WO2015027820A1 - 人Nectin-2蛋白的应用 - Google Patents

人Nectin-2蛋白的应用 Download PDF

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WO2015027820A1
WO2015027820A1 PCT/CN2014/084265 CN2014084265W WO2015027820A1 WO 2015027820 A1 WO2015027820 A1 WO 2015027820A1 CN 2014084265 W CN2014084265 W CN 2014084265W WO 2015027820 A1 WO2015027820 A1 WO 2015027820A1
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protein
nectin
thr
obesity
val
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李自强
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北京伟德杰生物科技有限公司
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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
    • C07K14/70503—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

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  • the present invention relates to the field of biomedicine, and in particular to the use of human Nectin-2 protein. Background technique
  • Obesity is both an independent disease and a predisposing factor for diabetes, cardiovascular disease, hypertension, stroke and various cancers.
  • Obesity has a trend of explosive epidemic in the world. At present, there are more than 270 million overweight people in China and more than 9.6 million obese people.
  • Obesity-induced Many diseases include: Diabetes mellitus, nonalcoholic fatty liver, gallstones, cardiovascular disease, and dementia, and some are even the main predisposing factors for cancer.
  • Obesity refers to a certain degree of overweight and excessively thick fat layer.
  • weight-loss drugs are mainly used to control appetite and inhibit nutrient absorption.
  • Many of the weight-loss drugs on the face have obvious side effects, or the effect is unstable and easy to rebound. Therefore, it is extremely urgent to use an effective method to quickly control the spread of obesity.
  • Brown fat is rich in mitochondria and consumes energy directly in the form of heat.
  • Recent research suggests that increasing energy consumption through brown fat is likely to be a new treatment for obesity and diabetes, as opposed to traditional anti-obesity treatments such as appetite control and nutrient absorption.
  • Brown fat-specific expression of uncoupling protein 1 (UCP1) is an important protein involved in energy metabolism.
  • the brown fat is uncoupled by oxidative phosphorylation, and all the energy generated by oxidation becomes heat energy, which is a key role played by the uncoupling protein UCP1.
  • UCP1 is a long-chain fatty acid-activated protein found in the mitochondrial inner membrane.
  • UCP1 can consume a portion of the protons produced during oxidative phosphorylation, causing the production of adenosine triphosphate to decrease and energy to be released in the form of heat. Therefore, up-regulating the expression of UCP1 can theoretically treat obesity. There are no reports of brown fat secreted proteins for the treatment of diabetes, insulin resistance, obesity and obesity-related diseases.
  • Nectin-2 is a member of the Nectin family of members of the immunoglobulin superfamily. Nectin-2 is structurally similar to other members of the family, Nectin-1, Nectin-3, and Nectin-4, both of which are single-pass transmembrane proteins with an immunoglobulin superfamily V-like domain and two immunizations. G-like superfamily C-like domain. Studies have shown that Nectin-2 is widely expressed in various tissues and cell lines, acts as an adhesion molecule in endothelial cells and epithelial cells, and participates in cell-cell interactions and connections.
  • Nectm-2 can interact with or interact with different species, that is, the Nectin-2 extracellular domain on the cell membrane can interact with molecules of the same type or different types of molecules on different cell surfaces. It has been reported that there are many genes related to hyperlipidemia, such as ApoE, ApoCl and Tomm40, which are located on the same chromosome as these genes, and whose genetic distance is close to these genes and is highly expressed in brown fat. Nectin-2 has not been reported on the function of fat metabolism. It is speculated that Nectin-2 may be involved in fatty acid metabolism. Summary of the invention
  • the object of the present invention is to provide a novel use of human Nectm-2 protein in promoting energy consumption of the body from a new perspective of promoting energy metabolism, thereby achieving a new use for treating diabetes, insulin resistance, obesity and related diseases.
  • the present invention first provides a fusion protein consisting of three consecutive immunoglobulin-like domains (Ig-like domains) (Fig. 1), respectively One Ig-like V domain at the end and two Ig-like C2-type 1 and Ig-like C2-type 2 domains at the C-terminus, the three domains are connected by two Linkers (Linkerl and Linker2).
  • Each Linker is made up of 1-10 amino acids in series.
  • the amino acid sequence of Linkerl is RVIAK
  • the amino acid sequence of Linker2 is VRYP.
  • Linker can also be other flexible amino acid sequences, such as repeats of GGGGS or GGGGS.
  • the aforementioned fusion protein is the ECD region of the human Nectin-2 protein (i.e., the extracellular region, Fig. 1), which is:
  • the invention also provides a gene encoding the above fusion protein.
  • the present invention also provides vectors, engineered bacteria or transgenic cell lines containing a gene encoding the above fusion protein.
  • the present invention also provides the use of a human Nectin-2 protein (containing an ECD region of a human Nectin-2 protein) for the preparation of a medicament for treating diabetes, insulin resistance, obesity or obesity-related diseases.
  • a human Nectin-2 protein containing an ECD region of a human Nectin-2 protein
  • the present invention also provides the use of a human Nectin-2 protein (containing an ECD region of a human Nectin-2 protein) for the preparation of a medicament for improving energy metabolism or promoting fever.
  • a human Nectin-2 protein containing an ECD region of a human Nectin-2 protein
  • the present invention also provides a pharmaceutical composition for treating diabetes, obesity or obesity-related diseases, the pharmaceutical composition comprising the fusion protein or human Nectin-2 protein, or
  • the modified or engineered proteins optionally include pharmaceutically acceptable microcarriers, nanoparticles and/or adjuvants and the like.
  • the invention also provides a pharmaceutical composition for increasing energy metabolism or promoting fever, the pharmaceutical composition comprising an effective amount of the fusion protein or human Nectin-2 protein, or a modified or engineered protein thereof, optionally Pharmaceutically acceptable microcarriers, nanoparticles, and/or adjuvants and the like are included.
  • the modified protein of the present invention includes, but is not limited to, the fusion protein or the human Nectin-2 protein is subjected to acetylation, pegylation, glycosylation or binding to BSA, and the like.
  • the engineered proteins described in the present invention include, but are not limited to, the fusion protein or the fusion protein of the human Nectin-2 protein and the immunoglobulin Fc fragment or with other proteins which do not alter the activity of the Nectin-2 protein.
  • the obesity-related disease described in the present invention is diabetes or insulin resistance.
  • the diabetes is type I or type II diabetes.
  • the pharmaceutical composition of the present invention may be in a sustained release dosage form.
  • the pharmaceutically acceptable carrier or adjuvant in the present invention includes, but is not limited to, microcarriers, nanoparticles, and/or adjuvants and the like.
  • the fusion protein and/or human Nectin-2 protein provided by the present invention and the derived protein thereof or the modified protein thereof can effectively inhibit obesity or be used for the treatment of diseases related to obesity, lower blood sugar, and increase energy metabolism of the body. Level, improve the body's glucose tolerance, enhance tissue insulin sensitivity, and enhance the body's adaptability to cold stimuli by activating body heat, thus opening new possibilities for the treatment of diabetes, insulin resistance, obesity and obesity-related diseases. way.
  • FIG. 1 is a schematic view showing the structure of the ECD region (i.e., extracellular region) of the nectin-2 protein of the present invention.
  • 2 is a fusion protein and immunoglobulin Fc (control) overexpressing human nectin-2 protein and immunoglobulin Fc fragment in HEK293 cells according to Example 1 of the present invention, and SDS-PAGE gel electrophoresis results after purification of the protein .
  • Figure 3 is a graph showing the effect of Nectin-2 protein on the body weight of mice in Example 2; wherein, IgG represents an immunoglobulin Fc control protein group in obese mice, and Nectm-2 represents an injection of human Nectm-2 protein in obese mice. A fusion protein set of immunoglobulin Fc fragments.
  • Figure 4 is a graph showing the effect of Nectin-2 protein on fasting blood glucose in mice according to Example 3 of the present invention; wherein IgG represents an immunoglobulin Fc control protein group in obese mice, and Nectm-2 represents an injection of human Nectm-2 protein in obese mice. A fusion protein set with an immunoglobulin Fc fragment.
  • FIG. 5 is a diagram showing the effect of Nectin-2 protein on energy metabolism in mice according to Example 4 of the present invention; wherein IgG represents an immunoglobulin Fc control protein group in obese mice, and Nectm-2 represents an injection of human Nectm-2 protein in obese mice. A fusion protein set with an immunoglobulin Fc fragment.
  • Figure 6 is a graph showing the effect of Nectin-2 protein on the dietary dose of mice in Example 4 of the present invention; wherein IgG represents an immunoglobulin Fc control protein group in obese mice, and Nectm-2 represents an injection of human Nectm-2 protein in obese mice. A fusion protein set with an immunoglobulin Fc fragment.
  • Figure 7 is a graph showing the effect of Nectin-2 protein on the exercise volume of mice in Example 4 of the present invention; wherein IgG represents an immunoglobulin Fc control protein group in obese mice, and Nectm-2 represents an injection of human Nectm-2 protein in obese mice. A fusion protein set of immunoglobulin Fc fragments.
  • Figure 8 is a diagram showing the expression of human adipocyte-specific genes UCP1 (A) and PPARa (B) in human ecto-2 protein according to Example 5 of the present invention; wherein IgG is a human immunoglobulin Fc control protein group, and Nectin-2 is a human Nectin The fusion protein group of the -2 protein and the immunoglobulin Fc fragment; the ordinate is the relative expression amount, and the relative expression amount of IgG is defined as 1.
  • the C57BL/6J mice used in the following examples were purchased from Beijing Vital Lihua Experimental Animal Technology Co., Ltd. unless otherwise specified.
  • the reagents used are of analytical grade grade and are commercially available from regular sources. Insulin was purchased from Novo Nordisk Pharmaceutical Co., Ltd.
  • Example 1 Obtainment of human Nectin-2 protein
  • HEK293 cells are first cultured in 293 FreeStyle medium (GIBCO), cultured under 5% CO 2 , and the speed of carbon dioxide shaker (INFORS) is 125- 135 rpm.
  • the final concentration of the cells at the time of transfection expression was 1 X 10 6 cells/mL, and the transfection reagent was 293Fectin (Invitrogen).
  • 293 FreeStyle medium supplemented with 30% of the original transfection volume was added.
  • the culture supernatant was collected by centrifugation at 2500 rpm, NaCl was added thereto to a final concentration of 3 M, then Tris was added to a final concentration of 50 mM, and the pH was adjusted to 8.5 after fully dissolved, and then centrifuged at 6000 rpm for 10 min. Finally, the supernatant was filtered through a 0.22 ⁇ m filter, and then purified on an AKTA Purifier (GE) protein purification system with 5 mL of Protein A (ToyoScreen AF-rProtein A-650F, TOSOH).
  • GE AKTA Purifier
  • mice Six-week-old C57BL/6J male mice were fed a 60 Kcal% high fat diet. (esearch diet, D12451), weekly weight recorded. After induction for 8 weeks, the high-fat diet-induced obesity group was randomly divided into a control group (IgG, 10) and an experimental group (Nectm-2, 10); then the experimental group was treated with Nectin-2 (Example Treatment in 1), subcutaneously injected at a dose of 5 mg/kg body weight/week; and the control group was treated with the same dose of IgG (immunoglobulin Fc as a control protein), administered by subcutaneous injection, administered weekly. The average daily intake of the animals in each experimental group was measured 2 weeks before the administration, and the body weight was recorded every week of administration. After 14 weeks of continuous administration, the mice were sacrificed by cervical dislocation, and the liver, epididymal fat and subcutaneous fat were taken out and weighed.
  • IgG immunoglobulin Fc as a control protein
  • Nectm-2 treatment can effectively inhibit obesity, specifically 50.85 ⁇ 0.5g in the control group and 46.85 ⁇ 1.lg (p ⁇ 0.05) in the experimental group.
  • mice of the ninth week of administration in Example 2 were further subjected to the following tests: The mice after 9 weeks of administration in Example 2 were fasted overnight to measure fasting blood glucose levels.
  • Nectin-2 protein increases the body's oxygen consumption (p ⁇ 0.05), indicating that Nectin-2 protein can improve the body's overall energy metabolism.
  • Nectin-2 had no significant effect on feed intake ( Figure 6) and exercise ( Figure 7).
  • Nectin-2 protein can increase the energy metabolism level of the body.
  • Example 5 Application of human Nectin-2 protein in up-regulating the expression of UCP1 and PPARa in sputum fat cell-specific genes
  • C3H10T1/2 cells were cultured in brown adipocyte differentiation medium (DMEM + 10% FBS + 20 nM insulin + 1 ⁇ ⁇ ⁇ 3 ) for 3 days.
  • the base (DMEM + 10% FBS + 0.125 mM indomethacin + 2 g / ml dexamethasone + 0.125 mM IBMX) was induced for 2 days.
  • brown fat cells were again cultured in the above differentiation medium, and the cultured cells were harvested on the 10th day.
  • Human Nectin protein and human IgG control protein were added from day 1 of cell culture until the last day.
  • Figure 8 shows that different concentrations of nectin (100 ng/ml, 1000 ng/ml) significantly up-regulated the expression of UCP1 and PPARa genes (* indicates p ⁇ 0.05) relative to control IgG treatment.
  • the present invention provides a novel use of the human nectin-2 protein.
  • the Nectin-2 protein and its derived protein or modified protein thereof can effectively inhibit obesity and diabetes, or be used for the treatment of diseases associated with obesity, including but not limited to hypertension, hyperlipidemia and the like.
  • Nectm-2 can not only lower blood sugar, but also increase the body's energy metabolism, increase the body's glucose metabolism, enhance tissue insulin sensitivity, and enhance the body's adaptability to cold stimuli by activating body fat metabolism, for diabetes, insulin.
  • the treatment of resistance, obesity and related diseases has opened up new avenues.
  • Gao X et al. Prevalence and trend of hepatitis C virus infection among blood donors in Chinese mainland: a systematic review and meta-analysis. BMC Infect Dis 2011; 11 : 88.
  • Nectin-2 reveals determinants of homophilic and heterophilic interactions that control cell -cell adhesion [J]. Proc Natl Acad Sci USA , 2012 , 109 ( 37 ): 14836 -14840.
  • the human poliovirus receptor related 2 protein is a new hematopoietic/endothelial homophilic adhesion molecule [J]. Blood , 1998 , 92 ( 12 ): 4602-4611.
  • Nectin PRR an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin, a PDZ domain-containing protein [J]. J Cell Biol , 1999, 145 (3): 539-549.
  • Ala Arg lie Ser Trp Leu Ser Ser Leu Asp Trp Glu Ala Lys Glu Thr

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Abstract

本发明提供了一种人Nectin-2蛋白的用途。Nectin-2蛋白、其衍生蛋白或其经过修饰的蛋白可以有效地抑制肥胖和糖尿病，也可用于与肥胖相关疾病的治疗，包括高血压、高脂血症等。Nectin-2蛋白不仅可以降低血糖，还通过增加机体的能量代谢水平，提高机体的葡萄糖代谢，增强组织的胰岛素灵敏性，并通过激活机体脂肪酸代谢来增强机体对寒冷刺激的适应性。

Description

人 Nectin-2蛋白的应用 技术领域
本发明涉及生物医药领域，具体地说， 涉及人 Nectin-2蛋白的应用。 背景技术
肥胖的发生是机体能量代谢失调的结果，即当机体能量摄入超过 能量消耗时， 余下的能量几乎完全转化为脂肪， 储藏于全身白色脂肪 中。肥胖既是一种独立的疾病， 又是 Π型糖尿病、心血管病、高血压、 中风和多种癌症的诱发因素。肥胖在世界范围内呈现出爆发性流行的 趋势， 目前我国超重人群 2.7亿以上， 肥胖者 9.6千万以上。 肥胖诱发 很多疾病包括： Π型糖尿病、 非酒精脂肪肝、 胆结石、 心血管病和老 年痴呆症， 有些甚至是癌症的主要诱发因素。 肥胖是指一定程度的明 显超重与脂肪层过厚， 由于食物摄入过多或机体代谢的改变而导致体 内脂肪积聚过多造成体重过度增长并引起人体病理、生理改变的一种 表现。 与正常体质者相比， 肥胖本身并没有太多明显特征， 但是肥胖 会带来很多严重的并发症， 包括高血压、 脑卒中、 糖尿病、 心血管疾 病、 脂代谢异常、 肿瘤和其他代谢性疾病的发生率明显增加， 而且肥 胖与 II型糖尿病并存， 表现为胰岛素抵抗、 糖耐量减低。 肥胖已经成 为 21世纪影响人健康的主要危险因素之一，严重危害人的身体健康及 生活质量。
然而， 传统的减肥药物主要用来控制食欲和抑制营养吸收， 而巿 面上的很多减肥药物大多副作用明显， 或者效果不稳定， 容易反弹。 因此利用行之有效的方法迅速控制肥胖的蔓延已迫在眉睫。
人体内除了白色脂肪之外， 还存在棕色脂肪。 棕色脂肪中含有丰 富的线粒体， 以直接产生热量的方式来消耗能量。 研究结果表明， 棕 色脂肪的活性与身体质量指数呈显著性负相关，而且有活性的棕色脂 肪可以消耗大量的脂肪和血糖，进一步提示棕色脂肪在能量代谢过程 中起着非常重要的作用。但迄今尚没有任何直接的证据表明棕色脂肪 参与机体的能量代谢。 最新研究结果提示， 相对于控制食欲和抑制营 养吸收等传统的抗肥胖疗法，通过棕色脂肪增加机体的能量消耗很可 能成为治疗肥胖和糖尿病的新方法。 棕色脂肪特异表达解偶联蛋白 1 ( UCP1 ), 是参与能量代谢的重要蛋白质。 棕色脂肪通过氧化磷酸化 解偶联， 氧化产生的能量全部变为热能， 是由解偶联蛋白 UCP1发挥 的关键作用。 UCP1是长链脂肪酸激活的蛋白， 存在于线粒体内膜。
UCP1可以消耗一部分氧化磷酸化过程中产生的质子， 使三磷酸腺苷 的制造量下降， 能量以热的形式释放， 因此上调 UCP1的表达从理论 上可以治疗肥胖症。 目前尚没有将棕色脂肪分泌的蛋白用于治疗糖尿 病、 胰岛素抵抗、 肥胖症及肥胖症相关疾病的相关报道。
Nectin-2是免疫球蛋白超家族成员 Nectin家族中的一员。 Nectin-2 与该家族的其它成员 Nectin-1、 Nectin-3和 Nectin-4结构相似， 均属于 单次跨膜蛋白， 其膜外具有 1个免疫球蛋白超家族 V样结构域和 2个免 疫球蛋白超家族 C样结构域。研究表明， Nectin-2在各种组织和细胞系 中广泛表达， 在内皮细胞和上皮细胞起黏附分子的作用， 参与细胞间 的相互作用和连接。 Nectm-2作为细胞黏附分子， 能够亲同种或与异 种相互作用， 即细胞膜上的 Nectin-2胞外域可与不同细胞表面上的同 类分子或不同类分子相互作用。 已有报道中， 对于与高血脂有关的 ApoE、 ApoCl和 Tomm40等基因研究较多， 而与这些基因位于同一个 染色体上， 且遗传距离与这些基因较近的、 并在棕色脂肪里高表达的 Nectin-2对脂肪代谢功能的研究尚未见报道。推测 Nectin-2有可能参与 脂肪酸代谢。 发明内容
本发明的目的是从促进能量代谢的全新角度出发， 提供人 Nectm-2蛋白在促进机体能量消耗中的功能， 从而达到治疗糖尿病、 胰岛素抵抗、 肥胖以及相关疾病的全新用途。 为了实现本发明目的， 本发明首先提供一种融合蛋白， 所述融合 蛋白由三个连续的免疫球蛋白样结构域 （ Immunologlobin-like domain, Ig-like domain )组成 （图 1 )， 分别是 N端的一个 Ig-like V结 构域和 C端的两个 Ig-like C2-type 1和 Ig-like C2-type 2结构域， 三个结 构域之间由两段 Linker ( Linkerl和 Linker2 )连接。 其中， N端的 Ig-like V结构域、 中间的 Ig-like C2-type 1结构域以及 C端的 Ig-like C2-type 2 结构域的氨基酸序列分别如 SEQ ID No.1-3所示。 每段 Linker由 1-10个 氨基酸串联而成。 例如， Linkerl的氨基酸序列为 RVIAK， Linker2的 氨基酸序列为 VRYP。 Linker也可为其他柔性氨基酸序列， 例如 GGGGS或 GGGGS的重复片段等。
前述的融合蛋白为人 Nectin-2蛋白的 ECD区 （即胞外区， 图 1 )， 其为：
1 ) 由 SEQ ID No.4所示的氨基酸序列构成的蛋白； 或
2 ) SEQ ID No.4所示氨基酸序列经取代、 缺失和 /或添加一个或 几个氨基酸且同等功能的由 1 )衍生的蛋白； 或
3 )与 SEQ ID No.4所示氨基酸序列的同源性在 90%以上的氨基酸 序列构成的蛋白。
本发明还提供编码上述融合蛋白的基因。
本发明还提供含有编码上述融合蛋白的基因的载体、工程菌或转 基因细胞系。
本发明还提供人 Nectin-2蛋白（包含人 Nectin-2蛋白的 ECD区 )在 制备用于治疗糖尿病、胰岛素抵抗、 肥胖或肥胖相关疾病的药物中的 应用。
本发明还提供人 Nectin-2蛋白（包含人 Nectin-2蛋白的 ECD区 )在 制备用于提高能量代谢或促进发热的药物中的应用。
本发明还提供一种用于治疗糖尿病、肥胖或肥胖相关疾病的药物 组合物， 所述药物组合物包括所述融合蛋白或人 Nectin-2蛋白， 或它 们经过修饰或改造的蛋白， 任选包括药学上可接受的微载体、 纳米颗 粒和 /或辅料等。
本发明还提供一种用于提高能量代谢或促进发热的药物组合物， 所述药物组合物包括有效量的所述融合蛋白或人 Nectin-2蛋白， 或它 们经过修饰或改造的蛋白， 任选包括药学上可接受的微载体、 纳米颗 粒和 /或辅料等。
本发明中所述经过修饰的蛋白包括但不限于，所述融合蛋白或人 Nectin-2蛋白经过乙酰化、 聚乙二醇化、 糖基化或与 BSA结合等。
本发明中所述经过改造的蛋白包括但不限于，所述融合蛋白或人 Nectin-2蛋白与免疫球蛋白 Fc片段或与其他蛋白融合构成的不改变 Nectin-2蛋白活性的融合蛋白。
本发明中所述肥胖相关疾病为糖尿病或胰岛素抵抗。 优选地， 所 述糖尿病为 I型或 II型糖尿病。
本发明中所述药物组合物可为缓释剂型。
本发明中所述药学上可接受的载体或辅料包括但不限于， 微载 体、 纳米颗粒和 /或辅料等。
本发明提供的融合蛋白和 /或人 Nectin-2蛋白， 以及它们的衍生蛋 白或它们经过修饰的蛋白可以有效地抑制肥胖或用于与肥胖相关疾 病的治疗， 降低血糖， 通过增加机体的能量代谢水平， 提高机体的葡 萄糖耐受量， 增强组织的胰岛素灵敏性， 并通过激活机体产热来增强 机体对寒冷刺激的适应性， 从而为糖尿病、 胰岛素抵抗、 肥胖以及肥 胖相关疾病的治疗开辟了新途径。 附图说明
图 1为本发明 Nectin-2蛋白 ECD区 （即胞外区） 的结构示意图。 图 2为本发明实施例 1中在 HEK293细胞中分别过表达人 Nectin-2 蛋白与免疫球蛋白 Fc片段的融合蛋白、 免疫球蛋白 Fc (对照）， 蛋白 纯化后的 SDS-PAGE凝胶电泳结果。 图 3为本发明实施例 2中 Nectin-2蛋白对小鼠体重的影响； 其中， IgG代表肥胖小鼠注射免疫球蛋白 Fc对照蛋白组， Nectm-2代表肥胖小 鼠注射人 Nectm-2蛋白与免疫球蛋白 Fc片段的融合蛋白组。
图 4为本发明实施例 3中 Nectin-2蛋白对小鼠空腹血糖的影响； 其 中， IgG代表肥胖小鼠注射免疫球蛋白 Fc对照蛋白组， Nectm-2代表肥 胖小鼠注射人 Nectm-2蛋白与免疫球蛋白 Fc片段的融合蛋白组。
图 5为本发明实施例 4中 Nectin-2蛋白对小鼠能量代谢的影响； 其 中， IgG代表肥胖小鼠注射免疫球蛋白 Fc对照蛋白组， Nectm-2代表肥 胖小鼠注射人 Nectm-2蛋白与免疫球蛋白 Fc片段的融合蛋白组。
图 6为本发明实施例 4中 Nectin-2蛋白对小鼠饮食量的影响；其中， IgG代表肥胖小鼠注射免疫球蛋白 Fc对照蛋白组， Nectm-2代表肥胖小 鼠注射人 Nectm-2蛋白与免疫球蛋白 Fc片段的融合蛋白组。
图 7为本发明实施例 4中 Nectin-2蛋白对小鼠运动量的影响；其中， IgG代表肥胖小鼠注射免疫球蛋白 Fc对照蛋白组， Nectm-2代表肥胖小 鼠注射人 Nectm-2蛋白与免疫球蛋白 Fc片段的融合蛋白组。
图 8为本发明实施例 5中人 Nectm-2蛋白上调棕色脂肪细胞特异性 基因 UCPl ( A )、 PPARa ( B ) 的表达； 其中， IgG为人免疫球蛋白 Fc 对照蛋白组， Nectin-2为人 Nectin-2蛋白与免疫球蛋白 Fc片段的融合蛋 白组； 纵坐标为相对表达量， IgG的相对表达量定义为 1。 具体实施方式
以下实施例用于说明本发明， 但不用来限制本发明的范围。 若未 特别指明， 实施例均按照常规实验条件， 如 Sambrook等分子克隆实验 手册 ( Sambrook J & Russell DW, Molecular cloning: a laboratory manual 2001 )， 或按照制造厂商说明书建议的条件。
除非特别指明，以下实施例中使用的 C57BL/6J小鼠均购自北京维 通利华实验动物技术有限公司。 所用试剂均为分析纯级别的试剂， 且 可从正规渠道商购获得。 胰岛素购自诺和诺德制药有限公司。 实施例 1 人 Nectin-2蛋白的获得
根据 GenBank中公布的人 Nectin-2mRNA序歹 'J ( Accession No.： C 456818.1 )， 将第 1-1035位的核苷酸序列与免疫球蛋白 Fc片段的基 因序列进行拼接， 拼接后的基因序列如 SEQ ID No .5所示。
通过人工合成上述序列成后， 克隆到哺乳动物表达载体 pcDNA3.1中， 构建好的表达载体瞬时转入 HEK293细胞中， 实现融合 蛋白的表达， 纯化目的蛋白。 蛋白纯化后的 SDS-PAGE凝胶电泳结果 如图 2所示。
细胞转染、细胞培养及蛋白纯化的具体过程如下：首先将 HEK293 细胞悬浮培养于 293 FreeStyle培养基中 （GIBCO ), 在 5%C02条件下 培养， 二氧化碳摇床（INFORS ) 的转速为 125-135 rpm。 转染表达时 细胞的终浓度为 1 X 106个细胞 /mL，转染试剂为 293Fectin (Invitrogen)。 转染后 48h和 96h，补加原始转染总体积 30%的 293FreeStyle培养基。 转 染后 6-7天， 2500rpm离心收集培养液上清， 向其中加入 NaCl至终浓度 为 3M，然后加入 Tris使其终浓度为 50mM，充分溶解后调节 pH值至 8.5， 然后 6000rpm离心 10min， 最后将上清液用 0.22μπι滤膜过滤， 然后用 5mL的 Protein A (ToyoScreen AF-rProtein A-650F, TOSOH)在 AKTA Purifier (GE)蛋白纯化系统上进行纯化。先用平衡液（ 50mM Tris/HCl， 3.0M NaCl， pH 8.5 )平衡柱子 5个柱体积后， 上样并继续用平衡液洗 柱子直至紫外吸收到基线， 然后用洗脱液（25mM柠檬酸， 4.5mM柠 檬酸钠， pH 3.0 ) 洗脱， 在紫外吸收大于 5mAU时收集流出液。 然后 加入 5%v/v中和液（ 2M Tris-HCl, pH 9.0 )进行中和， 对纯化的目的 蛋白进行 SDS-PAGE凝胶电泳。
实施例 2 人 Nectin-2蛋白在抑制肥胖中的应用
为了验证实施例 1中制备的 Nectin-2蛋白的抗肥胖作用，在本实施 例中构建如下高脂饲料诱导肥胖组小鼠模型：
取 6周龄的 C57BL/6J雄性小鼠， 饲喂 60 Kcal%的高脂饲料 ( esearch diet, D12451 ), 每周记录体重。 诱导 8周待其肥胖后将高 脂饮食诱导肥胖组随机分为对照组（IgG， 10只）和实验组组（Nectm-2， 10只）； 然后将实验组， 用 Nectin-2 (实施例 1中制备）处理， 以 5mg/kg 体重 /周的剂量皮下注射； 并将对照组用同样剂量的 IgG (免疫球蛋白 Fc作为对照蛋白）处理， 以皮下注射的方式给药， 每周给药， 给药前 2周检测各实验组动物每天平均摄食量， 并在给药的每周记录体重。 连续给药 14周后， 断颈处死小鼠， 取出肝脏、 附睾脂肪和皮下脂肪， 称重。
从图 3可以看出， Nectm-2处理可以有效地抑制肥胖， 具体为对照 组 50.85±0.5g， 实验组 46.85士 l .lg ( p<0.05 )。
实施例 3 人 Nectin-2蛋白在降低空腹血糖中的应用
已有研究结果表明， 肥胖往往伴随着严重的胰岛素抵抗。 在本实 施例中， 进一步对实施例 2中给药第 9周的小鼠， 进行了以下试验： 将实施例 2中给药 9周后的小鼠禁食过夜测空腹血糖水平。
结果如图 4所示， 高脂饮食诱导的对照组 （IgG ) 的空腹血糖 ( 5.84mmol ) 显著高于 Nectin-2处理的实验组空腹血糖 （ 5.2mmol， p<0.05 )。
实施例 4 人 Nectin-2蛋白在促进能量消耗中的应用
取实施例 2中给药第 10周后的小鼠， 用 T SE公司的 LabMaster检测 其耗氧量。从图 5可以看出， Nectin-2蛋白增加机体的耗氧量（p<0.05 )， 说明 Nectin-2蛋白能够提高机体的整体能量代谢。但是 Nectin-2对采食 量 （图 6 )和运动量（图 7 ) 没有显著影响。
由此可见， Nectin-2蛋白能够增加机体的能量代谢水平。
实施例 5 人 Nectin-2蛋白在上调椋色脂肪细胞特异性基因 UCP1、 PPARa表达中的应用
利用棕色脂肪细胞分化培养基（DMEM+10% FBS+20nM胰岛素 +1ηΜ Τ3 )培养 C3H10T1/2细胞 3天， 等细胞长满之后， 利用诱导培养 基 （ DMEM+10% FBS+0.125mM indomethacin+2 g/ml dexamethasone+0.125mM IBMX )诱导 2天。 第 6天重新将棕色脂肪细 胞置于上述分化培养基中培养， 第 10天收获培养的细胞。 人 Nectin蛋 白和人 IgG对照蛋白从细胞培养第 1天开始添加， 直至最后 1天。 从收 获的细胞样品中提取 RNA反转录成 cDNA， 然后利用荧光定量 PCR检 测 Nectin蛋白处理对棕色脂肪特异性基因 UCP1、 PPARa的影响。 图 8 结果显示， 相对于对照的 IgG处理， 不同浓度的 Nectin ( 100ng/ml、 lOOOng/ml )可显著上调 UCP1、 PPARa基因的表达量（*表示 p<0.05 )。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详 尽的描述， 但在本发明基础上， 可以对之作一些修改或改进， 这对本 领域技术人员而言是显而易见的。 因此， 在不偏离本发明精神的基础 上所做的这些修改或改进， 均属于本发明要求保护的范围。 工业实用性 本发明提供人 Nectin-2蛋白的新用途。 Nectin-2蛋白及其衍生蛋 白或其经过修饰的蛋白可以有效地抑制肥胖和糖尿病，或用于与肥胖 相关疾病的治疗， 包括但不限于高血压、 高脂血症等。 Nectm-2不仅 可以降低血糖， 还通过增加机体的能量代谢水平， 提高机体的葡萄糖 代谢， 增强组织的胰岛素灵敏性， 并通过激活机体脂肪酸代谢来增强 机体对寒冷刺激的适应性， 为糖尿病、 胰岛素抵抗、 肥胖以及相关疾 病的治疗开辟了新途径。
参考文献
1、 Gao X, et al. Prevalence and trend of hepatitis C virus infection among blood donors in Chinese mainland: a systematic review and meta-analysis. BMC Infect Dis 2011; 11 :88.
2、 Samanta D , Ramagopal UA , Rubinstein R , et al. Structure of Nectin-2 reveals determinants of homophilic and heterophilic interactions that control cell -cell adhesion [J]. Proc Natl Acad Sci U S A , 2012 , 109 ( 37 )： 14836-14840.
3、 Lopez M ,Aoubala M Jordier F ,et al. The human poliovirus receptor related 2 protein is a new hematopoietic/endothelial homophilic adhesion molecule [J]. Blood , 1998 , 92 ( 12 )： 4602-4611.
4、 Takahashi K , Nakanishi H , Miyahara M , et al. Nectin PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin , a PDZ domain-containing protein [J]. J Cell Biol , 1999 , 145 ( 3 )： 539-549.
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Claims

权 利 要 求 书
1. 一种融合蛋白， 其特征在于， 由三个连续的免疫球蛋白样结 构域组成， 分别是 N端的一个 Ig-like V结构域和 C端的两个 Ig-like C2-type 1和 Ig-like C2-type 2结构域， 三个结构域之间由两段 Linker连 接， 每段 Linker由 1-10个氨基酸串联而成；
其中， N端的 Ig-like V结构域、 C端的两个 Ig-like C2-type 1和 Ig-like C2-type 2结构域的氨基酸序列分别如 SEQ ID No.1-3所示。
2. 根据权利要求 1所述的融合蛋白， 其为：
1 ) 由 SEQ ID No.4所示的氨基酸序列构成的蛋白； 或
2 ) SEQ ID No.4所示氨基酸序列经取代、 缺失和 /或添加一个或 几个氨基酸且具有同等功能的由 1 ) 的衍生蛋白； 或
3 )与 SEQ ID No.4所示氨基酸序列的同源性在 90%以上的氨基酸 序列构成的蛋白。
3. 编码权利要求 1或 2所述融合蛋白的基因。
4. 含有权利要求 3所述基因的工程菌或转基因细胞系。
5. 人 Nectin-2蛋白或编码该蛋白的基因在制备用于治疗糖尿病、 胰岛素抵抗、 肥胖以及相关疾病的药物中的应用。
6.人 Nectin-2蛋白或编码该蛋白的基因在制备用于提高能量代谢 或促进发热的药物中的应用。
7. 一种用于治疗糖尿病、 胰岛素抵抗、 肥胖以及相关疾病的药 物组合物， 其特征在于， 所述药物组合物包括权利要求 1或 2所述的融 合蛋白或人 Nectin-2蛋白， 或它们经过修饰或改造后的蛋白， 任选包 括药学上可接受的微载体、 纳米颗粒和 /或辅料。
8. 根据权利要求 7所述的药物组合物， 其特征在于， 所述经过修 饰的蛋白是指蛋白经过包括但不限于乙酰化、 聚乙二醇化、 糖基化或 与 BSA结合。
9. 根据权利要求 7所述的药物组合物， 其特征在于， 所述经过改 造后的蛋白是指， 包括但不限于， 所述蛋白与免疫球蛋白 Fc片段或与 其他蛋白融合构成的融合蛋白。
10. 根据权利要求 7-9任一项所述的药物组合物， 其特征在于， 其为缓释剂型。
PCT/CN2014/084265 2013-08-29 2014-08-13 人Nectin-2蛋白的应用 WO2015027820A1 (zh)

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