CN103936835B - PD-L1IgV targeted affinity peptides having anti-tumor activity of D1 - Google Patents

PD-L1IgV targeted affinity peptides having anti-tumor activity of D1 Download PDF

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CN103936835B
CN103936835B CN201410176396.5A CN201410176396A CN103936835B CN 103936835 B CN103936835 B CN 103936835B CN 201410176396 A CN201410176396 A CN 201410176396A CN 103936835 B CN103936835 B CN 103936835B
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skin
d1
affinity peptides
affinity
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CN103936835A (en
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高艳锋
刘蓓媛
祁元明
李国栋
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郑州大学
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Abstract

本发明属于生物制药技术领域,具体涉及一种具有抗肿瘤活性的靶向PD-L1? The present invention belongs to the pharmaceutical field of biotechnology, particularly to targeted PD-L1 having anti-tumor activity? IgV的D-构型亲和肽D1产品及其制备和应用。 The D- configuration IgV affinity peptides D1 products and their preparation and use. 该亲和肽D1特异性结合于PD-L1? The D1 affinity peptides specifically binding to PD-L1? IgV区,其氨基酸序列为NYSKPTDRQYHF,分子量为1554.7。 IgV region, which amino acid sequence is NYSKPTDRQYHF, a molecular weight of 1554.7. 亲和肽D1通过Fomc固相多肽合成法制备,在制备抗结肠癌药物中作为主要活性成分发挥作用。 D1 affinity peptides were prepared by solid phase peptide synthesis Fomc, in manufacture of a medicament against colon carcinoma as the primary active ingredient to play a role. 本发明所提供的亲和肽D1通过利用镜像噬菌体展示肽库筛选技术,以PD-L1? The present invention provides affinity peptides image D1 by using phage display peptide library screening techniques to PD-L1? IgV为靶点进行高通量筛选获得。 IgV targeting high-throughput screening to obtain. 发明人通过小鼠体内荷瘤实验,证明了亲和肽D1具有较好的抗肿瘤活性,能明显抑制小鼠体内肿瘤的增长,从而为基于PD-L1的药物研究和开发提供新的思路和理论基础。 Experiments by the inventors of tumor bearing mice, demonstrated affinity peptides D1 has good anti-tumor activity in vivo can significantly inhibit tumor growth in mice, thereby providing a new way for drug research and development on PD-L1 and theoretical basis.

Description

具有抗肿瘤活性的祀向PD-LI IgV亲和化Dl Si has antitumor activity to PD-LI IgV Dl Affiliationalizing

技术领域 FIELD

[0001] 本发明属于生物制药技术领域,具体设及一种具有抗肿瘤活性多肤产品的筛选、 制备及应用,更具体的,本发明设及一种具有抗肿瘤活性的祀向PD-LlIgV的D-构型亲和肤Dl产品及其制备和应用。 [0001] The present invention belongs to the pharmaceutical field of biotechnology, and specifically provided for screening, and the preparation of antitumor activity with multiple skin products, and more particularly, the present invention is disposed and having an antitumor activity to PD-LlIgV Si the affinity and the D- configuration Dl skin products and their preparation and use.

背景技术 Background technique

[0002] 近年来,肿瘤的防控形势非常严峻。 [0002] In recent years, cancer prevention and control situation is very grim. 随着临床诊断、手术治疗、化疗和放疗等水平的提高,使得一部分病人能够得到早发现、早治疗,并且获得较好的预后,但是,寻找新的治疗手段和治疗药物一直是全球范围内的研究热点。 With the improvement of clinical diagnosis, surgery, chemotherapy and radiotherapy level, so that some patients can get early detection and early treatment, and to obtain a better prognosis, however, to find new treatments and drugs has been a worldwide research focus. 与传统的治疗方法相比,肿瘤免疫治疗能够激活或者诱导肿瘤患者建立起对肿瘤抗原的特异性免疫应答,清除原发的肿瘤细胞, 并且建立免疫记忆,阻止肿瘤的复发和转移。 Compared with traditional methods of treatment, tumor immunotherapy cancer patients can be activated to establish or induce tumor antigen-specific immune responses, clear primary tumor cells, and the establishment of immunological memory, to prevent recurrence and metastasis.

[0003] 在肿瘤免疫治疗过程中,负性共刺激分子主要介导免疫耐受和逃逸,而前人在肿瘤免疫治疗过程中遇到的最大挑战就是由于肿瘤免疫耐受和逃逸所导致的疗效不佳。 [0003] In the process of tumor immunotherapy, the main negative costimulatory molecules mediating immune tolerance and escape, while the greatest challenges faced in previous course of tumor immunotherapy is due to tumor escape immune tolerance and resulting efficacy poor. 因此,探讨通过抑制负性共刺激分子所介导的信号通路W打破机体已经建立的对肿瘤细胞的免疫耐受具有重要的理论意义和应用价值。 Thus, tolerance Discussion of great theoretical and practical value by suppressing the negative costimulatory molecules mediated signaling pathways W break immune body against tumor cells has been established.

[0004] T细胞的激活需两个来自细胞外的信号刺激,即淋己细胞活化的双信号作用。 [0004] T cell activation needs two signals from extracellular stimuli, i.e. leaching has double signal effect cell activation. 细胞活化的第一信号主要来自细胞抗原识别受体(TCR )与MHC分子抗原肤复合物的特异性结合,此过程为抗原识别。 A first main signal cell activation from antigen recognition cell receptor (TCR) with MHC molecules of the antigen-specific binding complex skin, this process for antigen recognition. 细胞活化的第二信号又称共刺激信号,是由抗原递呈细胞(APC)和细胞表面的粘附分子提供。 The second signal, also known as cell activation costimulatory signal, by antigen presenting cells (APC) and cell surface adhesion molecules provided. 运些粘附分子被称为共刺激分子,是一类细胞膜表面分子,可为T、B细胞的活化提供辅助信号,从而调节细胞的增殖、活化及分化。 These adhesion molecules are known transport costimulatory molecules, are a class of cell surface molecules, the auxiliary signal may be provided to the activation of T, B cells, thereby regulating cell proliferation, activation and differentiation. 根据产生的效应不同, 可将共刺激分子分为正性共刺激分子和负性共刺激分子。 Depending on the effect produced, costimulatory molecules can be divided into positive and negative costimulatory molecules, costimulatory molecules. 正性共刺激分子包括CD28、IC0S、 4-1BB等分子,而负性共刺激分子则包括CTLA-4、PD-1、TIM-3等分子。 Positive costimulatory molecules include CD28, IC0S, 4-1BB and other molecules, and the negative costimulatory molecules including CTLA-4, PD-1, TIM-3 molecule and the like.

[0005] PD-I /PD-Ll作为B7/CD28家族成员,已被证实通过抑制T细胞的活化和增殖来负调控免疫应答,并在调节免疫耐受、肿瘤免疫逃逸中发挥重要作用。 [0005] PD-I / PD-Ll as B7 / CD28 family, has been shown to be negatively regulated by the activation and proliferation of T cell immune response, and in the regulation of immune tolerance of tumor immune escape plays an important role. 因而利用PD-Ll的阻断剂作为肿瘤免疫治疗药物或佐剂具有很好的应用前景和安全性,而现有技术中,尚缺少较好的PD-Ll的阻断剂产品。 Thus the use of PD-Ll blocking agent as a tumor immunotherapeutic drugs or adjuvants have good prospects and security, and the prior art, better still lack of PD-Ll blocking agent products.

发明内容 SUMMARY

[0006] 本发明目的在于提供一种具有较好抗肿瘤活性的祀向PD-LlIgV的D-构型亲和肤Dl产品,可W特异性利用PD-Ll蛋白IgV区(D-PD-LlIgV)作为结合祀点,从而阻断PD-I / PD-Ll信号通路,解除对T细胞的活化和增殖的抑制,发挥抗肿瘤作用。 [0006] The object of the present invention to provide Si having a good anti-tumor activity of the D- affinity to PD-LlIgV Dl and skin products, W specificity using PD-Ll protein IgV region (D-PD-LlIgV ) Si as a binding site, thereby blocking the PD-I / PD-Ll signaling pathway, activation and release of the inhibition of T cell proliferation, anti-tumor effect.

[0007] 具体而言,本发明采用的技术方案如下: [0007] Specifically, the present invention employs the following technical solutions:

[0008] -种具有抗肿瘤活性祀向PD-Ll IgV亲和肤D1,属于D-构型,特异性结合于PD-Ll IgV 区,其氨基酸序列为:Asn-Tyr-Ser-Lys-Pro-T虹-Asp-Arg-Gln-Tyr-His-Phe,即NYSKPTDRQYHF,分子量为1554. 7。 [0008] - Si species having anti-tumor activity to PD-Ll IgV affinity skin D1, belonging to the D- configuration, that specifically binds to PD-Ll IgV region, the amino acid sequence: Asn-Tyr-Ser-Lys-Pro -T rainbow -Asp-Arg-Gln-Tyr-His-Phe, i.e. NYSKPTDRQYHF, a molecular weight of 1554.7.

[0009] 所述具有抗肿瘤活性祀向PD-LlIgV亲和肤D1,添加药学上可接受的载体或/和添加剂后,在制备抗结肠癌(CT26)药物中作为主要活性成分发挥作用。 After the [0009] Si having anti-tumor activity and affinity to PD-LlIgV skin D1, adding pharmaceutically acceptable carriers and / or additives, function as the main active ingredient in the preparation of anti-colon carcinoma (of CT26) medicament.

[0010] 所述具有抗肿瘤活性祀向PD-LlIgV亲和肤D1,通过化me固相多肤合成法人工合成制备。 [0010] The preparation of me by solid phase synthesis multiple synthetic skin to skin PD-LlIgV affinity worship D1 have antitumor activity.

[0011] 现有技术中对PD-1/PD-L1复合物的晶体结构研究认为,与受体PD-I结合的部位主要是PD-Ll的胞外段IgV区,因此发明人通过构建人PD-Ll IgV区的原核表达载体,得到人PD-Ll胞外段IgV区蛋白,并W其为祀点来筛选亲和肤,进而对其作为肿瘤免疫治疗药物和佐剂的可能性进行研究。 [0011] The prior art crystal structures of PD-1 / PD-L1 that complex, binding to PD-I receptor site is mainly of the extracellular domain of PD-Ll IgV region, thus constructed by the inventors of the human prokaryotic expression vector IgV region of PD-Ll, PD-Ll human give extracellular IgV region protein segment, and W as a sacrificial skin affinity screening point, and further study its possibility as a therapeutic agent and an adjuvant tumor immunity.

[0012] 在对祀点进行高通量筛选亲和肤过程中,发明人采用了库容量大、操作简便的隧菌体展示肤库技术进行了筛选。 [0012] In point of worship affinity and high throughput screening process of the skin, the inventors employed a large capacity, easy to operate tunneling skin cells display library screening techniques. 隧菌体展示肤库技术是将外源蛋白或多肤序列插入在M13 隧菌体衣壳蛋白P III的N末端,通过蛋白的融合表达将插入的随机多肤序列展示在隧菌体表面。 Skin cells display library technology tunnel is an exogenous protein or polypeptide sequence is inserted at the N-terminus of M13 cells tunneling P III capsid protein, the fusion protein expressed by the inserted random sequences are displayed on multiple tunnel skin cell surface. 由于展示多肤位于P III的N末端,运些小肤通常能够保持较为独立的空间结构,从而能够模拟配体与特异性受体祀标相互作用。 Since the N-terminus show multiple skin located P III, the skin is usually some small transport can be maintained more independent spatial structure, it is possible to simulate a ligand to worship marked specific receptor interactions. 由于通过隧菌体展示肤库筛选所得到的多肤均为天然氨基酸残基所组成,易被酶降解且体内半衰期较短。 Multi skin since the natural amino acid residues are screened by tunneling showing skin cells resulting library composed of, susceptible to enzymatic degradation and the short half-life in vivo. 镜像隧菌体筛选技术即W筛选祀蛋白的D-构型镜像分子为祀标,通过隧菌体展示肤库技术筛选能够与其结合的多肤配基,再合成运些多肤配基的镜像分子一D-构型多肤配基。 Mirror tunneling cell screening Screening i.e. W Si protein molecule is D- configuration Si standard image, showing skin cells by tunneling Screening library bound thereto can be multi-ligand skin, skin resynthesis operation of such multiple-ligand mirror a molecule multiple skin D- configuration ligand. 由于筛选得到的天然多肤配基能特异性结合D-构型祀蛋白,根据镜像对称关系,D构型多肤配基也能特异性结合天然构型的祀蛋白,因此该D-构型多肤即为天然祀蛋白的镜像配基。 Since the multi-screened natural skin ligand capable of specifically binding protein Si D- configuration, according to a mirror-symmetrical relationship, D-configuration multi skin ligand can specifically binding to native protein configuration Si, so that the D- mirroring is the natural ligand Quito worship skin proteins. 因此,发明人选用由D-构型氨基酸人工合成的PD-LlIgV区蛋白作为祀点,通过镜像隧菌体筛选技术得到D-构型的亲和肤,从而大幅度提高其抵抗酶降解的能力W延长体内作用半衰期。 Accordingly, the invention selects a region protein PD-LlIgV the D- amino acid as the synthetic point of Si to give D- configuration and affinity of skin cells by mirroring tunneling screening techniques to dramatically improve their resistance to enzymatic degradation W increased in vivo half-life.

[0013] 本发明通过利用镜像隧菌体展示肤库筛选技术,W D-PD-LlIgV为祀点进行高通量的筛选,人工合成了具有抗肿瘤活性的D-构型亲和肤D1。 [0013] In the present invention, W D-PD-LlIgV high-throughput screening of cells by using tunneling image display library screening technology skin Si point, the D- configuration synthetic skin affinity and D1 have antitumor activity. 发明人进一步通过小鼠体内荷瘤实验,证明了本发明所提供的祀向PD-LlIgV亲和肤Dl具有较好的抗肿瘤活性,能明显抑制小鼠体内肿瘤的增长,从而为基于PD-Ll的药物研究和开发提供新的思路和理论基础。 Further experiments by the inventors tumor bearing mice, demonstrated Si provided by the invention have better antitumor activity and affinity to PD-LlIgV Dl skin, can significantly inhibit in vivo tumor growth in mice, and thus based on PD- Ll drug research and development of new ideas and theoretical basis.

附图说明 BRIEF DESCRIPTION

[0014] 图1是亲和肤Dl的质谱鉴定图; [0014] FIG. 1 is an affinity mass spectrometry of FIG Dl skin;

[0015] 图2是亲和肤Dl的瘤旁给药对荷CT26结肠癌小鼠体重变化的影响结果图; [0015] FIG. 2 is next to the skin affinity and Dl of intratumoral administration on body weight in mice bearing CT26 colon FIG impact results;

[0016] 图3是亲和肤Dl的瘤旁给药对荷CT26结肠癌小鼠瘤体积变化的影响结果图; [0016] FIG. 3 is a side Dl affinity of skin tumors in mice administered to tumor bearing colon carcinoma CT26 volume change results in FIG effect;

[0017] 图4是亲和肤Dl的瘤旁给药对荷CT26结肠癌小鼠瘤重图; [0017] FIG. 4 is a skin and peritumoral administration Dl affinity of tumor weight in mice bearing CT26 colon carcinoma FIG;

[0018] 图5是亲和肤Dl的腹腔给药对荷CT26结肠癌小鼠体重变化的影响结果图; [0018] FIG. 5 is administered intraperitoneally Dl affinity of the skin on body weight in mice bearing CT26 colon FIG impact results;

[0019] 图6是亲和肤Dl的腹腔给药对荷CT26结肠癌小鼠瘤体积变化的影响结果图; [0019] FIG. 6 is administered intraperitoneally Dl affinity of skin tumors in mice bearing CT26 colon volume change results in FIG effect;

[0020] 图7是亲和肤Dl的腹腔给药对荷CT26结肠癌小鼠生存期影响结果图; [0020] FIG. 7 is an affinity skin affect the results of intraperitoneal administration of Dl versus survival of CT26 colon carcinoma bearing mice;

[0021] 图8是亲和肤Dl酶降解稳定性结果图。 [0021] FIG. 8 is a skin affinity and enzymatic degradation stability results Dl FIG.

具体实施方式 Detailed ways

[0022] 下面结合实施例对本发明做进一步的解释说明。 [0022] The following embodiments in conjunction with embodiments of the present invention further explanation.

[0023] 实施例1 [0023] Example 1

[0024] 本发明所提供的具有抗肿瘤活性的祀向PD-Ll IgV的D-构型亲和肤D1,特异性结合于PD-LlIgV区,是利用镜像隧菌体展示肤库技术筛选得到的,其氨基酸序列为:Asn-T yr-Ser-Lys-Pro-T虹-Asp-Arg-Gln-Tyr-Hi S-Phe,即NYSKPTDRQYHF,分子量为1554. 7。 [0024] The present invention provides a sacrificial having anti-tumor activity to PD-Ll IgV affinity of the D- and skin D1, specifically binding to PD-LlIgV region, using the tunnel mirror bacterial display library technology skin screened , the amino acid sequence: Asn-T yr-Ser-Lys-Pro-T rainbow -Asp-Arg-Gln-Tyr-Hi S-Phe, i.e. NYSKPTDRQYHF, a molecular weight of 1554.7.

[00巧]由于通过常规隧菌体展示肤库筛选所得到的多肤均为天然氨基酸残基所组成,易被酶降解且体内半衰期较短。 [Qiao 00] Since the skin by a multi-natural amino acid residues are conventional tunneling skin cells display library screening the resulting composition, and susceptible to enzymatic degradation in vivo half-life is short. 因此,我们选用由D-构型氨基酸人工合成的PD-Ll IgV区蛋白作为祀点,通过镜像隧菌体展示技术来筛选得到D-构型的括抗肤,从而大幅度提高其抵抗酶降解的能力W延长体内作用半衰期。 Thus, we use the D- amino acid of the synthetic PD-Ll IgV region protein as Si point, cells were screened by image display technology has been tunneling skin comprises an anti-D- configuration, and to dramatically improve its resistance to enzymatic degradation W's ability to extend in vivo half-life. 为便于本领域技术人员实施本发明,对其筛选过程简要说明如下: To facilitate those skilled embodiment of the present invention, a brief description of its selection process is as follows:

[0026] 筛选过程中所用培养基和主要溶液的配制说明如下: [0026] As used in the screening process and the main medium is formulated solutions described as follows:

[0027] LB液体培养基:称取膜蛋白腺10 g,酵母粉5 g,氯化钢5 g,加超纯水定容至1以12rC高压蒸汽灭菌20 min,冷却后室溫胆存备用。 [0027] LB liquid medium: Weigh membrane protein of adenovirus 10 g, yeast extract 5 g, steel chloride 5 g, ultrapure water was added to 1 volume 12rC autoclaved 20 min, cooled to room temperature after storage bile spare.

[0028] LB固体培养基:1 L LB液体培养基中加入15 g琼脂粉,加热使琼脂粉充分溶解, 揽拌均匀并分装为100血/瓶,12rC灭菌20 min,冷却至室溫后胆存备用。 [0028] LB solid medium: 1 L LB broth was added 15 g agar, agar powder by heating to fully dissolve, mix well and dispensed embrace of 100 blood / bottle, 12Rc sterilized 20 min, cooled to room temperature bile after storage backup.

[0029] LB^PTG/Xgal平板:将100 mL LB固体培养基用微波炉加热溶解,冷却至60°C左右时,加入75 y L IPTG/Xgal,小屯、混匀,避免产生气泡,倾倒入六孔板。 [0029] LB ^ PTG / Xgal plate: A 100 mL LB solid medium is heated and dissolved in a microwave oven, cooled to 60 ° C or so, was added 75 y L IPTG / Xgal, Xiaotun, mix, to avoid air bubbles, poured into six-well plates. 平板于4°C冰箱避光胆存备用。 Plates at 4 ° C in the dark refrigerator bile memory backup. IPTG/Xgal按W下配方制备:称取0. 5 g IPTG和0. 2g Xgal溶于5血DMF (N,N-二甲基甲酯胺)中,混匀后分装为300 iiL小份,于-20°C避光胆存。 IPTG / Xgal formulation prepared according to the W: Weigh 0. 5 g IPTG is dissolved in 5 0. 2g Xgal and blood DMF (N, N- dimethyl ester amine), and after mixing packaging in small portions to 300 iiL at -20 ° C in the dark bile memory.

[0030] 上层琼脂:称取膜蛋白腺10 g,酵母粉5 g,氯化钢5 g,MgClz • 6&0 1 g,琼脂糖7邑,加超纯水定容至比,微波炉煮沸3次,使琼脂糖充分溶解,冷却至60°C左右,分装成60 HiL的小份,121°C高压蒸汽灭菌20 min,冷却后室溫存放备用。 [0030] The top agar: Weigh membrane protein gland 10 g, yeast extract 5 g, steel chloride 5 g, MgClz • 6 & 0 1 g, 7 Yap agarose, ultra pure water added to volume ratio, microwave boiled three times, agarose fully dissolved, cooled to about 60 ° C, 60 HiL aliquoted into small parts, 121 ° C autoclaved 20 min, cooled backup storage at room temperature.

[003。 [003. LB-Tet (四环素)平板:微波炉加热溶解100血LB固体培养基,冷却至60°C左右时,加入100 y L Tet胆液,混匀后倾倒六平板,待冷却凝固后4°C避光胆存,一周内使用完。 LB-Tet (tetracycline) plates: 100 blood microwave LB solid medium solution was cooled to 60 ° C or so, was added 100 y L Tet bile, after mixing was poured six plates, to be cooled and solidified 4 ° C in the dark gall deposit, used up within a week. 四环素(Tet)胆液按W下配方制备:称取200 mg盐酸四环素,溶于10 mL的无水乙醇中,分装为300 iiL小份,于-20°C避光胆存。 Tetracycline (the Tet) bile formulation was prepared according to the W: Weigh 200 mg of tetracycline hydrochloride, was dissolved in 10 mL of absolute ethanol, 300 iiL dispensing of aliquots, stored at -20 ° C in the dark bile.

[0032] 包被液-0. 1 M NaHC〇3缓冲液(pH 8. 6):称取NaHCO 3 0. 84g用80血S蒸水溶解后用化OH调PH至8. 6,定容至100血,121°C高压蒸汽灭菌20 min,冷却后4°C存放备用。 [0032] The coating solution was NaHC〇3 -0 1 M buffer (pH 8. 6):. Weigh NaHCO 3 0. 84g distilled with 80 S blood of OH dissolved in water with PH adjusted to 8.6, volume blood 100, 121 ° C autoclaved 20 min, 4 ° C after cooling backup storage.

[0033] 封闭液-0.1 MN址C〇3 (抑8. 6)、5 mg/血BSA :称取化肥〇3 0. 84 g、BSA 0. 5 g 用80血S蒸水溶解后用化OH调抑至8. 6,定容至100血,过滤除菌,分装为5血小份,4°C胆存备用。 [0033] blocking buffer -0.1 MN address C〇3 (suppression 8. 6), 5 mg / blood BSA: Weigh fertilizer 〇3 0. 84 g, the BSA 0. 5 g with distilled water was dissolved with 80 of blood S OH adjusted to 8.6 suppressors, blood volume to 100, filter sterilized, aliquoted to 5 parts by platelets, 4 ° C bile memory backup.

[0034] 洗脱液-0. 2 M甘氨酸-肥1缓冲液(pH 2. 2)、1 mg/血BSA :量取50血0. 2M甘氨酸溶液和44血0.2M肥1溶液,加入200 mg BSA,加水定容至200血,过滤除菌,4°C备用。 [0034] -0 2 M glycine eluent - 1 fertilizer buffer (pH 2. 2), 1 mg / blood BSA:. Amount of blood 50 44 0. 2M glycine solution and blood fat 0.2M solution 1, was added 200 mg BSA, add water to 200 blood, filter sterilized, 4 ° C standby.

[0035] 中和液-IM Tris-HCl缓冲液(pH 9. 1):称取12. 114 g Tris碱,适量超纯水溶解, 肥1调抑至9.1,定容、过滤除菌,分装备用。 [0035] The neutralized solution -IM Tris-HCl buffer (pH 9. 1): Weigh 12. 114 g Tris base, dissolving an appropriate amount of ultrapure water, a fat suppression adjusted to 9.1, volume, filter sterilized, divided equipment use.

[0036] TBS缓冲液-50 ml Tris-肥UPH7. 5)、150mM化Cl :按W下步骤制备,第一步先称取6.055 g Trisbase,用少量双蒸水(300~500ml)溶解,再用浓肥1将抑调至7. 5,最后加双蒸水至1000 ml,此步骤制备得Tris缓冲液(50 ml,PH7. 5);第二步称取化Cl 8. 766邑, 先W少量双蒸水溶解化Cl,再加入第一步所制备的化is缓冲液(50 ml,抑7. 5) 100ml,最后加双蒸水至1000ml,充分摇匀,高压灭菌即得。 . [0036] TBS buffer -50 ml Tris- fertilizer UPH7 5), 150mM of Cl: prepared according to the step W, the first step was weighed 6.055 g Trisbase, dissolved in a small amount of double distilled water (300 ~ 500ml), then 1 with concentrated fertilizer was adjusted to 7.5 suppressors, and finally adding distilled water to 1000 ml, this preparation step to obtain a Tris buffer (50 ml, PH7 5.); a second step of weighing Cl 8. 766 eup, first W small amount of double distilled water was dissolved Cl, added is the first step of the prepared buffer solution (50 ml, suppression 7. 5) 100ml, and finally adding distilled water to 1000ml, shake it well, that was autoclaved.

[0037] 洗涂液(TBST) :0. 1% (v/v) Tween-20 TBS,TBS 即前述TBS 缓冲液。 [0037] The wash coating solution (TBST):. 0 1% (v / v) Tween-20 TBS, TBS i.e. the TBS buffer.

[0038] 阳G/化Cl :称取阳G-8000 20 g,化Cl 14. 61 g,加超纯水定容至100血,12TC高压蒸汽灭菌30 min,冷却至室溫,4°C胆存备用。 [0038] Yang G / of Cl: Weigh the male G-8000 20 g, of Cl 14. 61 g, ultrapure water was added to 100 blood volume, 12TC autoclaved 30 min, cooled to room temperature, 4 ° C bile memory backup.

[00測筛选步骤: [00 test screening step:

[0040] (1)包被:采用片段拼接技术用D-构型的氨基酸化学合成PD-Ll蛋白的IgV区, 经过尿素浓度梯度复性后得到目的蛋白D-PD-LlIgV (溶于0.1 M pH 8. 6的胞肥〇3溶液)。 [0040] (1) Coating: splicing technique employed fragment IgV region amino acid chemical synthesis of D- configuration PD-Ll protein, the renaturation of urea concentration gradient obtained through protein D-PD-LlIgV (dissolved in 0.1 M pH 8.6 intracellular solution of fertilizer 〇3). 用目的蛋白包被96孔板,15 yg/孔(150^以100^邑/1111〇,密闭湿盒4°(:解育过夜; With the protein coated 96-well plate, 15 yg / hole (150 ^ 100 ^ ap / 1111〇 confined wet box 4 ° (: Solutions incubated overnight;

[0041] (2)封闭:弃去包被液,用枪头吸出剩余残液并迅速加满封闭液,密闭湿盒4°C解育3 h; [0041] (2) Blocking: The coating solution was discarded, the remaining residue was aspirated with a pipette and rapidly fill blocking solution, wet sealed sterile cartridge 4 ° C Solution 3 h;

[0042] (3)洗涂:TBST洗涂6次,每次不少于2 min,注意无菌操作,动作要快W免微孔板干燥; [0042] (3) washcoated: TBST wash coating 6 times less than 2 min, attention to aseptic be quick drying microplate Free W;

[0043] (4)结合:快速加入预先用TBST稀释至滴度为2 X 1〇11的文库的隧菌体100 y L/ 孔,室溫溫和摇动Ih ;在运个过程中,与祀蛋白具有亲和力的隧菌体会结合在蛋白上; [0043] (4) in combination: rapid pre-diluted in TBST was added to a library titer of 2 X 1〇11 tunneling cells 100 y L / hole, Ih is gentle shaking at room temperature; in a transport process, and Si protein experience tunneling bacteria having binding affinity at the protein;

[0044] (5)洗涂:TBST洗涂10次,洗去未结合的隧菌体; [0044] (5) washcoated: TBST wash coating 10, to wash away unbound cells tunneling;

[004引(6 )洗脱海孔加入100 y L洗脱液,室溫溫和摇动20min ; [004 lead (6) to give 100 y L sea well of eluent, 20min with gentle shaking at room temperature;

[004引(7)中和:用移液枪小屯、吸出洗脱的隧菌体,转移至预先已加好15化中和液的灭菌离屯、管中,溫和吹打混匀; In [primer 004 (7): with a pipette Xiaotun aspirated eluted tunneling cells, transferred to 15 well in advance of sterilization has been added and the liquid from the village, tubes, mixed by gentle pipetting;

[0047] (8)扩增:把第一轮筛选得到的隧菌体与大肠杆菌邸2738加入LB液体培养基(TetO中共同培养,进行扩增和纯化,得到次级肤库; [0047] (8) amplification: the first round of screening the E. coli cells obtained tunneling Di 2738 LB broth (TetO in co-culture, amplification and purification, to give the skin a secondary library was added;

[0048] (9)滴度测定:分别对各轮筛选得到的隧菌体W及扩增后的次级肤库在LB/IPTG/ Xgal平板上进行隧菌体滴度测定,并进行回收率计算,隧菌体回收率=[洗脱隧菌体数/ 投入隧菌体数]X 100% ; [0048] (9) titer: separately for each wheel W screened and tunneling bacteria after amplification of the secondary skin cells tunneling library titer determination on LB / IPTG / Xgal plates, and recovery calculating, tunnel cells recovery = [number of cells tunneling eluted / number of cells into tunneling] X 100%;

[0049] (10)重复筛选:将扩增得到的隧菌体投入下一轮筛选,重复上述筛选过程。 [0049] (10) repeating Screening: The bacterial cells obtained amplified tunnel into the next round of screening, the screening process described above is repeated. 经5轮亲和筛选,即可使得含目的多肤的隧菌体得到高度富集; 5 by affinity selection, such that tunneling can containing the target cells were highly enriched plurality of skin;

[0050] (11)测序:挑取第五轮筛选后的滴度测定平板上的隧菌斑进行扩增,取200 iiL 新鲜扩增的隧菌体胆存液送往金唯智测序公司进行全自动测序,测序引物为-96 g III测序引物:5' -CCC TCA TAG TTA GCG TAA CG-3'。 [0050] (11) Sequencing: titer picked after the fifth round of screening assays for tunneling on the plate plaque amplification, take 200 iiL fresh bile bacterial amplification the reservoir tunneling sent Goldvision chi Company full sequencing automated sequencing, sequencing primers -96 g III as a sequencing primer: 5 '-CCC TCA TAG TTA GCG TAA CG-3'.

[0051] 多肤序列分析:根据DNA测序结果推导其所编码的氨基酸序列,得到括抗肤的氨基酸序列。 [0051] Sequence analysis of multiple skin: its deduced amino acid sequence encoded by DNA sequencing according to the result, comprising the amino acid sequence of an anti-skin.

[00閲筛选结果: [00 reading Filter results:

[0053] (1)分别对各轮筛选得到的隧菌体洗脱液进行滴度测定,并计算投入隧菌体回收率,结果见下表。 [0053] (1) respectively the eluate tunneling cell was subjected to rounds of screening titer determination, and calculates the tunnel into the recovery cell, results in the table below.

Figure CN103936835BD00061

[00巧](2)我们对于筛选得到的非特异性序列和与祀蛋白D-PD-LlIgV亲和力不高的序列进行了淘汰,选择十二肤库对D-PD-LlIgV蛋白进行了镜像筛选,得到两条具有重复克隆的D肤序列,其中包括了Dl肤,具体结果如下表。 [00 Qiao] (2) We conducted a phase-out for non-specific sequences screened and worship protein with D-PD-LlIgV not high affinity sequence, select the twelve skin library for D-PD-LlIgV protein mirrored screening, D has obtained two clone sequence repeats skin, including skin Dl specific results in the following table.

[0056] [0056]

[0057] 实施例2 [0057] Example 2

Figure CN103936835BD00071

[0058] 所述具有抗肿瘤活性的PD-Ll IgV的D-构型亲和肤Dl采用化me固相多肤合成法进行合成。 D- configuration skin affinity and Dl [0058] PD-Ll IgV having anti-tumor activity of the synthesized using a solid phase multi-me skin synthesis.

[0059] 合成过程中所用到的主要试剂有: [0059] Reagents used in the synthesis process are:

[0060] 封头液:乙酸酢/化晚溶液(1:1 v/v); [0060] Liquid Head: Health acetate / night of a solution (1: 1 v / v);

[0061] 巧检试剂:A.巧S酬/乙醇溶液巧% w/v),B.苯酪/乙醇(4:1 w/v),C.氯化钟/ 化晚(2% v/v); [0061] Qiao test agent: A clever paid S / ethanol solution Qiao% w / v), B casein benzene / ethanol (4:... 1 w / v), C bell chloride / LATE (2% v / v);

[006引脱保护液:赃晚/DMF溶液(20% v/v); [006 was deprotected primer: stolen Night / DMF solution (20% v / v);

[006引切割试剂:按体积含量计,TFA (82. 5%),&0 (5%),苯酪(5%),苯甲硫酸(5%),乙二硫醇(2. 5%)。 [006 cited cleavage agent: by volume content of, TFA (82. 5%), & 0 (5%), benzene casein (5%), benzoic acid (5%), ethanedithiol (2.5%) .

[0064] 合成步骤简要描述如下: [0064] Brief description of the synthetic steps are as follows:

[0065] (1)溶胀树脂、加第一个氨基酸 [0065] (1) to swell the resin the first amino acid added to

[006引A.溶胀树脂:取0. 3~0. 5 g化nk树脂巧树脂相连的肤的C末端氨基酸为酷胺) 置于洗干净且干燥的多肤合成仪内,加入适量的DMF,浸泡30 min左右使树脂充分溶胀,真空累抽出合成仪中的DMF。 [006 A. swollen resin primer: Take 0. 3 ~ 0 5 g of skin nk connected resins clever cool amine of the C-terminal amino acid) and placed in a clean dry skin plurality synthesizer, an appropriate amount of DMF was added , soak 30 min the resin fully swollen in vacuo tired out synthesizers DMF.

[0067] B.洗涂:步骤如下,加入适量的DMF,震荡洗涂2min共2次;加入适量的MeOH,震荡洗涂2min共3次;加入适量的DCM,震荡洗涂2 min共3次;加入适量的DMF,再震荡洗涂2min共2次。 [0067] B. washcoated: the steps of adding an appropriate amount of DMF, shock washcoated 2min 2 times; adding an appropriate amount of MeOH, shaking three times washcoated 2min; adding an appropriate amount of DCM, wash coating shaking 3 times 2 min ; adding an appropriate amount of DMF, then washcoated shock 2min 2 times.

[006引C.加第一个氨基酸:通过公式计算应该加入的首个氨基酸、化化及DIC的量,计算公示如下: [006 C. primers plus the first amino acid: by formula should be added to the first amino acid, and the quantization of DIC, the public is calculated as follows:

[0069] 氨基酸质量=树脂质量X2. 5倍当量X带有保护基团的氨基酸的相对分子质量, [0069] The amino resin mass = mass X2. 5 equivalents relative molecular mass of X having the amino protecting groups,

[0070] HOBT质量=树脂质量X 2. 5倍当量XHOBT的相对分子质量, [0070] HOBT molecular mass = mass of the resin mass XHOBT X 2. 5 equivalents of

[0071] DIC用量=树脂质量X 2. 5倍当量XDIC的相对分子质量, [0071] DIC = mass of the resin used in an amount X 2. 5 equivalents relative molecular mass of XDIC,

[0072] 天平砰取D-Fmoc-氨基酸、HOBT于50血烧杯,用少量的DMF溶解后加入合成仪中,再用移液枪吸取DIC加入合成仪,将合成仪置于摇床中,室溫下反应化,使氨基酸与树脂发生禪联反应。 [0072] D-Fmoc- balance bang taken amino acid, HOBT blood beaker at 50, with a small amount of DMF was added to dissolve the synthesizer, and then was added DIC suction pipette synthesizer, the synthesizer placed in a shaker, chamber temperature of the reaction, an amino acid resin with Zen-linking reaction.

[007引D.洗涂:同步骤B。 [Cited D. washcoated 007: same as step B.

[0074] E.测取代值:由于在290nm下,巧甲氧幾基有很强的紫外吸收,因此通过测定它的吸光值,用比色法可W来测定第一个氨基酸与树脂结合的情况。 [0074] E. measured substituted Found: Since at 290nm, a few clever methoxy group there is a strong ultraviolet absorption, so by measuring its absorbance value W can be determined colorimetrically first amino acid to the resin bound Happening. 挑取步骤D中反应后树脂1~1. 5 mg,烘干,用电子分析天平准确称量树脂质量;然后将树脂装入5mL的离屯、管中,加入3血的脱保护液震荡IOmin ;随后转移进入比色皿中于290皿测定样品OD值;另一比色皿中加入3mL的脱保护液为空白对照,按公式计算取代值,计算公示如下: . Step D picked reaction resin 1 ~ 1 5 mg, dry, weighed by electronic balance accurate mass of the resin; and then the resin is charged from the village 5mL tube was added 3 blood deprotection solution shock IOmin ; then transferred into the sample OD measured at 290 cuvette dish; further added to the cuvette solution 3mL deprotected as blank control value is calculated according to the formula substituted, publicity calculated as follows:

[007引取代值=样品OD值/1. 65 X树脂质量。 [007 incorporated substituent sample OD value = / 1. 65 X resin mass.

[0076] 取代值在0. 4~0. 6时最佳,若取代值小于0. 4,则表明氨基酸与树脂结合的量过少,应该重复上述过程加第一个氨基酸。 [0076] substituted at the value 0.4 ~ 0.6 best, if the value is less than 0.4 substituents, it means that the amount of amino acids bound to the resin is too small, the above-described process should be repeated adding a first amino acid.

[0077] F.封头:向合成仪加入适量的封头液震摇20min共两次。 [0077] F. Head: adding an appropriate amount to the synthesizer head was shaken twice with a total of 20min.

[007引G.洗涂:同步骤B。 [G. wash coating primer 007: same as step B.

[0079] (2)加第二个氨基酸及后续肤链延伸 [0079] (2) applying a second amino acid chain extension and subsequent skin

[0080] H.脱保护:合成仪中加入适量的脱保护液,摇床反应20 min共2次。 [0080] H. Deprotection: The deprotection solution was added an appropriate amount of the synthesizer, the reaction shaker for 2 times 20 min.

[00引]I.洗涂:同步骤B。 [00 primer] washcoated the I: with step B.

[008引J.巧检:挑取步骤I中的少量树脂放入巧检管,分别滴加巧检试剂A液1滴、B液2滴、C液1滴,盖好巧检管,沸水浴2min观察树脂颜色;若为蓝色(Pro、His、Ser脱保护后巧检颜色为红栋色),则脱保护正常;否则须重复脱保护过程。 [008 Qiao J. cited subject: step I picked small amount of resin into coincidence pigs were added dropwise Qiao sample liquid 1 drop of Reagent A, B solution 2 drops, C 1 drop of solution, capped sample tube Qiao, boiling water observation resin bath 2min color; if blue (Pro, His, Ser after deprotection Qiao dong subject color is red color), the normal deprotection; otherwise be repeated deprotection.

[0083] K.加氨基酸:计算并将称量好的氨基酸W及册BT用少量DMF溶解后加入多肤合成仪,再用移液枪加入DIC,置于摇床中,室溫下反应2. 5 h。 [0083] K. Add amino acids: amino acids weighed and calculated volumes and W BT After addition of a small amount of DMF was dissolved plurality skin synthesizer pipette then added DIC, placed on a shaker at room temperature for 2 . 5 h.

[0084] L.洗涂:同步骤B。 [0084] L. washcoated: same as step B.

[0085] M.巧检:过程同步骤J,若树脂为亮黄色,无蓝斑,则氨基酸缩合完全,可进行下一步反应。 [0085] M. Qiao subject: procedure is the same procedure J, when the resin is bright yellow, no blue spots, the full amino acid condensation can be carried out in the next reaction. 若为蓝色,则氨基酸缩合不完全,需重复进行此氨基酸的缩合反应,直至巧检树脂完全呈亮黄色。 If it is blue, the amino acid is not completely condensed, this is repeated for the condensation reaction of amino acids, until the resin was completely coincidence detecting bright yellow.

[0086] N.重复步骤HM至完成整条肤的缩合反应。 [0086] N. Repeat steps HM the condensation reaction to completion of the entire skin.

[0087] (3)多肤切割 [0087] (3) Multi-cut skin

[008引0.脱保护:同步骤H。 [008 0. deprotection primer: with Step H.

[008引P.巧检:同步骤J。 [008 P. skillfully seized the lead: with step J.

[0090] Q.洗涂:同步骤B。 [0090] Q. washcoated: same as step B.

[0091] R.切割:在通风楓中配置切割试剂并倒入合成仪,将揽拌器放入合成仪,开始切割反应,揽拌器缓慢揽拌切割2.5 h左右。 [0091] R. Cutting: maple arranged in the air and poured into the cleavage agent synthesizer embrace stirrer into the synthesizer, the cleavage reaction starts and takes stirrer was slowly stirred embrace cutting around 2.5 h. 反应完毕后,用真空抽滤累抽滤出切割试剂,再用DCM洗涂几遍揽拌奖和树脂,并将抽滤得到的切割液装入圆底烧瓶。 After completion of the reaction, vacuum suction filtration tired a cleavage agent, coating wash several times with DCM and the resin embrace stirred award, and the cutting fluid suction obtained a round bottom flask.

[009引S.旋转蒸发:用旋转蒸发仪将切割液中的TFA去除,旋转蒸发仪溫度调整为65。 [009 rotary evaporator S. primer: cutting the TFA was removed in a rotary evaporator, rotary evaporator temperature was adjusted to 65. 旋转蒸发10~20min。 Rotary evaporator 10 ~ 20min. 之后再加入冰乙酸,继续旋转蒸发,10~20min,此步重复6次,由于多肤序列中含有精氨酸和赖氨酸,需进行冰切,W去除侧链的保护集团。 Was added followed by glacial acetic acid, continued rotary evaporation, 10 ~ 20min, this step was repeated 6 times, since the sequence contains multiple skin arginine and lysine, the need for cutting ice, W removal of side-chain protecting groups. 向瓶中加入5mL TFA, 置于冰中,摇床上震摇30min。 Was added to the bottle 5mL TFA, placed on ice, shaken shaker 30min. 冰切完毕后,继续置于旋转蒸发仪中,旋转蒸发10~20min。 After completion of the ice cut, placed on a rotary evaporator and continues in a rotary evaporator 10 ~ 20min.

[0093] T.沉淀:旋转蒸发完毕后,将切割液倒入装有50mL冰乙酸的烧杯中,冰浴静置30min,使多肤沉淀。 [0093] T. precipitate: after rotary evaporation was complete, the solution was poured into a beaker containing cut 50mL of glacial acetic acid, the ice bath was allowed to stand for 30min, so that multiple skin precipitate.

[0094] U.离屯、:吸管吸取烧杯底部乙酸沉淀混合液,20(K)r/min,离屯、2min,弃上清收集沉淀,冰乙酸重悬洗涂沉淀5次。 [0094] U. from Tun,: Pipette bottom of the beaker acetate precipitation mixture, 20 (K) r / min, from the village, 2min, the precipitate was collected supernatant was discarded, glacial acetic acid, the precipitate was resuspended washcoated 5 times.

[0095] V.烘干:将收集的多肤沉淀于37°C烘箱烘干,电子分析天平称重,计算粗肤产率, 将其密封保存于-20°C备用。 [0095] V. Drying: multiple skin collected precipitate dried in an oven at 37 ° C, electronic analytical balance weighing, calculating skin crude yield, which was sealed and stored at -20 ° C for use.

[0096] (4) RP-HPLC分析和制备纯化多肤 [0096] (4) RP-HPLC analysis and purification of multiple skin preparation

[0097] RP-HPLC分析中流动相溶液配制: [0097] RP-HPLC analysis, the mobile phase was prepared:

[0098] 流动相A液(1%。TFA溶液):移液管量取I血TFA,加入超纯水定容至1000血, 超声波仪器超声除气去泡; [0098] Mobile phase A solution (1% .TFA solution): I pipette amount of blood TFA, ultrapure water was added up to 1000 blood, ultrasonic defoaming degassing ultrasonic instrument;

[0099] 流动相B液(乙腊):使用色谱纯级乙腊溶液作为流动相B液,注意用超声波仪器超声除气去泡。 [0099] Mobile phase B liquid (wax B): Class B December chromatography using as a mobile phase solution B solution, attention ultrasonic instrument using ultrasonic defoaming degassing.

[0100] 多肤纯化制备采用梯度洗脱分离纯化: [0100] Preparation of Multi skin Purification was purified by gradient elution:

[010。 [010. W.提前开机30 min预热,打开软件操作界面,参数设置:波长设置228皿,流速设置5 mL/min ; W. advance 30 min warm boot, to open the software interface, parameter settings: wavelength 228 disposed dish, set flow rate 5 mL / min;

[0102] X.平衡:平衡约30 min ; [0102] X. balance: the balance about 30 min;

[010引Y.粗肤制备:称取粗肤约20 mg,用含特定浓度乙腊的0. 1% TFA水溶液(4血)溶解,0.45 ym微孔滤膜过滤后进样。 [010 primer Y. crude skin preparation: Weigh rough skin about 20 mg, containing a particular concentration of wax B 0. 1% TFA solution (4 serum) was dissolved, 0.45 ym filter membrane backward like. 收集主峰产物置于-80°C过夜冻存。 The main peak was collected product was placed in frozen overnight at -80 ° C.

[0104] Z.冻干:将冻好的主峰产物样品置于冻干机中冻干2地,之后收集并称量精肤,-20°C密封保存备用。 [0104] Z. lyophilization: the main peak of the frozen product was freeze-dried samples were placed in a lyophilizer to 2, then collected and weighed fine skin, -20 ° C sealed for use.

[0105] (5)质谱鉴定 [0105] (5) mass spectrometry

[0106] 取少量精肤,通过电喷雾电离质谱分析鉴定多肤分子质量,质谱鉴定结果如图1 所示,鉴定结果符合预期。 [0106] A small amount of fine skin, skin identified multiple molecular mass by electrospray ionization mass spectrometry, mass spectrometry results shown in Figure 1, the identification results in line with expectations.

[0107] 实施例3 [0107] Example 3

[0108] W实施例2制备的具有抗肿瘤活性的祀向PD-Ll IgV的D-构型亲和肤Dl为例, 发明人做了进一步的荷瘤小鼠体内实验,具体实验过程如下: [0108] W Example 2 Si having antitumor activity prepared in Example affinity to the D- and Dl PD-Ll IgV skin, the inventors made further in vivo tumor-bearing mice, the specific experimental procedure is as follows:

[0109] (1)亲和肤Dl对荷CT26结肠癌小鼠移植瘤生长抑制实验 [0109] (1) the affinity of the skin Dl mice bearing CT26 colon carcinoma tumor growth inhibition assay

[0110] 选择24只实验用Ba化/c小鼠,将鼠源结肠癌(CT26)细胞用生理盐水(NS)将细胞浓度调整至5 X IO6个细胞/mU常规消毒后将0. 1血细胞悬液(含5 X 10 5个细胞)接种于每只Ba化/c小鼠右前肢蔽窝皮下,持续观察皮下瘤体生长情况。 After [0110] Twenty-four of the experiments with Ba / c mice, the murine colon carcinoma (of CT26) cells the cell concentration was adjusted to 5 X IO6 cells / mU conventional disinfection with saline (NS) 0. 1 Blood Cells suspension (containing 5 X 10 5 cells) were seeded in each of the Ba / c mice were subcutaneously Right forelimb shield socket for subcutaneous tumor growth was observed.

[0111] 将实施例2制备的亲和肤Dl溶于生理盐水中,制成多肤药物,-20°C分装保存,备用。 Affinity skin Dl [0111] Preparation Example 2 was dissolved in physiological saline embodiment, the skin is made of multiple drugs, -20 ° C Aliquot standby. 接种鼠源结肠癌(CT26)细胞9天后,将小鼠按瘤体积分组,每组6只,分为阴性对照组(NS),阳性对照组(5-FU),多肤药物实验组。 Inoculation murine colon carcinoma (of CT26) cells 9 days, the mice were tumor integrating groups of six, divided into the negative control group (the NS), positive control group (5-FU), drug testing multiple skin group.

[0112] 为确定给药方式不同对抗肿瘤效果的影响,发明人进行了前后两次荷瘤实验,两次实验采取不同的给药方式。 [0112] In order to determine the mode of administration combat the effects of different tumor effect, the inventors conducted before and after the two tumor-bearing experiments, two experiments take a different mode of administration. 具体实验设置如下: Specific experimental set up as follows:

[0113] 第一次荷瘤实验所有实验组均采用瘤旁给药。 [0113] The first tumor-bearing experimental tumors beside all experimental groups were administered using. 多肤药物分为高剂量组(2 mg/kg) 和低剂量组(0. 5 mg/kg)实验组每3天给药一次,共给药7天;5-化阳性对照组每天给药一次,连续给药四天结束。 Multi Drug skin into the high-dose group (2 mg / kg) and low dose (0. 5 mg / kg) is administered once every 3 days for the experimental group were administered 7 days; 5- daily administration of positive control group once the end of four days continuous administration. 实验期间小鼠自由进食和饮水,每日测量小鼠体重及肿瘤的长(a) 短(b)径,并按公式瘤体积=(31/6)X a X b2计算肿瘤体积并绘制肿瘤生长曲线。 Mice free food and water during the experiment, body weight of mice were measured daily and tumor length (a) Short (b) path, and press the formula tumor volume = (31/6) X a X b2 tumor volumes were calculated and plotted tumor growth curve. 给药结束后将小鼠处死取出肿瘤并称重。 After the completion of the administration mice were sacrificed tumors removed and weighed.

[0114] 第二次荷瘤实验采取腹腔给药方式,给药组分为高剂量组(5. 0 mg/kg)和低剂量组(2.0 mg/kg),每天给药一次,连续给药12天。 [0114] The second experiment Tumor take intraperitoneal administration, high dose administration group divided into groups (5. 0 mg / kg) and low dose (2.0 mg / kg), administered once daily, continuous administration 12 days. 实验期间小鼠自由进食和饮水,每日测量小鼠体重及肿瘤的长(a)短(b)径,并按公式计算肿瘤体积并绘制肿瘤生长曲线。 Mice free food and water during the experiment, body weight of mice were measured daily and tumor length (a) Short (b) path, and press the formula tumor volume and tumor growth curve. 给药结束后将小鼠处死取出肿瘤并称重。 After the completion of the administration mice were sacrificed tumors removed and weighed.

[0115] 实验结果见图2~图6。 [0115] The results shown in Figure 2 to 6. 从图2、图5我们可W看出多肤药物组实验小鼠体重正常, 阳性对照组因5-RJ的毒副作用较大而出现消瘦情况。 From FIG. 2, FIG. 5 we can see multiple skin pharmaceutical W mice of normal weight, a positive control group due to toxicity of 5-RJ where large and weight loss occurs. 由此可见,亲和肤Dl没有明显毒副作用。 Thus, pro and skin Dl no obvious side effects. 从图3、图4我们可W看出采用瘤旁给药方式时,药物组实验小鼠肿瘤体积和瘤重都比阴性对照组小。 From FIG 3, FIG 4 we can see that when using W peritumoral administration, the tumor volume of drug group and experimental mice tumor weight smaller than the negative control group. 高剂量和低剂量差距不大,推测是由于亲和肤Dl不易降解,或者剂量相对偏高,因而没有表现出剂量依赖性。 High and low dose gap is not, presumably due to the skin affinity and Dl resistant to degradation, or the dose is relatively high, and therefore did not show dose-dependent. 从图6我们可W看出采用腹腔给药方式时,药物组实验小鼠肿瘤体积比阴性对照组小。 When W in FIG. 6 we can see that by intraperitoneal administration, the tumor volume of mice pharmaceutical smaller than the negative control group. 此时,低剂量比高剂量给药组的实验小鼠瘤体积增长相对缓慢,说明2mg/kg剂量的Dl能更好的抑制荷瘤小鼠肿瘤的生长。 At this time, the volume of tumor growth in mice administered low-dose than the high dose group is relatively slow, described 2mg / kg dose of Dl better growth inhibition of tumor bearing mice.

[011引(2)生存期实验 [011 lead (2) survival test

[0117] 选择24只实验用Ba化/c小鼠,将鼠源结肠癌(CT26)细胞用生理盐水(NS)将细胞浓度调整至5 X IO6个细胞/mU常规消毒后将0. 1血细胞悬液(含5 X 10 5个细胞)接种于每只Ba化/c小鼠右前肢蔽窝皮下,持续观察皮下瘤体生长情况。 After [0117] Twenty-four of the experiments with Ba / c mice, the murine colon carcinoma (of CT26) cells the cell concentration was adjusted to 5 X IO6 cells / mU conventional disinfection with saline (NS) 0. 1 Blood Cells suspension (containing 5 X 10 5 cells) were seeded in each of the Ba / c mice were subcutaneously Right forelimb shield socket for subcutaneous tumor growth was observed.

[0118] 将实施例2制备的亲和肤Dl溶于生理盐水中,制成多肤药物,-20°C分装保存,备用。 Affinity skin Dl [0118] Preparation Example 2 was dissolved in physiological saline embodiment, the skin is made of multiple drugs, -20 ° C Aliquot standby.

[0119] 接种鼠源结肠癌(CT26)细胞9天后,将小鼠按瘤体积分组,每组6只,分为阴性对照组(NS),阳性对照组(5-FU),多肤药物实验组。 [0119] inoculated with murine colon carcinoma (of CT26) cells 9 days, the mice were tumor integrating groups of six, divided into the negative control group (the NS), positive control group (5-FU), drug testing multiple skin group. 各组均采用瘤旁给药,每天给药一次,共给药12天,观察小鼠生存状况,记录小鼠死亡时间。 Each group uses peritumoral administration, administered once daily administration for 12 days to observe survival of mice, dead mice record time.

[0120] 实验结果见图7。 [0120] The results shown in Figure 7. 从图7我们可W看出Dl药物组实验小鼠生存期(2mg/kg组37d 至53d,5mg/kg组36d至52d)与阴性对照组(17d至36d)相比显著延长。 From Figure 7 we can see that W Dl pharmaceutical survival of mice (2mg / kg group 37d to 53d, 5mg / kg group 36d to 52d) and the negative control group (17d to 36d) significantly prolonged compared. 推测是由于Dl肤不易降解,或者剂量相对偏高,因而高剂量组小鼠的生存期与低剂量组相比并没有优势。 Presumably due Dl skin resistant to degradation, or the dose is relatively high, and thus the survival of the high dose group mice, and no advantage compared to the low dose group.

[0121] 实施例4 [0121] Example 4

[0122] 为检验本发明所提供的亲和肤Dl的稳定性,发明人进一步做了酶降解稳定性实验,具体实验过程如下: [0122] Dl skin stability and affinity for testing of the present invention is provided, the inventors made a further study on the biodegradation stability of the enzyme, the specific test procedure is as follows:

[0123] 将IOuL 10 2M Dl肤和对照L肤(W PD-LlIgV蛋白为祀标筛选隧菌体十二肤库得到的k构型亲和十二肤)母液加入190UL血清(用PBS十倍稀释)中,立即震荡混匀,取出20uL混合液,加入离屯、管中,此时计时为Omin,余下的于37°C下继续解育,并分别在不同的时间点(Omin,15min,30min,60min,120min,240min,480min)取出20uL。 [0123] The IOuL 10 2M Dl skin and control skin L (W PD-LlIgV filter subscript k Si protein affinity tunnel configuration libraries skin cells obtained twelve and twelve skin) stock was added 190UL serum (ten times with PBS dilution), and vortexed immediately removed 20uL mixture was added Tun from the tube, in which case the timing is Omin, continued under sterile solution remaining in 37 ° C, respectively, and at different time points (Omin, 15min, 30min, 60min, 120min, 240min, 480min) removed 20uL.

[0124] 将取出的样品加入90uL乙腊震荡混匀W终止酶解过程,样品置于冰上5 min, 再取出90 Ul 0.5%的冰乙酸稀释W确保酶解过程停止。 [0124] The extracted sample is added to 90uL of acetic December vortexed W terminate enzymatic processes, samples were placed on ice for 5 min, then remove 90 Ul 0.5% glacial acetic acid diluted W ensure hydrolysis process is stopped. 13000 g,15 min离屯、,收集上清,-80 °C冻存,至RP-HPLC分析。 13000 g, 15 min ,, the supernatant was collected from the village, -80 ° C frozen to RP-HPLC analysis.

[01巧]从图8可W看出Dl在480min后仍然不会被降解,而作为对照的L肤在120 min 时已经降解了一半。 [Qiao 01] W can be seen from FIG. 8 Dl still will not be degraded after 480min, and the skin as L in 120 min when the control has been degraded by half. 由此可知,Dl肤在体内环境不易被降解,能够有效延长其作用时间,从而达到更好的治疗效果。 It can be seen, Dl skin is not easily degraded in vivo environment, it can effectively prolong the duration of action, so as to achieve better therapeutic effect.

Claims (3)

1. 一种具有抗肿瘤活性靶向ro-LlIgV亲和肽D1,其特征在于,该亲和肽D1属于D-构型,特异性结合于FO-LlIgV区,其氨基酸序列为:Asn-Tyr-Ser-Lys-Pr〇-Thr-Asp-Arg-Gl n-Tyr-His-Phe,SPN-YSKPTDRQYHF,分子量为1554. 7Da。 An anti-tumor activity with ro-LlIgV targeting peptide D1 affinity, wherein the affinity peptides D1 belonging D- configuration, that specifically binds to FO-LlIgV region, the amino acid sequence: Asn-Tyr -Ser-Lys-Pr〇-Thr-Asp-Arg-Gl n-Tyr-His-Phe, SPN-YSKPTDRQYHF, a molecular weight of 1554. 7Da.
2. 权利要求1所述具有抗肿瘤活性靶向H)-L1IgV亲和肽D1在制备抗结肠癌药物中的应用。 1 has the antitumor activity targeting H) -L1IgV affinity peptides D1 in colon cancer in the manufacture of a medicament according to claim.
3. 权利要求1所述具有抗肿瘤活性靶向H)-L1IgV亲和肽D1的制备方法,其特征在于,通过Fmoc固相多肽合成法制备,具体步骤如下: (1) 溶胀树脂、加第一个氨基酸; (2) 加第二个氨基酸及后续肽链延伸,至完成整条肽的缩合反应; (3) 多肽切割; (4) RP-HPLC分析和制备纯化多肽。 3. The preparation of claim 1 having antitumor activity targeting H) -L1IgV affinity peptides D1, characterized in that the polypeptide is prepared by Fmoc solid phase synthesis, the following steps: (1) to swell the resin add of an amino acid; (2) adding the second amino acid peptide chain elongation and subsequent to completion of the entire peptide condensation reaction; (3) cleavage polypeptide; (4) RP-HPLC analysis and purification of the polypeptide preparation.
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WO2010077634A1 (en) * 2008-12-09 2010-07-08 Genentech, Inc. Anti-pd-l1 antibodies and their use to enhance t-cell function
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Publication number Priority date Publication date Assignee Title
WO2010077634A1 (en) * 2008-12-09 2010-07-08 Genentech, Inc. Anti-pd-l1 antibodies and their use to enhance t-cell function
CN103304638A (en) * 2013-07-08 2013-09-18 郑州大学 PD-L1 affinity peptide with anti-tumour activity and application for same

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