WO2019161684A1 - Ccr4的n端重组蛋白及其用途 - Google Patents

Ccr4的n端重组蛋白及其用途 Download PDF

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WO2019161684A1
WO2019161684A1 PCT/CN2018/115442 CN2018115442W WO2019161684A1 WO 2019161684 A1 WO2019161684 A1 WO 2019161684A1 CN 2018115442 W CN2018115442 W CN 2018115442W WO 2019161684 A1 WO2019161684 A1 WO 2019161684A1
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recombinant protein
ccr4
cancer
derivative
disease
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杨鹏远
高雅楠
游茂军
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中国科学院生物物理研究所
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Priority to EP18907350.5A priority patent/EP3663313B1/en
Priority to JP2020534529A priority patent/JP7043605B2/ja
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Definitions

  • the present invention relates to the field of biomedical technology, and in particular to N-terminal recombinant protein of CCR4 and derivative compounds thereof, derivatives or compositions of recombinant protein antibodies or the like, alone or in combination with other compounds or compositions having related uses for treatment, prevention, inhibition or Use in improving cancer and diseases or conditions associated with CCR4 signaling.
  • CCR4 CC chemokine receptor 4
  • CKR4 CKR4, CMKBR4, ChemR13, K5-5, etc.
  • CKR4 CKR4, CMKBR4, ChemR13, K5-5, etc.
  • CKR4 CKR4, CMKBR4, ChemR13, K5-5, etc.
  • -p21.3 region a 7-transmembrane G protein-coupled receptor (GPCR), expressed mainly in various lymphocytes and tissues, high expression of CCR4 and various inflammatory diseases, autoimmune diseases, hematological tumors, and Invasion, metastasis and prognosis of malignant solid tumors.
  • GPCR 7-transmembrane G protein-coupled receptor
  • CCR4 There are two known high-affinity ligands for CCR4, namely Thymus and activation regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22).
  • TARC/CCL17 Thymus and activation regulated chemokine
  • MDC/CCL22 macrophage-derived chemokine
  • CCR4 exerts immune effects by different types of T cells, such as Th2 cells in asthma and respiratory allergic diseases, by chemotaxis of CCR4 expressed on the surface of Th2 cells, Th17 cells and regulatory T cells (Treg) and its ligand CCL22/CCL17.
  • Th17 cells induce inflammatory enteritis such as Crohn's disease, ulcerative colitis and autoimmune diseases
  • CCR4 on the surface of Treg cells chemots Treg cells by binding to its ligand CCL22/CCL17, causing Immune escape, resulting in adverse clinical consequences. Therefore, compounds targeting CCR4 are expected to become new strategies for the treatment of related diseases.
  • CCL22/CCL17-CCR4 signaling pathway Inhibiting the CCL22/CCL17-CCR4 signaling pathway, alone or in combination with other compounds or compositions of related use for the treatment, prevention, inhibition or amelioration of cancer and diseases or conditions associated with CCR4 signaling, such as inflammatory diseases including allergic Disease, inflammatory bowel disease, inflammatory skin disease, asthma, autoimmune diseases, graft rejection, blood system tumors such as leukemia, lymphoma and other blood-borne cancers such as cutaneous T-cell lymphoma, acute lymphocytic leukemia, etc.
  • inflammatory diseases including allergic Disease, inflammatory bowel disease, inflammatory skin disease, asthma, autoimmune diseases, graft rejection
  • blood system tumors such as leukemia, lymphoma and other blood-borne cancers such as cutaneous T-cell lymphoma, acute lymphocytic leukemia, etc.
  • cancer including solid tumors and metastatic diseases such as gastric cancer, liver cancer, kidney cancer, colon cancer, pancreatic cancer, lung cancer, bladder cancer, breast cancer, ovarian cancer, cervical cancer, melanoma, thyroid cancer, prostate cancer, nervous system tumors, etc. It has become a new strategy for immunological targeted therapy of related diseases and has a good clinical application prospect.
  • CCR4 monoclonal antibody for treating tumors is mogamulizumab, which has good therapeutic effects in the treatment or research of cutaneous T-cell lymphoma (CTCL), mycosis fungoides, Sézary syndrome, non-Hodgkin's lymphoma and other diseases.
  • CCR4 receptor antagonists have good therapeutic effects in the study of asthma, rhinitis, dermatitis, thrombotic diseases, autoimmune diseases and other diseases.
  • the present invention found that the N-terminal protein sequence of CCR4 can be efficiently analyzed by a two-step model, and the N-terminal protein structure of CCR4 can be efficiently synthesized, and the N-terminal recombinant protein of CCR4 and its derivative triad (N-CCR4) 3 can be efficiently synthesized.
  • the N-terminal recombinant protein of CCR4 and the derived tripin can bind to the chemokine CCL22 and significantly inhibit the chemotaxis of Treg.
  • the N-terminal recombinant protein of the chemokine receptor CCR4 provided by the present invention, a derivative compound or a derivative thereof, or a derivative or a recombinant protein antibody or the like, which is used alone or in combination, has other related compounds or compositions for use in the treatment, prevention, and inhibition. Or have a good clinical application prospect in improving cancer and diseases or conditions associated with CCR4 signaling.
  • the present invention provides a N-terminal recombinant protein of CCR4, a derivative compound or derivative thereof, a recombinant protein antibody or the like, or a composition or composition thereof, for use in the treatment, prevention, inhibition or amelioration of cancer, and other compounds or compositions having related uses, alone or in combination Use of a disease or condition associated with CCR4 signaling.
  • the present invention provides a N-terminal recombinant protein of CCR4, a derivative thereof or a derivative thereof, a recombinant protein antibody or the like, or a composition thereof, including but not limited to C-terminal N-terminal recombinant protein (N-CCR4), and a derivative compound thereof a derivative compound such as a ligated or triple-linked N-terminal recombinant protein (N-CCR4) n , a recombinant protein antibody (N-CCR4) n- Fc or other structural analog.
  • N-CCR4 protein sequence described therein is:
  • N-terminal recombinant protein of CCR4 of the present invention and derivatives or recombinant derivatives thereof, or derivatives or compositions thereof, can be used alone or in combination with other compounds or compositions having related uses.
  • N-terminal recombinant protein of CCR4 of the present invention and derivative compounds, recombinant protein antibodies and the like derivatives or compositions thereof can be administered orally, parenterally, such as intramuscularly, intravenously, subcutaneously or by implantation, inhalation, Administration by topical administration or the like may be formulated, either singly or together, in a suitable dosage unit formulation containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles suitable for the respective routes of administration.
  • therapeutic agents which can be administered simultaneously or sequentially or separately with the compounds or compositions of the present invention include, but are not limited to, conventional inflammatory disease therapeutic agents such as anti-inflammatory drugs, hormonal drugs, immunosuppressive agents, antihistamines, and the like, conventional Cancer treatment drugs such as radiotherapy and chemotherapy drugs, conventional first-line drugs (such as cisplatin, vincristine, sorafenib, etc.), immunological checkpoint inhibitors (PD-1/PD-L1, CTLA-4, 4-1BB, CD40/CD40L, etc. and other tumor immunotargeting drugs (VEGF/VEGFR, EGF, etc.).
  • conventional inflammatory disease therapeutic agents such as anti-inflammatory drugs, hormonal drugs, immunosuppressive agents, antihistamines, and the like
  • conventional Cancer treatment drugs such as radiotherapy and chemotherapy drugs
  • conventional first-line drugs such as cisplatin, vincristine, sorafenib, etc.
  • immunological checkpoint inhibitors PD-1/PD-L1, CTLA-4, 4-1
  • the compounds of the invention are useful for treating, preventing, inhibiting or ameliorating cancer and diseases or conditions associated with CCR4 signaling.
  • the treatment includes, but is not limited to, 1) an allergic disease, such as a systemic allergic or hypersensitivity reaction; 2) inflammatory bowel disease, such as Crohn's disease, ulcerative colitis, ileitis, and Enteritis, etc.; 3) psoriasis and inflammatory skin diseases such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, dermatomyositis and urticaria; 5) vasculitis; 6) spondyloarthropathy 7) scleroderma; 8) asthma and respiratory allergic diseases, such as allergic asthma, exercise-induced asthma, allergic rhinitis, hypersensitivity lung disease, etc.; 9) autoimmune diseases such as arthritis, multiple sclerosis Disease, systemic lupus erythematosus, diabetes, nephritis, etc.; 10) graft rejection, such as xenograft rejection and graft versus host disease; 11) leukemia, lymph
  • the compounds of the invention may be administered simultaneously or sequentially or separately with other therapeutic agents that may be used in combination for the treatment, prevention, inhibition or amelioration of cancer and diseases or conditions associated with CCR4 signaling.
  • the invention provides a pharmaceutical composition that can be formulated as an injection, tablet or capsule.
  • the present invention provides the following:
  • a pharmaceutical composition comprising the recombinant protein according to any one of items 1 to 5 or a derivative thereof.
  • the disease or condition associated with CCR4 signaling comprises 1) an allergic disease, such as a systemic allergic or hypersensitivity reaction; 2) inflammatory bowel disease, such as Crohn's disease, ulcer Colitis, ileitis and enteritis; 3) psoriasis and inflammatory skin diseases such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, dermatomyositis and urticaria; 5) vasculitis; 6) spondyloarthropathy; 7) scleroderma; 8) asthma and respiratory allergic diseases such as allergic asthma, exercise-induced asthma, allergic rhinitis, hypersensitivity lung disease; 9) autoimmune diseases such as arthritis , multiple sclerosis, systemic lupus erythematosus, diabetes, nephritis; 10) graft rejection, like xenograft rejection and graft versus host disease; 11) leukemia, lymphoma and other blood
  • the N-terminal protein of CCR4, N-CCR4, is a hydrophobicity alignment of CCR4 and CXCR4 protein sequences (http://www.bioinfo.mpg.de/AlignMe/AlignMe_MSA.html) and the Gibbs freedom to calculate the CCR4 protein sequence. It can be predicted by ⁇ G ( ⁇ G predictor sever v1.0, http://dgpred.cbr.su.se) (Fig. 1A, B).
  • the N-CCR4 protein sequence is:
  • the (N-CCR4) 3 DNA codon-optimized sequence was cloned into the pET22b(+) vector (Novagen, Cat. No. 69744-3) by NdeI and BamHI restriction sites, and transformed into Transetta competent cells (full version). Gold, item number CD801-01).
  • the overnight shaken solution was inoculated to a new LB medium at a ratio of 1:100.
  • a final concentration of 1 mM IPTG (amresco, cat: 0487-1G) was added, and the expression was induced at 25 rpm, 220 rpm/min. hour.
  • the cells were disrupted by a high-pressure bacteria-breaking apparatus (JN-02C low-temperature ultra-high pressure continuous flow cell disrupter), and a nickel column (BBI, Cat. No. C600793) was combined and washed with a buffer containing 100 mM imidazole Tris-HCl (pH 8.0). Depurified and dialyzed twice in 2 L PBS.
  • JN-02C low-temperature ultra-high pressure continuous flow cell disrupter JN-02C low-temperature ultra-high pressure continuous flow cell disrupter
  • BBI nickel column
  • the murine kappa III signal peptide-(N-CCR4) 3- Fc DNA sequence was cloned into the pCEP4 vector by KpnI and BamHI, wherein the murine kappa III signal peptide is a secretion signal peptide and Fc is a human IgG1 Fc.
  • the constructed plasmid was transfected into 293FT cells with PEI (Shanghai Qishen Material Reagent Co., Ltd., Cat. No. 24765-2), and the third day of transfection was purified by protein A prepackaged gravity column (BBI, Cat. No. C600951). The eluted Elution sample was run on a 10% SDS-PAGE gel (Fig. 1D).
  • the GST-pulldown assay can verify whether two proteins interact, so it was verified by GST-pulldown experiments that (N-CCR4) 3 interacts with CCL22.
  • the CCL22 DNA sequence was codon optimized and CCL22 and GST were cloned into the pET22b vector by restriction enzyme sites NdeI, BamHI and HindIII. Transetta competent cells were transformed.
  • the overnight shaken solution was inoculated to a new LB medium at a ratio of 1:100.
  • a final concentration of 1 mM IPTG (amresco, cat: 0487-1G) was added, and the expression was induced at 20 ° C, 210 rpm/min. hour.
  • the cells were disrupted by a high-pressure bacteria-killing apparatus, and GST agarose (BBI, Cat. No.
  • the pGEX-6P-1 plasmid (GE, Cat. No. 27-4597-01) was transformed into Transetta competent cells.
  • the overnight shaken solution was inoculated to a new LB medium at a ratio of 1:100.
  • a final concentration of 1 mM IPTG (amresco, cat: 0487-1G) was added, and the expression was induced at 25 ° C, 210 rpm / min. hour.
  • the cells were disrupted by a high-pressure bacteria-breaking instrument, and GST agarose (BBI, Cat. No.
  • Chemotaxis assays are routinely observed models of chemotactic migration of cells, and thus were used to verify (N-CCR4) 3 inhibition of Treg cell chemotaxis.

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Abstract

本发明提供重组蛋白或其衍生物，所述重组蛋白具有CC趋化因子受体4(CCR4)的N端部分的氨基酸序列。本发明还提供所述重组蛋白或其衍生物在治疗或预防与CCR4信号传导相关的疾病或状况中的用途。

Description

CCR4的N端重组蛋白及其用途 技术领域
本发明涉及生物医药技术领域，具体涉及CCR4的N端重组蛋白及其衍生化合物、重组蛋白抗体等衍生物或组合物单独或联合具有相关用途的其他化合物或组合物用于治疗、预防、抑制或改善癌症和与CCR4信号传导相关的疾病或状况中的应用。
背景技术
CC趋化因子受体4(CC chemokine receptor 4，CCR4)又称CKR4、CMKBR4、ChemR13、K5-5等，属于CC趋化因子受体家族，含360个氨基酸残基，定位于3号染色体p24-p21.3区域，为7次跨膜G蛋白偶联受体(GPCR)，主要表达于各淋巴细胞和组织，CCR4的高表达与多种炎症性疾病、自身免疫性疾病、血液系统肿瘤以及恶性实体瘤的浸润、转移和预后相关。已知的CCR4高亲和力配体有两个，分别为胸腺活化调节趋化因子(Thymus and activation regulated chemokine，TARC/CCL17)和巨噬细胞衍生趋化因子(macrophage-derived chemokine，MDC/CCL22)。CCR4主要通过表达在Th2细胞、Th17细胞及调节性T细胞(Treg)表面的CCR4与其配体CCL22/CCL17的结合趋化不同类型T细胞发挥免疫效应，如Th2细胞在哮喘和呼吸变应性疾病等发挥重要促炎作用；Th17细胞诱发炎性肠炎如克罗恩病、溃疡性结肠炎及自身免疫性疾病等；Treg细胞表面的CCR4通过与其配体CCL22/CCL17的结合趋化Treg细胞，引起免疫逃逸，从而导致不良临床后果。因此靶向CCR4的化合物有望成为相关疾病治疗的新策略。
目前还没有CCR4和CCL22晶体结构的报道，且CCR4与其配体相互作用的分子机制未知。两步法模型是现在广为接受的GPCR与配体结合并激活的机制，其认为GPCR的N端在识别配体过程中发挥了非常重要的作用。因此通过明析CCR4的N端蛋白序列，解析CCR4的N端蛋白结构，可以有效合成CCR4N端重组蛋白，并通过对其重组蛋白功能研究及其衍生化合物或抗体开发，有望通过靶向CCR4竞争性抑制 CCL22/CCL17-CCR4信号通路，单独或联合具有相关用途的其他化合物或组合物用于治疗、预防、抑制或改善癌症和与CCR4信号传导相关的疾病或状况，如炎性疾病包括变态反应性疾病、炎性肠炎、炎性皮肤病、哮喘、自身免疫性疾病等、移植物排异、血液系统肿瘤如白血病、淋巴瘤和其它血源性癌症如皮肤T细胞淋巴瘤、急性淋巴细胞白血病等和癌症包括实体瘤及转移性疾病如胃癌、肝癌、肾癌、肠癌、胰腺癌、肺癌、膀胱癌、乳腺癌、卵巢癌、宫颈癌、黑色素瘤、甲状腺癌、前列腺癌、神经系统肿瘤等，成为相关疾病免疫靶向治疗的新策略，具有良好的临床应用前景。
目前上市的治疗肿瘤的CCR4单克隆抗体为mogamulizumab，在皮肤T细胞淋巴瘤(CTCL)、蕈样霉菌病、Sézary综合征、非霍奇金淋巴瘤等疾病治疗或研究中均有良好的治疗效果。而CCR4受体拮抗剂在哮喘、鼻炎、皮炎、血栓性疾病、自身免疫性疾病等疾病的研究中均有良好的治疗效果。
但迄今为止，尚未见CCR4的N端重组蛋白及其衍生化合物、重组蛋白抗体等衍生物或组合物单独或联合具有相关用途的其他化合物或组合物用于治疗、预防、抑制或改善癌症和与CCR4信号传导相关的疾病或状况的报道。
发明内容
本发明研究发现，通过两步法模型明析CCR4的N端蛋白序列，解析CCR4的N端蛋白结构，可以有效合成CCR4的N端重组蛋白及其衍生三联蛋白(N-CCR4) 3，实验证明CCR4的N端重组蛋白及衍生三联蛋白能够与趋化因子CCL22结合，并显著性抑制Treg的趋化作用。
本发明提供的趋化因子受体CCR4的N端重组蛋白及其衍生化合物、重组蛋白抗体等衍生物或组合物单独或联合具有相关用途的其他化合物或组合物，在用于治疗、预防、抑制或改善癌症和与CCR4信号传导相关的疾病或状况中具有良好的临床应用前景。
在一个方面，本发明提供CCR4的N端重组蛋白及其衍生化合物、重组蛋白抗体等衍生物或组合物单独或联合具有相关用途的其他化合物或组合物用于治疗、预防、抑制或改善癌症和与CCR4信号传导相关的疾病 或状况的用途。
在另一个方面，本发明提供CCR4的N端重组蛋白及其衍生化合物、重组蛋白抗体等衍生物或组合物，包括但不限于CCR4的N端重组蛋白(N-CCR4)，其衍生化合物如二连、三联等多联N端重组蛋白等(N-CCR4) n、其重组蛋白抗体(N-CCR4) n-Fc或其他结构类似物等衍生化合物。
其中所述的N-CCR4蛋白序列为：
Figure PCTCN2018115442-appb-000001
其中所述的(N-CCR4) 3蛋白序列为：
Figure PCTCN2018115442-appb-000002
其中所述的(N-CCR4) 3-Fc蛋白序列为：
Figure PCTCN2018115442-appb-000003
在另一个方面，本发明的CCR4的N端重组蛋白及其衍生化合物、重组蛋白抗体等衍生物或组合物可单独或联合具有相关用途的其他化合物或组合物使用。
在另一个方面，本发明的CCR4的N端重组蛋白及其衍生化合物、重组蛋白抗体等衍生物或组合物可通过口服、胃肠外如肌肉内、静脉内、皮下注射或植入、吸入、或局部给药等途径施用，可以单独或一起配制成含 有适合各给药途径的常规无毒药学上可接受载体、佐剂和运载体的合适剂量单位制剂。
在另一个方面，用于联合使用的其他化合物或组合物可通过其常用的途径和用量施用，与本发明的化合物或组合物同时或顺序或分开施用。可与本发明化合物或组合物连用同时或顺序或分开施用的治疗剂包括但不限于常规炎症性疾病治疗药物如抗炎类药物、激素类药物、免疫抑制剂、抗组胺类药物等，常规癌症治疗药物如放化疗类药物、常规一线药物(如顺铂、长春新碱、索拉菲尼等)、免疫检查点抑制剂(PD-1/PD-L1，CTLA-4，4-1BB，CD40/CD40L等)及其他肿瘤免疫靶向药物等(VEGF/VEGFR，EGF等)。
在另一个方面，本发明的化合物可用于治疗、预防、抑制或改善癌症和与CCR4信号传导相关的疾病或状况。
在另一个方面，所述治疗包括但不限于1)变应性疾病，如全身过敏性或超敏性反应等；2)炎性肠炎，如克罗恩病、溃疡性结肠炎、回肠炎和肠炎等；3)银屑病和炎性皮肤病，如皮炎、湿疹、特应性皮炎、变应性接触性皮炎、皮肌炎和荨麻疹等；5)脉管炎；6)脊椎关节病；7)硬皮病；8)哮喘和呼吸变应性疾病，如过敏性哮喘、运动诱导哮喘、过敏性鼻炎、超敏性肺病等；9)自身免疫性疾病，如关节炎、多发性硬化症、系统性红斑狼疮、糖尿病、肾炎等；10)移植物排异，如同种异体移植物排异和移植物抗宿主病等；11)白血病、淋巴瘤和其它血源性癌症如皮肤T细胞淋巴瘤、急性淋巴细胞白血病等；12)需要抑制不良炎症的其他疾病，如动脉粥样硬化、肌炎、神经变性疾病、脑炎、脑膜炎、肝炎、肾炎等；13)癌症，包括实体瘤及转移性疾病如胃癌、肝癌、肾癌、肠癌、胰腺癌、肺癌、膀胱癌、乳腺癌、卵巢癌、宫颈癌、黑色素瘤、甲状腺癌、前列腺癌、神经系统肿瘤等。
在另一个方面，本发明的化合物与可联合使用的其他治疗剂可同时或顺序或分开施用，用于治疗、预防、抑制或改善癌症和与CCR4信号传导相关的疾病或状况。
在另一个方面，本发明提供药物组合物，其可制成注射剂、片剂或胶囊剂。
更具体地，本发明提供以下各项：
1.重组蛋白或其衍生物，所述重组蛋白具有CC趋化因子受体4(CCR4)的N端部分的氨基酸序列。
2.根据1的重组蛋白或其衍生物，其中所述重组蛋白的氨基酸序列如SEQ ID NO.1所示。
3.根据1的重组蛋白或其衍生物，其中所述衍生物是所述重组蛋白的多联体或所述重组蛋白与免疫球蛋白Fc片段的融合蛋白。
4.根据1的重组蛋白或其衍生物，其中所述衍生物是所述重组蛋白的三联体，并且其氨基酸序列如SEQ ID NO.2所示。
5.根据1的重组蛋白或其衍生物，其中所述衍生物是所述重组蛋白与免疫球蛋白Fc片段的融合蛋白，并且其氨基酸序列如SEQ ID NO.3所示。
6.药物组合物，所述药物组合物包含根据1-5中任一项的重组蛋白或其衍生物。
7.根据1-5中任一项的重组蛋白或其衍生物或根据6的药物组合物在制备药物中的用途，所述药物用于抑制CCR4与CCR4的配体的结合。
8.根据1-5中任一项的重组蛋白或其衍生物或根据6的药物组合物在制备药物中的用途，所述药物用于治疗或预防与CCR4信号传导相关的疾病或状况。
9.根据8的用途，其中所述与CCR4信号传导相关的疾病或状况包括1)变应性疾病，如全身过敏性或超敏性反应；2)炎性肠炎，如克罗恩病、溃疡性结肠炎、回肠炎和肠炎；3)银屑病和炎性皮肤病，如皮炎、湿疹、特应性皮炎、变应性接触性皮炎、皮肌炎和荨麻疹；5)脉管炎；6)脊椎关节病；7)硬皮病；8)哮喘和呼吸变应性疾病，如过敏性哮喘、运动诱导哮喘、过敏性鼻炎、超敏性肺病；9)自身免疫性疾病，如关节炎、多发性硬化症、系统性红斑狼疮、糖尿病、肾炎；10)移植物排异，如同种异体移植物排异和移植物抗宿主病；11)白血病、淋巴瘤和其它血源性癌症如皮肤T细胞淋巴瘤、急性淋巴细胞白血病；12)需要抑制不良炎症的其他疾病，如动脉粥样硬化、肌炎、神经变性疾病、脑炎、脑膜炎、肝炎、肾炎；13)癌症，包括实体瘤及转移瘤，如胃癌、肝癌、肾癌、肠癌、胰腺癌、肺癌、膀胱癌、乳腺癌、卵巢癌、宫颈癌、黑色素瘤、甲状腺癌、 前列腺癌、神经系统肿瘤。
10.根据8的用途，其中所述与CCR4信号传导相关的疾病或状况是癌症。
11.根据1-5中任一项的重组蛋白或其衍生物或根据6的药物组合物用于抑制CCR4与CCR4的配体的结合的用途。
12.根据1-5中任一项的重组蛋白或其衍生物或根据6的药物组合物用于治疗或预防与CCR4信号传导相关的疾病或状况的用途。
附图说明
图1.CCR4的N端蛋白N-CCR4预测及其衍生蛋白(N-CCR4) 3、(N-CCR4) 3-Fc纯化。
图2.(N-CCR4) 3与CCL22相互作用实验。
图3.(N-CCR4) 3抑制Treg细胞的趋化作用实验。
具体实施方式
实施例1：预测CCR4的N端蛋白N-CCR4
1.CCR4的N端蛋白N-CCR4是通过CCR4与CXCR4蛋白序列疏水性比对(http：//www.bioinfo.mpg.de/AlignMe/AlignMe_MSA.html)及计算CCR4蛋白序列的吉布斯自由能ΔG(ΔG predictor sever v1.0，http：//dgpred.cbr.su.se)预测的(图1A、B)。
N-CCR4蛋白序列为：
Figure PCTCN2018115442-appb-000004
2.N-CCR4衍生蛋白(N-CCR4) 3纯化
将(N-CCR4) 3DNA密码子优化后的序列通过NdeI和BamHI酶切位点克隆到pET22b(+)载体(Novagen，Cat.No.69744-3)上，转化Transetta感受态细胞(全式金，货号CD801-01)。按照1∶100接种过夜摇菌菌液至新的LB培养基中，待菌液OD600为0.6时，加入终浓度1mM IPTG(amresco，cat：0487-1G)，25度，220rpm/min诱导表达10小时。PBS重悬细胞后通过高压破菌仪(JN-02C低温超高压连续流细胞破碎仪)破菌，镍柱(BBI， 货号C600793)结合并用含有100mM咪唑Tris-HCl(pH8.0)缓冲液洗脱纯化，2L PBS透析两次。取诱导前ctrl样，诱导后lys样，穿透液Ft样，洗涤w样，30mM咪唑洗脱样，100mM咪唑洗脱样，500mM咪唑洗脱样，跑12％SDS-PAGE胶检测(图1C)。
(N-CCR4) 3蛋白DNA密码子优化后序列为：
Figure PCTCN2018115442-appb-000005
(N-CCR4) 3蛋白序列为：
Figure PCTCN2018115442-appb-000006
3.N-CCR4衍生抗体(N-CCR4) 3-Fc纯化
将鼠的κIII信号肽-(N-CCR4) 3-Fc DNA序列通过KpnI和BamHI克隆到pCEP4载体，其中鼠的κIII信号肽为分泌信号肽，Fc为人源IgG1 Fc。并用PEI(上海起发生物试剂有限公司，货号24765-2)将构建的质粒转染293FT细胞，转染第三天收上清用protein A预装重力柱(BBI，货号C600951)纯化。取洗脱Elution样跑10％SDS-PAGE胶检测(图1D)。
(N-CCR4) 3-Fc DNA序列：
Figure PCTCN2018115442-appb-000007
Figure PCTCN2018115442-appb-000008
(N-CCR4) 3-Fc蛋白序列：
Figure PCTCN2018115442-appb-000009
Figure PCTCN2018115442-appb-000010
实施例2：(N-CCR4) 3与CCL22相互作用实验
GST-pulldown实验可以验证两个蛋白是否有相互作用，因此通过GST-pulldown实验验证(N-CCR4) 3与CCL22有相互作用。
1.CCL22-GST蛋白表达
CCL22 DNA序列进行密码子优化，并通过酶切位点NdeI，BamHI和HindIII将CCL22及GST克隆到pET22b载体上。转化Transetta感受态细胞。按照1∶100接种过夜摇菌菌液至新的LB培养基中，待菌液OD600为0.6时，加入终浓度1mM IPTG(amresco，cat：0487-1G)，20℃，210rpm/min诱导表达10小时。PBS重悬细胞后通过高压破菌仪破菌，GST琼脂糖(BBI，货号C600031)结合过夜后，用150mM NaCl，1mM DTT(amresco，货号0281-5G)10mM Tris-HCl(pH8.0)缓冲液洗涤10个柱体积，并用10mM还原型谷胱甘肽(BBI，货号70-18-8)150mM NaCl，1mM DTT Tris-HCl(pH8.0)缓冲液洗脱纯化，2L PBS透析两次。
CCL22-GST DNA序列：
Figure PCTCN2018115442-appb-000011
Figure PCTCN2018115442-appb-000012
CCL22-GST蛋白序列：
Figure PCTCN2018115442-appb-000013
2.GST蛋白表达
pGEX-6P-1质粒(GE，货号27-4597-01)转化Transetta感受态细胞。按照1∶100接种过夜摇菌菌液至新的LB培养基中，待菌液OD600为0.6时，加入终浓度1mM IPTG(amresco，cat：0487-1G)，25℃，210rpm/min诱导表达10小时。PBS重悬细胞后通过高压破菌仪破菌，GST琼脂糖(BBI，货号C600031)结合过夜后，用10mM Tris-HCl，150mM NaCl，1mM DTT(amresco，货号0281-5G)缓冲液洗涤10个柱体积，并用10mM还原型谷胱甘肽(BBI，货号70-18-8)150mM NaCl，1mM DTT Tris-HCl(pH8.0)缓冲液洗脱纯化，2L PBS透析两次。
3.GST pulldown
分别将120ng CCL22-GST，GST蛋白与360ng(N-CCR4) 3混合，分 别加入20ul GST琼脂糖，用PBS补足液体至600ul；4℃旋转结合，4h；PBS+0.1％Triton-100洗3次，PBS洗3次；30ul 1x loading buffer溶解beads上的蛋白，煮沸3分钟，高速离心，取1ul上清跑12％SDS PAGE胶，银染(碧云天，快速银染试剂盒，货号P0017S)检测，如图2所示，(N-CCR4) 3与CCL22有相互作用。
实施例3：(N-CCR4) 3抑制Treg细胞的趋化作用实验
趋化实验是常规观测细胞趋化迁移的模型，因此用其验证(N-CCR4) 3对Treg细胞趋化的抑制作用。
分选Naive CD4+T淋巴细胞，加入anti-CD3(2μg/mL)、anti-CD28(1μg/mL)、TGFβ(1ng/mL)和IL-2(4ng/mL)刺激2天，收取诱导好的Treg细胞，2*10 5/孔置于趋化小室上室中，下室分别加入肿瘤细胞培养上清及0.05μM、0.1μM和1μM(N-CCR4) 3重组蛋白，4小时后，收取下室中的Treg细胞并计数统计分析，如图3所示，加入(N-CCR4) 3可显著抑制Treg细胞的趋化运动。

Claims (10)

  1. 重组蛋白或其衍生物，所述重组蛋白具有CC趋化因子受体4(CCR4)的N端部分的氨基酸序列。
  2. 权利要求1的重组蛋白或其衍生物，其中所述重组蛋白的氨基酸序列如SEQ ID NO.1所示。
  3. 权利要求1的重组蛋白或其衍生物，其中所述衍生物是所述重组蛋白的多联体或所述重组蛋白与免疫球蛋白Fc片段的融合蛋白。
  4. 权利要求1的重组蛋白或其衍生物，其中所述衍生物是所述重组蛋白的三联体，并且其氨基酸序列如SEQ ID NO.2所示。
  5. 权利要求1的重组蛋白或其衍生物，其中所述衍生物是所述重组蛋白与免疫球蛋白Fc片段的融合蛋白，并且其氨基酸序列如SEQ ID NO.3所示。
  6. 药物组合物，所述药物组合物包含权利要求1-5中任一项的重组蛋白或其衍生物。
  7. 权利要求1-5中任一项的重组蛋白或其衍生物或权利要求6的药物组合物在制备药物中的用途，所述药物用于抑制CCR4与CCR4的配体的结合。
  8. 权利要求1-5中任一项的重组蛋白或其衍生物或权利要求6的药物组合物在制备药物中的用途，所述药物用于治疗或预防与CCR4信号传导相关的疾病或状况。
  9. 权利要求8的用途，其中所述与CCR4信号传导相关的疾病或状况包括1)变应性疾病，如全身过敏性或超敏性反应；2)炎性肠炎，如克罗恩病、溃疡性结肠炎、回肠炎和肠炎；3)银屑病和炎性皮肤病，如皮炎、湿疹、特应性皮炎、变应性接触性皮炎、皮肌炎和荨麻疹；5)脉管炎；6)脊椎关节病；7)硬皮病；8)哮喘和呼吸变应性疾病，如过敏性哮喘、运动诱导哮喘、过敏性鼻炎、超敏性肺病；9)自身免疫性疾病，如关节炎、多发性硬化症、系统性红斑狼疮、糖尿病、肾炎；10)移植物排异，如同种异体移植物排异和移植物抗宿主病；11)白血病、淋巴瘤和其它血源性癌症如皮肤T细胞淋巴瘤、急性淋巴细胞白血病；12)需要抑制不良炎症 的其他疾病，如动脉粥样硬化、肌炎、神经变性疾病、脑炎、脑膜炎、肝炎、肾炎；13)癌症，包括实体瘤及转移瘤，如胃癌、肝癌、肾癌、肠癌、胰腺癌、肺癌、膀胱癌、乳腺癌、卵巢癌、宫颈癌、黑色素瘤、甲状腺癌、前列腺癌、神经系统肿瘤。
  10. 权利要求8的用途，其中所述与CCR4信号传导相关的疾病或状况是癌症。
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