptide chain, inhibiting the synthesis of bacterial proteins, and acting as a bacteriostatic agent.
  • Adverse reactions include 1) gastrointestinal reactions: diarrhea, nausea, vomiting, stomach cramps, mouth and tongue pain, decreased appetite, etc. The incidence and response are related to the dose. 2) Allergic reactions: there may be urticaria and drug fever. It is characterized by drug fever, rash, and eosinophilia. 3) Liver damage: can cause liver damage, such as elevated serum alanine aminotransferase, jaundice and the like.
  • Quinolone antibiotics target bacterial DNA (DNA) and cause bacterial cells to no longer divide.
  • the main adverse reactions of this class of drugs are: 1 gastrointestinal reactions: nausea, vomiting, discomfort, pain, etc.; 2 central response: headache, dizziness, poor sleep, etc., and can cause mental symptoms; 3 due to this class of drugs can inhibit ⁇ - the role of aminobutyric acid, therefore can induce epilepsy, with a history of epilepsy with caution; 4 such drugs can affect cartilage development, pregnant women, underage children should be used with caution; 5 can produce crystallized urine, still in alkaline urine More likely to occur; 6 large doses or long-term application of this class of drugs are prone to liver damage.
  • Death receptor 5 is a member of the tumor necrosis factor receptor family and has high levels of expression in activated peripheral blood lymphocytes, inflammatory tissues, ischemic tissues, and some tumor cells.
  • DR5 receptor Tumor necrosis factor related apoptosis inducing ligand (TRAIL) leads to a series of signal cascades, which activate caspase-8 to induce apoptosis.
  • TRAIL Tumor necrosis factor related apoptosis inducing ligand
  • the human DR5 protein is a transmembrane protein composed of 411 amino acids. Among them, amino acids 1 to 55 are signal peptides, amino acids 84 to 179 are chain-like binding regions containing two cysteine-rich repeating functional regions, amino acids 184 to 206 are transmembrane regions, and intracellular regions contain death. Domain. Soluble DR5 (soluble DR5, sDR5) is a soluble form of DR5 that does not contain a transmembrane region and is secreted outside the cell due to the lack of transmembrane regions that cannot be expressed on the cell membrane.
  • sDR5 Although sDR5 retains its activity in combination with TRAIL ligands, it does not transmit apoptotic signals to cells and blocks TRAIL-DR5-mediated apoptosis. And sDR5 is a human body's own protein, which has the advantages of low toxicity and no immunogenicity, and has great potential as an important therapeutic drug for seminal vesiculitis. At present, whether sDR5 has a therapeutic effect on seminal vesiculitis has not been reported at home and abroad.
  • the sDR5-Fc fusion protein blocks the TRAIL-DR5 pathway-induced apoptosis by blocking the binding of TRAIL to the DR5 receptor on the cell membrane surface by binding to the TRAIL molecule.
  • seminal vesiculitis whatever the pathogenesis is, it will eventually involve cell apoptosis.
  • the TRAIL-DR5 pathway is involved in a combination of factors leading to seminal vesicles, so the range of indications is greater than that of antibiotics.
  • the sDR5-Fc fusion protein is derived from the body's own protein components and is safer than antibiotics. Compared with the traditional Chinese medicine used, the sDR5-Fc fusion protein has a clear mechanism of action, specific target, and less toxic side effects.
  • One aspect of the present invention provides a fusion protein, wherein the amino terminus of the fusion protein is a human death receptor 5 soluble fragment; and the carboxy terminus is an Fc fragment of human immunoglobulin 1 (IgG1).
  • the human death receptor 5 soluble fragment has the amino acid sequence set forth in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID No. 3 or SEQ ID No. 4, or has A protein having 95% or more homology to SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID No. 3 or SEQ ID No. 4 and having the same function.
  • amino acid sequence of the human death receptor 5 soluble fragment is as shown in SEQ ID NO.
  • the Fc fragment of human IgG1 has the amino acid sequence set forth in SEQ ID NO.
  • the amino acid sequence of the Fc fragment of human IgG1 is set forth in SEQ ID NO.
  • the human death receptor 5 soluble fragment is ligated or directly linked to the Fc fragment of human IgG1.
  • the linker is selected from the group consisting of amino acid sequences, and more preferably, the linker is selected from the group consisting of 1-100 amino acid sequences, or 1-10 amino acid sequences.
  • nucleotide sequence encoding the aforementioned fusion protein is provided.
  • the use of the aforementioned human sDR5-Fc recombinant fusion protein for the preparation of a medicament for treating or preventing inflammation is provided.
  • the inflammation is selected from seminal vesiculitis, prostatitis, and urethritis.
  • the present invention provides a use of a tumor necrosis factor-related apoptosis-inducing ligand antagonist for the preparation of a medicament for treating or preventing inflammation.
  • tumor necrosis factor-related apoptosis inducing ligand antagonist is selected from the aforementioned human sDR5-Fc recombinant fusion protein.
  • the inflammation is selected from the group consisting of seminal vesiculitis, prostatitis, and urethritis.
  • the present invention provides the use of a human sDR5-Fc recombinant fusion protein for the preparation of a medicament for treating or preventing blood or seminal vesicle swelling.
  • the invention discloses the above recombinant fusion protein, which can be combined with a pharmaceutically acceptable excipient to form a pharmaceutical preparation composition to exert a more stable therapeutic effect, and the preparation can be a suspension, a water needle, a lyophilized preparation and the like which are commonly used in the pharmaceutical field, preferably a water needle.
  • the pharmaceutically acceptable excipient comprises one or a combination of a surfactant, a solution stabilizer, an isotonicity adjusting agent and a buffer
  • Surfactants include nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (Tween 20 or 80); Triton; sodium dodecyl sulfate (SDS); poloxamer (eg poloxamer 188); Pluronics; Sodium sulfate; tetradecyl, linoleyl or octadecyl sarcosine, etc., should be added in such a way that the granulation tendency of the recombinant fusion protein is minimal, and the solution stabilizer can be a saccharide, including reducing sugars and non-reducing properties.
  • Sugar, amino acids include monosodium glutamate or histidine
  • alcohols include propylene glycol, polyethylene glycol, triol, higher sugar alcohol or a combination thereof
  • the amount of solution stabilizer should be added The final formed formulation is maintained in a stable state for a period of time that is considered stable by those skilled in the art.
  • the isotonicity adjusting agent may be one of sodium chloride and mannitol
  • the buffer may be TRIS, phosphate buffer, histidine buffer.
  • the dosage when the sDR5-Fc recombinant fusion protein is administered to an animal including a human, the dosage is different depending on the age and weight of the patient, the disease characteristics and severity, and the administration route, and the results of the animal experiment can be referred to. And in all cases, the total dose can not exceed a certain range. Specifically, the dose for intravenous injection is 0.01 to 3000 mg/day.
  • the above recombinant fusion protein and composition thereof disclosed in the present invention can also be administered in combination with other therapeutic drugs for seminal vesiculitis, prostatitis and urethritis for the treatment of seminal vesiculitis, prostatitis and urethritis.
  • These drugs include anti-infectives and anti-inflammatory drugs.
  • the recombinant fusion proteins and compositions thereof disclosed herein can be administered in combination with one or a combination of the above-described anti-infective and anti-inflammatory drugs.
  • the sDR5-Fc antibody fusion protein of the present invention can block the core signaling pathway of apoptosis, and the TRAIL-DR5 apoptosis signaling pathway is widely present in inflammation caused by various factors, and therefore, human sDR5-Fc antibody fusion protein treatment Seminal vesicles are extensive.
  • the human sDR5-Fc antibody fusion protein is derived from the human body's own protein, and the metabolite is an amino acid, so the safety is superior to other therapeutic drugs. And the mechanism of action is completely different from antibiotics and traditional Chinese medicines, which can be safely combined with other therapeutic drugs to enhance the efficacy and promote rehabilitation.
  • Figure 1A shows an additional 25 amino acid hydrophobicity analysis in SEQ ID NO.
  • Figure 1B - Figure 1C SEQ ID NO. 6 and SEQ ID NO. 8 are compared to human TRAIL protein affinity assay results, respectively.
  • Figure 2 sDR5-Fc inhibits the occurrence of seminal vesiculitis in mice.
  • Fig. 2A-2C shows the seminal vesicle congestion of the mice in the saline group, and the seminal vesicle is normal;
  • Fig. 2D The normal capsule of the 9 mg/kg sDR5-Fc administration group is normal.
  • FIG. 3 Comparison of the incidence of mouse seminal vesiculitis. Using t test, the incidence of seminal vesiculitis in the saline group was significantly higher than that in the sDR5-Fc group, p ⁇ 0.05.
  • the inventors constructed a recombinant fusion protein, which was expressed and purified in CHO cells.
  • the human DR5 soluble fragment was fused with human immunoglobulin IgG1 Fc fragment in various ways.
  • the results of mass spectrometry showed that the N-terminus of the target protein was unstable, and multiple amino acid cleavage occurred. Based on this, a plurality of amino acid sequences were designed and prepared from amino groups.
  • the human death receptor 5 soluble fragment (SEQ ID No. 1 to 4) and the carboxy terminal human IgG1 Fc fragment (SEQ ID NO. 5) are composed. Wherein the protein of SEQ ID NO.
  • the recombinant protein formed by SEQ ID NO. 1 and SEQ ID NO. 5 contains the lowest proportion of splicing isomers, 1% at 37 degrees, and SEQ ID NO. 2
  • the recombinant protein formed by SEQ ID NO. 5 contains 31% of the splicing isomer; SEQ ID NO. 3 is 49%; SEQ ID NO. 4 is 52%, both of which are significantly higher than SEQ ID NO.
  • the ratio of splicing isomers, so the recombinant proteins formed by SEQ ID NO. 1 and SEQ ID NO. 5 are the most optimal and most stable.
  • the recombinant protein in vitro activity assay was carried out using L929 cells, and the results showed that the protein activity of the recombinant protein formed by SEQ ID NO. 1 and SEQ ID NO. 5 was SEQ ID NO. 3 or SEQ ID NO. 4 and SEQ.
  • the protein activity of the recombinant protein formed by ID NO. 5 was not different, and was significantly higher than that of the recombinant protein formed by SEQ ID NO. 2 and SEQ ID NO.
  • the recombinant protein formed by SEQ ID NO. 2 and SEQ ID NO. 5 has a protein activity of 126.2% relative to the R&D sDR5-Fc protein activity, SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO.
  • the protein activity of the recombinant protein formed by ID NO. 5 is 300% to 400%, and the recombinant protein and TRAIL molecule formed by SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, respectively. Have a strong ability to combine.
  • the DR5 soluble fragment and the IgG1 Fc fragment may or may not contain a linking sequence.
  • the linker sequence is usually an amino acid sequence or a linker sequence comprising an acyl group, and the sequence of the amino acid sequence having a length of 1-100 amino acids, preferably 1-10 amino acids, is usually a sequence which does not affect two proteins.
  • the DR5 soluble fragment and the IgG1 Fc fragment do not contain a linker sequence as shown in SEQ ID NO.
  • nucleotide sequence encoding the fusion protein of SEQ ID NO. 6 is set forth in SEQ ID NO.
  • the fusion protein of the present invention can conveniently prepare by various known methods. These methods are, for example but not limited to, recombinant DNA methods, synthetic, and the like [see Murray KM, Dahl SLAnn; Pharmacother 1997 Nov; 31(11): 1335-8].
  • the gene encoding the fusion protein of the present invention has the sequence of SEQ ID NO. 7, and the sequence is particularly suitable for highly expressing the fusion protein of the present invention in eukaryotic cells, preferably CHO cells.
  • the nucleic acid encoding of the present invention can be conveniently prepared by a variety of known methods based on the nucleotide sequences described herein. These methods are, for example but not limited to, PCR, DNA synthesis, etc., and the specific method can be found in the Guide to Molecular Cloning.
  • any suitable eukaryotic expression vector can be used, and these vectors can be pcDNA3.1, pDR, pL101 and the like.
  • One skilled in the art can select a suitable expression vector based on the host cell.
  • a pL101 expression vector is employed.
  • the invention provides a host cell expressing a fusion protein of the invention comprising a coding sequence for a fusion protein of the invention.
  • the host cell is preferably a eukaryotic cell such as, but not limited to, a CHO cell, a 293 cell, a COS cell or the like.
  • the cell is a CHO cell which can express the fusion protein of the present invention well, and a fusion protein having good binding activity and good stability can be obtained.
  • sequence SEQ ID No. 8 has 25 amino acid sequences at the N-terminus than SEQ ID No. 6, and the sequence of SEQ ID NO. 8 is as follows:
  • the recombinant protein having the amino acid sequence of SEQ ID NO. 6 is more soluble relative to the recombinant protein having the amino acid sequence of SEQ ID NO.
  • SEQ ID No. 6 was reduced by only 25 amino acids at the N-terminus relative to SEQ ID No. 8, the inventors unexpectedly found that the recombinant protein of SEQ ID NO. 6 can be concentrated up to a high concentration of 100 mg/ml without Precipitation will occur, and it is easy to prepare a lyophilized powder for long-term stable storage.
  • the recombinant protein of SEQ ID NO. 8 will produce a macroscopic precipitate at a high concentration of 100 mg/ml, and is not suitable for a lyophilized powder dosage form.
  • the inventors tested the affinity of SEQ ID No. 6 and SEQ ID No. 8 for TRAIL protein, and the affinity of the recombinant protein of SEQ ID NO. 6 to human TRAIL protein (sino biological company, Cat. No. 10409-HNAE, SEQ)
  • the affinity of ID NO.8 with human TRAIL protein is 1 to 2 orders of magnitude higher ( Figures 1B and 1C).
  • Example 2 Human sDR5-Fc antibody fusion protein for the treatment of seminal vesicle.
  • mice Twenty C57BL/6 male mice were divided into two groups (saline group and 9 mg/kg fusion protein group SEQ ID NO. 6), 10 mice per group, and each mouse was injected with 15 mg/kg of peas in the tail vein. Protein (ConA), 1 hour later, each group of mice was given intravenous saline, 9 mg/kg fusion protein, respectively, and the administration volume was 10 ml/kg. ConA, saline and fusion protein groups were continuously injected for 15 weeks, then the mice were sacrificed and the seminal vesicles of the mice were observed. Compared with the saline group, the seminal vesicles of the 9 mg/kg fusion protein group were not congested and the appearance was normal (see Figure 2). .
  • ConA Protein
  • mice in the saline group had a high incidence of seminal vesiculitis, and the incidence of seminal vesiculitis was significantly lower in the administration group than in the saline group (p ⁇ 0.05).
  • the results are shown in Fig. 3.

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Abstract

一种人sDR5-Fc重组融合蛋白及其作为制备治疗生殖系统炎症的药物中的应用。具体地，构建了人DR5的可溶片段与人IgG1的Fc片段的融合蛋白，其人DR5的可溶片段氨基酸序列如SEQ ID NO.1、SEQ ID NO.2、SEQ ID No.3或SEQ ID No.4所示，人IgG1的Fc片段氨基酸序列如SEQ ID NO.5所示，或具有与SEQ ID NO.1、SEQ ID NO.2、SEQ ID No.3、SEQ ID No.4或SEQ ID NO.5 95％以上同源性并具有相同功能的重组融合蛋白。还提供所述人sDR5-Fc重组融合蛋白在制备治疗或预防炎症的药物中的应用。其中炎症选自精囊炎、前列腺炎、尿道炎。所述重组融合蛋白稳定性好，生物活性高，且能够高效安全地用于生殖系统炎症的治疗。

Description

人sDR5-Fc重组融合蛋白及其作为制备治疗生殖系统炎症的药物中的应用 技术领域
本发明涉及一种融合蛋白，具体涉及一种人sDR5-Fc重组融合蛋白及其作为制备治疗精囊炎的药物中的应用。
背景技术
精囊是藏精子的器官，有丰富的血管层，由于精囊、前列腺与泌尿道、直肠等器官紧密相邻，故很容易导致感染。感染后炎性物质的刺激，会导致精囊壁水肿而充血，当患者排精时，平滑肌猛烈收缩，导致小血管破裂而可出现血精。当射精管被炎症蔓延至时，将导致精囊射精管壶腹部及射精管开口狭窄，进一步加重炎症的反应，并且有助于结石的形成，相反结石又加重了管腔狭窄，造成感染的复发和迁延。精囊炎常与前列腺炎同时发生，致病菌多为溶血性链球菌、金黄色葡萄球菌及大肠杆菌等，分为急性和慢性精囊炎两类，如果急性期炎症未彻底控制，则易于转为慢性精囊炎。主要症状包括血精、会阴部坠胀、射精痛甚至部分患者伴有梗阻性无精子不育症，其病程较长。精囊炎会影响男性的生殖系统，可使患者出现不育症等并发症。
精囊炎的基本治疗原则是：急性期以抗生素治疗为主。治疗药物主要包括以下几种：
(1)头孢呋辛酯片
头孢呋辛酯片为第二代头孢菌素类抗生素。口服经胃肠道吸收后，在酯酶作用下迅速水解为头孢呋辛而发挥抗菌作用。头孢呋辛酯片的作用机制是抑制细菌细胞壁的合成。常见的不良反应都是胃肠道反应如恶心、呕吐、腹泻及稀便等。偶有伪膜性肠炎。
(2)红霉素
红霉素为大环内酯类抗生素，抗菌谱与青霉素近似，对革兰阳性菌有较强的抑制作用。作用机制主要是与核糖核蛋白体的50S亚单位相结合，抑制肽酰基转移酶，影响核糖核蛋白体的移位过程，妨碍肽链增长，抑制细菌蛋白质的合成，系抑菌剂。不良反应包括1)胃肠道反应：有腹泻、恶心、呕吐、胃绞痛、口舌疼痛、胃纳减退等，发生率、反应与剂量大小有关。2)过敏反应：可有荨麻疹及药物热。表现为药物热、皮疹、嗜酸粒细胞增多等。3)肝脏损害：可引起肝脏损害，如血清丙氨酸氨基转移酶升高，出现黄疸等。
(3)喹诺酮类药物抗感染治疗
喹诺酮类抗生素以细菌的脱氧核糖核酸(DNA)为靶，而使细菌细胞不再分裂。本类药物的不良反应主要有：①胃肠道反应：恶心、呕吐、不适、疼痛等；②中枢反应：头痛、 头晕、睡眠不良等，并可致精神症状；③由于本类药物可抑制γ-氨基丁酸的作用，因此可诱发癫痫，有癫痫病史者慎用；④本类药物可影响软骨发育，孕妇、未成年儿童应慎用；⑤可产生结晶尿，犹其在碱性尿中更易发生；⑥大剂量或长期应用本类药物易致肝损害。
由此可见，目前治疗精囊炎的药物种类单一，疗效有限，亟需开发新的治疗药物。
死亡受体5(Death receptor 5,DR5)是肿瘤坏死因子受体家族的成员，在活化的外周血淋巴细胞、炎症组织、缺血组织和一些肿瘤细胞中表达水平高。DR5受体与肿瘤坏死因子相关凋亡诱导配体(Tumor necrosis factor related apoptosis inducing ligand,TRAIL)相结合后引起一系列信号级联反应，激活caspase-8引发细胞凋亡。
人DR5蛋白是由411个氨基酸组成的跨膜蛋白。其中1～55位氨基酸是信号肽，84～179位氨基酸是含有2个富含半胱氨酸的重复功能区的链状结合区，184～206位氨基酸为跨膜区，胞内区含有死亡结构域。可溶性DR5(soluble DR5,sDR5)为DR5不含跨膜区域的可溶性形式，因缺乏跨膜区域不能在细胞膜上表达而被分泌至细胞外。sDR5虽然保持与TRAIL配体相结合的活性，但不能向细胞内传导凋亡信号，可阻断TRAIL-DR5介导的细胞凋亡反应。且sDR5是人体自身蛋白，具有毒性小、无免疫原性的优点，极具作为精囊炎的重要治疗药物的潜质。目前，sDR5对于精囊炎是否存在治疗作用，国内外尚无报道。
发明内容
探索和寻找其有效的治疗方法是目前临床研究的重点。目前用于治疗精囊炎的药物大多数为抗生素，不同的致病菌导致的精囊炎只能采用相对应的抗生素，且抗生素有很多的副作用，肝脏、肾脏毒性，胃肠道不良反应，甚至会造成抗生素滥用。
sDR5-Fc融合蛋白是通过与TRAIL分子结合，阻断TRAIL与细胞膜表面的DR5受体结合，从而阻断TRAIL-DR5通路诱导的细胞凋亡。在精囊炎中，无论致病机制是什么，必然最终会涉及细胞的凋亡。TRAIL-DR5通路参与多种因素导致的精囊炎，因此适应症范围比抗生素大。且sDR5-Fc融合蛋白来源于人体自身的蛋白组分，比抗生素更安全。相对临床上使用的中药，sDR5-Fc融合蛋白作用机理明确，作用靶点特异，毒副作用更小。
本发明一个方面提供了一种融合蛋白，所述的融合蛋白的氨基端是人死亡受体5可溶片段；羧基端是人免疫球蛋白1(IgG1)的Fc片段。
在另一优选例中，所述的人死亡受体5可溶片段具有如SEQ ID NO.1、SEQ ID NO.2、SEQ ID No.3或SEQ ID No.4所示的氨基酸序列，或具有与SEQ ID NO.1、SEQ ID NO.2、SEQ ID No.3或SEQ ID No.4 95％以上同源性并具有相同功能的蛋白。
较佳的，所述的人死亡受体5可溶片段的氨基酸序列如SEQ ID NO.1所示。
在另一优选例中，所述的人IgG1的Fc片段具有SEQ ID NO.5所示的氨基酸序列。较佳的，所述的人IgG1的Fc片段的氨基酸序列如SEQ ID NO.5所示。
在另一优选例中，人死亡受体5可溶片段与人IgG1的Fc片段之间以接头连接或直接连接。
较佳的，接头选自氨基酸序列，更佳的，接头选自1-100个氨基酸序列，或者1-10个氨基酸序列。
本发明的第二方面，提供了编码前述融合蛋白的核苷酸序列。
本发明再一个方面提供了前述的人sDR5-Fc重组融合蛋白在制备治疗或预防炎症的药物中的应用。
其中，炎症选自精囊炎、前列腺炎、尿道炎。
本发明再一个方面提供了一种肿瘤坏死因子相关凋亡诱导配体拮抗剂在制备治疗或预防炎症的药物中的应用。
其中，所述肿瘤坏死因子相关凋亡诱导配体拮抗剂选自前述的人sDR5-Fc重组融合蛋白。
其中，所述炎症选自精囊炎、前列腺炎、尿道炎。
本发明再一个方面提供了人sDR5-Fc重组融合蛋白在制备治疗或预防血精或精囊肿胀的药物中的应用。
本发明公开上述重组融合蛋白，可以和药学上可以接受的辅料一起组成药物制剂组合物从而更稳定地发挥疗效，制剂可为制药领域常用的混悬、水针、冻干等制剂，优选水针或冻干制剂，对于本发明公开的上述重组融合蛋白的水针或冻干制剂，药学上可以接受的辅料包括表面活性剂、溶液稳定剂、等渗调节剂和缓冲液之一或其组合，其中表面活性剂包括非离子型表面活性剂如聚氧乙烯山梨醇脂肪酸酯(吐温20或80)；Triton；十二烷基硫酸钠(SDS)；poloxamer(如poloxamer 188)；Pluronics；月桂硫酸钠；十四烷基、亚油基或十八烷基肌氨酸等，其加入量应使重组融合蛋白颗粒化趋势最小，溶液稳定剂可以为糖类，包括还原性糖和非还原性糖，氨基酸类包括谷氨酸单钠或组氨酸，醇类包括丙二醇、聚乙二醇、三元醇、高级糖醇之一或其组合，溶液稳定剂的加入量应该使最后形成的制剂在本领域的技术人员认为达到稳定的时间内保持稳定状态，等渗调节剂可以为氯化钠、甘露醇之一，缓冲液可以为TRIS、磷酸盐缓冲液、组氨酸缓冲液之一。
本发明中sDR5-Fc重组融合蛋白在对包括人在内的动物给药时，给药剂量因病人的年龄和体重，疾病特性和严重性，以及给药途径而异，可以参考动物实验的结果和种种情况， 总给药量不能超过一定范围。具体讲静脉注射的剂量是0.01～3000mg/天。
本发明公开的上述重组融合蛋白及其组合物还可以和其他的精囊炎、前列腺炎、尿道炎治疗药物联合给药，用于精囊炎、前列腺炎、尿道炎的治疗。这些药物包括抗感染类和抗炎类。本发明公开的重组融合蛋白及其组合物可以和上述的抗感染类和抗炎类药物之一或其组合联合用药。
有益效果
1、现有技术中治疗精囊炎的药物大多数是抗生素和中药，本发明为精囊炎的治疗提供了新的思路。
2、本发明人sDR5-Fc抗体融合蛋白能阻断细胞凋亡的核心信号通路，TRAIL-DR5凋亡信号通路广泛存在于多种因素导致的炎症中，因此，人sDR5-Fc抗体融合蛋白治疗精囊炎具有广泛性。
3、人sDR5-Fc抗体融合蛋白来源于人体自身的蛋白，代谢产物为氨基酸，因此安全性较其它治疗药物更优。且作用机理完全不同于抗生素和中药，能够安全的与其它治疗药物联用，增强疗效，促进康复。
附图说明
图1氨基酸序列SEQ ID NO.6与SEQ ID NO.8重组蛋白性质比较。
图1A SEQ ID NO.8中多出25个氨基酸疏水性分析。图1B-图1C SEQ ID NO.6与SEQ ID NO.8分别与人TRAIL蛋白亲和力检测结果比较。
图2sDR5-Fc抑制小鼠精囊炎的发生。图2A-图2C生理盐水组小鼠精囊充血，患精囊炎；图2D 9mg/kg sDR5-Fc给药组小鼠精囊正常。
图3小鼠精囊炎发生率比较。使用t检验，生理盐水组小鼠精囊炎的发生率显著性高于sDR5-Fc组，p<0.05。
具体实施方式
以下通过实施例对本发明作进一步的说明，但本发明的保护范围并不局限于此。
下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。实施例中所用到的各种试剂和样品，除另有说明均为市售产品。
实施例1重组sDR5-Fc表达序列的设计和重组
发明人经过长期的研究和大量实验，构建了重组融合蛋白，在CHO细胞中表达并纯化获得蛋白。将人DR5可溶性片段与人免疫球蛋白IgG1Fc片段进行多种方式的融合，质谱 分析结果显示目的蛋白N端不稳定，会出现多个氨基酸剪切，据此设计并制备多条氨基酸序列，由氨基端的人死亡受体5可溶片段(SEQ ID No.1～4)和羧基端的人IgG1的Fc片段(SEQ ID NO.5)组成。其中SEQ ID NO.1的蛋白氨基酸序列最短，由SEQ ID NO.1和SEQ ID NO.5形成的重组蛋白所含剪接异构体比例最低，在37度下为1％，而SEQ ID NO.2和SEQ ID NO.5形成的重组蛋白所含剪接异构体比例为31％；SEQ ID NO.3为49％；SEQ ID NO.4为52％，均显著性高于SEQ ID NO.1的剪接异构体比例，因此SEQ ID NO.1和SEQ ID NO.5形成的重组蛋白的纯度最优，最稳定。且用L929细胞进行重组蛋白体外活性检测(MTT实验)，结果显示由SEQ ID NO.1和SEQ ID NO.5形成的重组蛋白的蛋白活性与SEQ ID NO.3或SEQ ID NO.4和SEQ ID NO.5形成的重组蛋白的蛋白活性没有差异，均明显高于SEQ ID NO.2和SEQ ID NO.5形成的重组蛋白的蛋白活性。相对R&D公司sDR5-Fc蛋白活性，SEQ ID NO.2和SEQ ID NO.5形成的重组蛋白的蛋白活性为126.2％，SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.4和SEQ ID NO.5分别形成的重组蛋白的蛋白活性为300％～400％，SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.4和SEQ ID NO.5分别形成的重组蛋白和TRAIL分子有很强的结合能力。
SEQ ID NO.1：
Figure PCTCN2018082316-appb-000001
SEQ ID NO.2：
Figure PCTCN2018082316-appb-000002
SEQ ID NO.3：
Figure PCTCN2018082316-appb-000003
SEQ ID No.4：
Figure PCTCN2018082316-appb-000004
SEQ ID NO.5：
Figure PCTCN2018082316-appb-000005
Figure PCTCN2018082316-appb-000006
本发明所述的融合蛋白中，所述的DR5可溶片段与IgG1Fc片段之间，可以含有或不含连接序列。所述的连接序列通常是氨基酸序列或包含酰基基团的连接序列，氨基酸序列的长度为1-100个氨基酸，优选为1-10个氨基酸的序列通常是对两个蛋白不产生影响作用的序列。作为本发明的优选方式，所述的DR5可溶片段与IgG1Fc片段之间不含有连接序列，如SEQ ID NO.6所示：
SEQ ID NO.6
Figure PCTCN2018082316-appb-000007
编码SEQ ID NO.6融合蛋白的核苷酸序列如SEQ ID NO.7所示：
Figure PCTCN2018082316-appb-000008
Figure PCTCN2018082316-appb-000009
根据本发明提供的氨基酸序列，本技术领域人员可方便地用各种已知方法制得本发明的融合蛋白。这些方法如但不限于：重组DNA法，人工合成，等【参见Murray KM,Dahl SLAnn；Pharmacother 1997Nov；31(11):1335-8】。
在得知了本发明的融合蛋白的氨基酸序列后，本领域人员可以方便的根据所述的氨基酸序列获得编码本发明的融合蛋白的基因序列。
作为本发明的优选方式，本发明的融合蛋白的编码基因具有SEQ ID NO.7所示的序列，采用该序列，特别适合于在真核细胞(优选CHO细胞)中高表达本发明的融合蛋白。
根据本文所述的核苷酸序列，本技术领域人员可方便地用各种已知方法制得本发明的编码核酸。这些方法例如但不限于：PCR,DNA人工合成等，具体的方法可参见《分子克隆实验指南》。
对于本发明，任何合适的真核表达载体都可以使用，这些载体可以为pcDNA3.1，pDR，pL101等。本领域技术人员可以根据宿主细胞来选择合适的表达载体。作为本发明的优选方式，当在真核细胞特别是CHO细胞(中国仓鼠卵巢细胞)中进行表达时，采用pL101表达载体。
本发明提供了表达本发明融合蛋白的宿主细胞，其中含有本发明的融合蛋白的编码序列。所述的宿主细胞优选的是真核细胞，例如但不限于CHO细胞，293细胞，COS细胞等。作为本发明的优选方式，所述的细胞是CHO细胞，其可良好地表达本发明的融合蛋白，可获得结合活性良好，稳定性良好的融合蛋白。
序列SEQ ID No.8比SEQ ID No.6在N端多25个氨基酸序列，SEQ ID NO.8序列如下：
Figure PCTCN2018082316-appb-000010
氨基酸序列为SEQ ID NO.6的重组蛋白相对氨基酸序列为SEQ ID NO.8的重组蛋白可溶性更好。虽然SEQ ID No.6相对于SEQ ID No.8仅在N端减少了25个氨基酸，发明人却意外地发现SEQ ID NO.6的重组蛋白通过浓缩，可以达到100mg/ml的高浓度而不会产生沉淀，容易制成冻干粉剂长期稳定保存，而SEQ ID NO.8的重组蛋白在100mg/ml的 高浓度会产生肉眼可见沉淀，不适合采用冻干粉剂型。
此外，发明人测试了SEQ ID No.6和SEQ ID No.8与TRAIL蛋白的亲和力，SEQ ID NO.6的重组蛋白与人TRAIL蛋白(sino biological公司,货号10409-HNAE,)的亲和力比SEQ ID NO.8与人TRAIL蛋白的亲和力高1～2个数量级(如图1B和1C)。
实施例2：人sDR5-Fc抗体融合蛋白治疗精囊炎。
20只C57BL/6雄性小鼠，平均分成2组(生理盐水组和9mg/kg融合蛋白组SEQ ID NO.6)，每组10只小鼠，每只小鼠尾静脉注射15mg/kg刀豆蛋白(ConA)，1小时后，每组小鼠分别尾静脉注射给予生理盐水、9mg/kg融合蛋白，给药体积均为10ml/kg。连续注射ConA、生理盐水和融合蛋白组15周，然后处死小鼠，观察小鼠的精囊，与生理盐水组比较，9mg/kg融合蛋白组小鼠精囊明显没有充血，外观正常(见图2)。因此，生理盐水组小鼠显著性高发精囊炎，给药组与生理盐水组相比精囊炎的发生率显著降低(p<0.05)，结果详见图3。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (12)

  1. 一种人sDR5-Fc重组融合蛋白，其特征在于，所述的融合蛋白的氨基端是人死亡受体5可溶片段；羧基端是人免疫球蛋白IgG1的Fc片段。
  2. 如权利要求1所述的人sDR5-Fc重组融合蛋白，其特征在于，所述的人死亡受体5可溶片段具有如SEQ ID NO.1、SEQ ID NO.2、SEQ ID No.3或SEQ ID No.4所示的氨基酸序列，或具有与SEQ ID NO.1、SEQ ID NO.2、SEQ ID No.3或SEQ ID No.4 95％以上同源性并具有相同功能的重组融合蛋白。
  3. 如权利要求1所述的人sDR5-Fc重组融合蛋白，其特征在于，所述的人免疫球蛋白IgG1的Fc片段具有如SEQ ID NO.5所示的氨基酸序列。
  4. 编码权利要求1-3任一项所述的人sDR5-Fc重组融合蛋白的核苷酸序列。
  5. 权利要求1-3任一项所述的人sDR5-Fc重组融合蛋白在制备治疗或预防生殖系统炎症的药物中的应用。
  6. 根据权利要求5所述的应用，其中生殖系统炎症选自精囊炎、前列腺炎、尿道炎。
  7. 一种肿瘤坏死因子相关凋亡诱导配体拮抗剂在制备治疗或预防生殖系统炎症的药物中的应用。
  8. 根据权利要求7所述的应用，所述肿瘤坏死因子相关凋亡诱导配体拮抗剂选自权利要求1-3任一项所述的人sDR5-Fc重组融合蛋白。
  9. 根据权利要求7所述的应用，所述生殖系统炎症选自精囊炎、前列腺炎、尿道炎。
  10. 权利要求1-3任一项所述的人sDR5-Fc重组融合蛋白在制备治疗或预防血精或精囊肿胀的药物中的应用。
  11. 一种治疗精囊炎的药物组合物，其包含权利要求1-3任一项所述的人sDR5-Fc重组融合蛋白及其药学上可接受的辅料。
  12. 根据权利要求11所述的治疗精囊炎的药物组合物，其中还包含抗生素和/或中药。
PCT/CN2018/082316 2017-06-06 2018-04-09 人sDR5-Fc重组融合蛋白及其作为制备治疗生殖系统炎症的药物中的应用 WO2018223764A1 (zh)

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