WO2018153208A1 - 一种人pd-l1蛋白高亲和性肽及其应用 - Google Patents

一种人pd-l1蛋白高亲和性肽及其应用 Download PDF

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WO2018153208A1
WO2018153208A1 PCT/CN2018/074112 CN2018074112W WO2018153208A1 WO 2018153208 A1 WO2018153208 A1 WO 2018153208A1 CN 2018074112 W CN2018074112 W CN 2018074112W WO 2018153208 A1 WO2018153208 A1 WO 2018153208A1
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protein
seq
peptide
human
affinity
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朱乃硕
刘宝修
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朱乃硕
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Priority to RU2019129985A priority Critical patent/RU2725954C1/ru
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Priority to EP18757780.4A priority patent/EP3587440B1/en
Priority to US16/488,164 priority patent/US11377501B2/en
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • G—PHYSICS
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • the invention belongs to the field of biotechnology, and particularly relates to a human PD-L1 protein high affinity peptide and application thereof.
  • Tumors are a serious disease that threatens human health and are difficult to prevent effectively.
  • the main methods for treating tumors include radiotherapy, chemotherapy and surgical treatment, but the effect is general, and the 5-year survival rate after surgery is low.
  • the development of an effective drug for the treatment of tumors is a hot topic in current cancer research.
  • Immunological detection point therapy represented by anti-PD-1/PD-L1 is a new method for tumor treatment in recent years.
  • the target polypeptide is obtained by chemical synthesis and purification.
  • the dissociation constant of the polypeptide and PD-L1 protein was detected by ELISA, and the blocking effect of the peptide on PD-1/PD-L1 signaling pathway was detected.
  • the expression of PD on the surface of tumor cells was detected by flow cytometry.
  • the human PD-L1 protein high-affinity peptide (referred to as PPLC) provided by the present invention has the amino acid sequence of SEQ.ID.NO.1 and has the following functional characteristics: 1 can be measured with human PD-L1 protein.
  • the dissociation constant can reach 0.75 ⁇ M (the dissociation constant indicates the concentration of the ligand when half of the receptor is bound by the ligand, and the smaller the Kd value, the higher the affinity of the receptor to the ligand); 2 can effectively block PD- 1/PD-L1 protein affinity; 3 can effectively bind to cell surface expressed PD-L1 protein; 4 injection of tumor-inoculated animals can effectively inhibit tumor growth and prolong animal survival time.
  • the present invention also provides a gene encoding the human PD-L1 protein high-affinity peptide (consisting of respective degenerate codons of the corresponding amino acids), the nucleotide sequence of which is shown in SEQ.ID.NO.2.
  • the present invention also obtains a series of peptide sequences having different sequences, but having the same function as the peptide represented by SEQ.ID.NO.1, and the sequence thereof mainly comprises: SEQ.ID.NO.3, SEQ.ID. NO.4.
  • the present invention also provides a SEQ.ID.NO.1 comprising the amino acid sequence of the PD-L1 protein high affinity peptide, the amino acid sequence is SEQ.ID.NO.3 and the amino acid sequence is SEQ.ID.NO.4.
  • a peptide sequence which is a single or multiple repeated tandem or branched peptide molecular sequence, and a molecule containing these core sequence features (homology above 70%), the peptide amino acid sequence of which is SEQ.ID.NO.5 Shown.
  • the present invention also provides a peptide modified by biological and chemical groups, which comprises a PD-L1 protein high affinity peptide SEQ.ID.NO.1, SEQ.ID.NO.3 and SEQ.ID.NO.4
  • the present invention also provides a modified peptide comprising the above various PD-L1 protein high-affinity peptides, the PD-L1 protein high-affinity peptide plus a fluorescent group modification such as FITC, isotopic labeling, chemiluminescence group A molecule modified by a pellet or an enzyme reagent for detecting the PD-L1 protein.
  • a fluorescent group modification such as FITC, isotopic labeling, chemiluminescence group A molecule modified by a pellet or an enzyme reagent for detecting the PD-L1 protein.
  • the present invention also provides the use of the above various PD-L1 protein high affinity peptides for the preparation of PD-L1 protein antagonists.
  • the present invention also provides the use of the above various PD-L1 protein high-affinity peptides in the preparation of PD-L1 protein expression test substances and clinical tests.
  • the invention also provides the use of a peptide gene having the nucleotide sequence of SEQ. ID. NO. 2 for the preparation of a PD-L1 protein antagonist and for use in tracer detection.
  • the peptide of the present invention having the amino acid sequence of SEQ.ID.NO.1 can be used as an antagonist of the PD-L1 protein.
  • the peptide has strong affinity with PD-L1 protein, can block the negative regulation signaling pathway PD-1/PD-L1 of human immunity, thereby activating immunity, initiating T cell killing of tumor, and can be used as a potential tumor.
  • the peptide of the present invention having the amino acid sequence of SEQ.ID.NO.3 and SEQ.ID.NO.4 can also be used as an antagonist of the PD-L1 protein. These two peptides also have strong affinity with the PD-L1 protein, and can block the affinity of PD-1/PD-L1, and initiate immunity to kill tumor cells.
  • Affinity test results of the peptide and PD-L1 protein 2 ⁇ g/ml of PD-L1 protein was coated on a 96-well enzyme plate at 4 ° C overnight, and different concentrations of PPLC peptide with FITC label were added for 1 h, and added. HRP-labeled anti-FITC antibody was incubated for 1 h, then ABTS coloring solution was added, and the OD410 value was read by an M5 microplate reader, and GraphPad Prism 6 was plotted and analyzed. The results confirmed that PPLC has a strong affinity with the PD-L1 protein, and the dissociation constant is 0.75 ⁇ M (Fig. 1).
  • the peptide competes with PD-1 for the binding of PD-L1 protein: 2 ⁇ g/ml of PD-L1 protein was coated overnight on a 96-well enzyme plate at 4 ° C, and different concentrations of PPLC and 1 ⁇ g/ml of PD- were used. 1 protein was mixed, after incubation, rabbit anti-human PD-1 monoclonal antibody was added as primary antibody, HRP-labeled goat anti-rabbit IGg monoclonal antibody was used as secondary antibody, ABTS was developed, and OD410 was read with M5 microplate reader, GraphPad Prism 6 Drawing and analyzing. The results show that PPLC can effectively block the binding of PD-1/PD-L1 (Fig. 2).
  • the effect of the peptide on the tumor volume of tumor animal models the experimental animals were divided into PBS group and PPLC group, and the 6-week-old female Balb/c was inoculated subcutaneously. After two weeks, when the tumor reached 100 mm 3 , PPLC drug treatment was performed. . The size of the mouse tumor was measured daily and the difference was recorded. The peptide was found to significantly reduce the growth rate of mouse tumors (Fig. 5) and prolong the survival time of the mice (Fig. 6).
  • the peptide has a strong affinity for the PD-L1 protein, and the peptide can block the affinity of the PD-1/PD-L1 protein in the human body, thereby breaking the immune tolerance of the tumor and activating the immunity. , initiate T cell killing of tumors, and achieve the purpose of treating tumors. Therefore, the peptide can be used as a drug for tumor targeted therapy. Since the PPLC peptide has high affinity with the PD-L1 protein, it can be used to prepare a probe for detecting the PD-L1 protein, when the PPLC peptide is modified with a fluorescent group such as FITC, an isotopic label, a chemiluminescent group or an enzyme label. Reagent-modified molecules that can be used to quantify, characterize, and localize the presence of various biological samples and cellular PD-L1 proteins.
  • a fluorescent group such as FITC, an isotopic label, a chemiluminescent group or an enzyme label.
  • Figure 1 shows the affinity of PPLC for PD-L1 protein.
  • Figure 2 shows the binding of PPLC to block PD-1/PD-L1 protein.
  • Figure 3 shows the results of flow cytometry experiments. Among them, the left picture shows the results of PD-L1 monoclonal antibody (PD-L1-Ab), and the right picture shows the results of peptide PL-C detection. Gray shades were PD-L1 negative control cells and red and blue lines were PD-L1-positive CHO-PDL1 expressing cells.
  • PD-L1-Ab monoclonal antibody
  • peptide PL-C detection Gray shades were PD-L1 negative control cells and red and blue lines were PD-L1-positive CHO-PDL1 expressing cells.
  • Figure 4 shows the results of fluorescence confocal experiments. Among them, the upper right panel shows the results of observation of the FITC-labeled PPLC peptide with high affinity of PD-L1 expressed on the surface of CHO cells by confocal microscopy.
  • Figure 5 is a graph showing that PPLC inhibits tumor growth in mice.
  • Figure 6 shows PPLC prolonging mouse survival time.
  • the desired polypeptide sequence is artificially synthesized by a chemical method, and the sequence has an affinity for the PD-L1 protein and is capable of blocking the affinity of the PD-1/PD-L1 protein.
  • Lys (Dde)-WangResin was soaked in DCM for 10 min, then DCM was drained, then 25% by volume of 25% piperidine (piperidine/DMF) was added, and the piperidine was drained by bubbling with nitrogen for 20 min. DMF was added for 1 min, and after 6 cycles, it was drained, and the resin ninhydrin was detected to be blue.
  • the product was: H-Lys (Dde)-WangResin.
  • Three equivalents of Fmoc-Val-OH, HATU, DIEA in DMF were added to the resin.
  • the DMF reaction solution was drained, DMF was added and nitrogen was blown for 1 minute, and then drained, and the mixture was circulated three times, and the ninhydrin detection resin was transparent.
  • the product is: Fmoc-Val-Lys (Dde)-WangResin, and so on, to obtain a crude product. Purification was carried out on a Hanbang YCM C18 column using acetonitrile and Milli-Q water. Thereby, a polypeptide of interest with high specificity and high activity is obtained.
  • Affinity test results of the peptide and PD-L1 protein 2 ⁇ g/ml of PD-L1 protein was coated overnight on a 96-well enzyme label at 4 ° C, and different concentrations of PPLC peptide with FITC label were added for 1 h incubation. HRP-labeled anti-FITC antibody was added for 1 h, then ABTS coloring solution was added, and the OD410 value was read by M5 microplate reader, and GraphPad Prism 6 was plotted and analyzed. The results confirmed that PPLC has a strong affinity with the PD-L1 protein, and the dissociation constant is 0.75 ⁇ M.
  • mice Effect of the peptide on the tumor volume of tumor animal models: The experimental animals were divided into PBS group and PPLC group, and the female Balb/c was subcutaneously inoculated with tumor. Two weeks later, when the tumor reached 100 mm 3 , PPLC drug treatment was performed. The size of the mouse tumor was measured daily and the difference was recorded. The results showed that the peptide can significantly reduce the growth rate of mouse tumors and prolong the survival time of mice.
  • PPLC has a strong affinity with the PD-L1 protein and can block the affinity of the PD-1/PD-L1 protein at the molecular level.
  • Animal experiments showed that the tumor volume of mice treated with PPLC peptide was significantly smaller than that of the control group. The above results indicate that PPLC can break the tumor immune tolerance and improve the killing of tumor cells by T cells to achieve the purpose of treating tumors.
  • Tumor immunotherapy is a new method for treating tumors in recent years.
  • Immunological detection point blockade represented by PD-1/PD-L1 protein is an important component.
  • PD-1/PD-L1 antibody drugs have been approved by the US Food and Drug Administration, and have shown good results in clinical use and in diseases such as melanoma.
  • antibody treatment also faces problems of high immunogenicity, high cost, large side effects, and off-target.
  • the development of an anti-tumor drug with both targeting and small immunogenicity is an important direction in the development of immunology today.
  • the human PD-L1 protein high-affinity peptide provided by the invention can block the binding of PD-1/PD-L1 protein, break the tumor immune tolerance, and significantly inhibit the growth of tumor cells, so it can be used as a potential tumor target. To the treatment of the drug.
  • the modified PPLC peptide can detect the expression of human PD-L1 protein and is used as a detection reagent.

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Abstract

本发明提供了人PD-L1蛋白亲和多肽及其应用，所述多肽的氨基酸序列为SEQ ID NO：1、SEQ ID NO：3和SEQ ID NO：4所示，或者为SEQ ID NO：1、SEQ ID NO：3和SEQ ID NO：4单次重复或多次重复的串联或分支的肽，其氨基酸序列为SEQ ID NO：5所示。所述多肽对人PD-L1蛋白具有高亲和性，可以竞争性阻断PD-1/PD-L1蛋白的亲和，阻断人体肿瘤的负调控耐受通路；活化免疫，提高T细胞对肿瘤细胞的杀伤力。

Description

一种人PD-L1蛋白高亲和性肽及其应用 技术领域
本发明属于生物技术领域，具体涉及一种人PD-L1蛋白高亲和性肽及其应用。
背景技术
肿瘤是威胁人类健康的一种比较严重的疾病，且难以有效的预防。目前治疗肿瘤的方法主要有放疗、化疗和手术治疗等，但效果一般，手术后的5年生存率较低。开发出一种有效的治疗肿瘤的药物是当前肿瘤研究的热门课题。以抗PD-1/PD-L1为代表的免疫检测点疗法是近年来兴起的一种肿瘤治疗的全新手段，目前已经被FDA批准用于临床治疗的PD-1/PD-L1抗体类药物有三个，虽然都在一定程度上有效果，但也伴随着效率低、副作用及脱靶的表现。因此开发出一种高效、持久、具有广泛治疗作用的药物迫在眉睫。
采用化学的方法合成、纯化后得到目的多肽。采用ELISA的方法检测多肽与PD-L1蛋白的解离常数，并检测该多肽对PD-1/PD-L1信号通路的体外阻断作用；用流式细胞仪检测该多肽对肿瘤细胞表面表达PD-L1蛋白的亲和作用；最后再注射小鼠肿瘤动物模型，通过观察小鼠肿瘤大小的变化及生存时间的变化推断该多肽治疗肿瘤的潜在价值。
发明内容
本发明的目的是提供一种人PD-L1蛋白高亲和性肽及其在治疗肿瘤方面的应用。
本发明提供的人PD-L1蛋白高亲和性肽(记为PPLC)，其氨基酸序列为SEQ.ID.NO.1所示，具有如下功能特征：①可与人PD-L1蛋白，测得解离常数可达0.75μM(解离常数表示一半受体被配体结合时配体的浓度，Kd值越小代表受体与配体的亲和性越高)；②可有效阻断PD-1/PD-L1蛋白的亲和；③可有效的和细胞表面表达的PD-L1蛋白结合；④注射接种肿瘤的动物，可有效抑制肿瘤的生长，延长动物生存时间。
本发明还提供编码所述人PD-L1蛋白高亲和性肽的基因(由相应氨基酸各简 并密码子构成)，其核苷酸序列为SEQ.ID.NO.2所示。
通过生物技术改造，本发明还获得了一系列序列不同，但是具有SEQ.ID.NO.1所示肽相同功能的肽序列，其序列主要包括：SEQ.ID.NO.3、SEQ.ID.NO.4。
本发明还提供一种同时含有PD-L1蛋白高亲和性肽氨基酸序列的SEQ.ID.NO.1，氨基酸序列为SEQ.ID.NO.3和氨基酸序列为SEQ.ID.NO.4的肽序列，为单次重复或多次重复的串联或分支的肽分子序列，及含有这些核心序列特征(同源性在70％以上)的分子，其肽氨基酸序列为SEQ.ID.NO.5所示。
本发明还提供一种经过生物和化学基团修饰的肽，其含有PD-L1蛋白高亲和性肽SEQ.ID.NO.1，SEQ.ID.NO.3和SEQ.ID.NO.4作为核心序列的C端(或N端，或侧链)基团上加抗原或药物或PEG修饰或其他分子基团共价修饰的结构特征分子。
本发明还提供一种经过修饰的肽，包括上述各种PD-L1蛋白高亲和性肽，其PD-L1蛋白高亲和性肽的加FITC等荧光基团修饰、同位素标记、化学发光基团或酶标试剂修饰的分子，用于检测PD-L1蛋白。
本发明还提供上述各种PD-L1蛋白高亲和性肽，在制备PD-L1蛋白拮抗剂中的应用。
本发明还提供上述各种PD-L1蛋白高亲和性肽，在制备PD-L1蛋白表达检测物和临床检测物中的应用。
本发明还提供如核苷酸序列为SEQ.ID.NO.2的肽基因在制备表达PD-L1蛋白拮抗剂中的应用和在示踪检测中的应用。
本发明的氨基酸序列为SEQ.ID.NO.1的肽可作为PD-L1蛋白的拮抗药物。该肽与PD-L1蛋白具有较强的亲和性，可以阻断人体免疫的负调控信号通路PD-1/PD-L1，从而活化免疫，启动T细胞对肿瘤的杀伤，可作为潜在的肿瘤靶向治疗的药物。本发明的氨基酸序列为SEQ.ID.NO.3、SEQ.ID.NO.4的肽同样可以作为PD-L1蛋白的拮抗药物。这两条肽同样与PD-L1蛋白具有较强的亲和性，并且可阻断PD-1/PD-L1的亲和，启动免疫对肿瘤细胞的杀伤。
该肽与PD-L1蛋白亲和性实验结果：将2μg/ml的PD-L1蛋白4℃过夜包被96孔酶标版上，加入不同浓度的带有FITC标记的PPLC肽共孵育1h，加入HRP标记的抗FITC的抗体孵育1h，然后加入ABTS显色液，通过M5酶标仪读取OD410数值，GraphPad Prism 6作图并分析。结果证实PPLC与PD-L1蛋白具有极强的亲和力，解离常 数为0.75μM(图1)。
该肽与PD-1竞争性结合PD-L1蛋白的实验结果：将2μg/ml的PD-L1蛋白4℃过夜包被96孔酶标版上，将不同浓度的PPLC和1μg/ml的PD-1蛋白混合，孵育后加入兔抗人PD-1的单抗作为一抗，HRP标记的羊抗兔IGg的单抗作为二抗，ABTS显色后用M5酶标仪读取OD410，GraphPad Prism 6作图并分析。结果显示PPLC可有效阻断PD-1/PD-L1的结合(图2)。
该肽与细胞表面表达的PD-L1蛋白结合的实验结果：将重组人PD-L1蛋白重组质粒转染CHO细胞株，培养36小时后，加入PD-L1蛋白单抗或带有FITC标记的PPLC肽，4℃孵育30分钟，跑流式细胞仪检测(图3)；转染质粒后继续培养36小时，加入带有FITC标记的PPLC肽37℃孵育30分钟，荧光共聚焦检测(图4)。结果显示PPLC肽可以有效和细胞表面表达的PD-L1蛋白结合(图3/图4)。
该肽对肿瘤动物模型肿瘤体积大小的影响：将实验动物分为PBS组和PPLC组，对6周大的雌性Balb/c皮下接种肿瘤，两周后待肿瘤达到100mm 3时，进行PPLC药物治疗。每天量取小鼠肿瘤的大小，并记录差异。结果发现该肽可以显著的降低小鼠肿瘤的生长速度(图5)，延长小鼠的生存时间(图6)。
由此可见，该肽对PD-L1蛋白具有极强的亲和性，利用该肽可以阻断人体内的PD-1/PD-L1蛋白的亲和，从而打破肿瘤的免疫耐受，活化免疫，启动T细胞对肿瘤的杀伤，达到治疗肿瘤的目的。因此，可将该肽作为一种肿瘤靶向治疗的药物。由于PPLC肽具有与PD-L1蛋白的高亲和特性,可以用于制备检测PD-L1蛋白的探针,当将PPLC肽用FITC等荧光基团修饰、同位素标记、化学发光基团或酶标试剂修饰的分子，可用于定量、定性和定位检测各种生物样品和细胞PD-L1蛋白的存在。
附图说明
图1为PPLC对PD-L1蛋白的亲和性。
图2为PPLC阻断PD-1/PD-L1蛋白的结合。
图3为流式细胞实验结果。其中，左图为PD-L1单抗(PD-L1-Ab)检测结果，右图为多肽PL-C检测结果。灰色阴影为PD-L1阴性对照细胞，红色和蓝色的线为PD-L1阳性的CHO-PDL1表达细胞。
图4为荧光共聚焦实验结果。其中，右上图显示FITC标记的PPLC肽与CHO细胞表面表达的PD-L1高度亲和而用共聚焦显微镜观察检测的结果。
图5为PPLC抑制小鼠肿瘤的生长。
图6为PPLC延长小鼠生存时间。
具体实施方式
一、多肽序列的获得与改造
通过化学的方法人工合成所需的多肽序列，序列具有与PD-L1蛋白亲和的作用，并且能够阻断PD-1/PD-L1蛋白的亲和。
二、多肽的合成与纯化
Lys(Dde)-WangResin用DCM浸泡10min后抽干DCM，然后加入树脂体积三倍的25％哌啶(哌啶/DMF)，用氮气鼓吹20min后抽干哌啶。加入DMF鼓吹1min，循环6次后抽干，取树脂茚三酮检测呈蓝色。产物为：H-Lys(Dde)-WangResin。加入树脂3倍当量的Fmoc-Val-OH，HATU，DIEA在DMF中。用氮气鼓吹20min后抽干DMF反应液，加入DMF用氮气鼓吹1min后抽干，循环3次，茚三酮检测树脂呈透明。产品为：Fmoc-Val-Lys(Dde)-WangResin，依次类推，得到粗产品。用乙腈、Milli-Q水通过汉邦YCM C18柱子进行纯化。从而获得高特异性、高活性的目的多肽。
三、PPLC对PD-L1蛋白的亲和及动物实验结果
1、该肽与PD-L1蛋白亲和性实验结果：将2μg/ml的PD-L1蛋白4℃过夜包被96孔酶标版上，加入不同浓度的带有FITC标记的PPLC肽共孵育1h，加入HRP标记的抗FITC的抗体孵育1h，然后加入ABTS显色液，通过M5酶标仪读取OD410数值，GraphPad Prism 6作图并分析。结果证实PPLC与PD-L1蛋白具有极强的亲和力，解离常数为0.75μM。
2、该肽与PD-1竞争性结合PD-L1蛋白的实验结果：将2μg/ml的PD-L1蛋白4℃过夜包被96孔酶标版上，将不同浓度的PPLC和1μg/ml的PD-1蛋白混合，孵育后加入兔抗人PD-1的单抗作为一抗，HRP标记的羊抗兔IGg的单抗作为二抗，ABTS显色后用M5酶标仪读取OD410，GraphPad Prism 6作图并分析。结果显示PPLC可有效阻断PD-1/PD-L1的结合。
3、该肽与细胞表面表达的PD-L1蛋白结合的实验结果：将重组人PD-L1蛋白重组质粒转染CHO细胞株，培养36小时后，加入PD-L1蛋白单抗或带有FITC标记的PPLC肽，4℃孵育30分钟，跑流式细胞仪检测；转染质粒后继续培养36小时，加入带有FITC标记的PPLC肽37℃孵育30分钟，荧光共聚焦检测。结果显示PPLC肽可以有 效和细胞表面表达的PD-L1蛋白结合。
4、该肽对肿瘤动物模型肿瘤体积大小的影响：将实验动物分为PBS组和PPLC组，对雌性Balb/c皮下接种肿瘤，两周后待肿瘤达到100mm 3时，进行PPLC药物治疗。每天量取小鼠肿瘤的大小，并记录差异。结果发现该肽可以显著的降低小鼠肿瘤的生长速度，延长小鼠的生存时间。
结果(如图1-图6)发现，PPLC与PD-L1蛋白具有极强的亲和作用，并且能在分子水平上阻断PD-1/PD-L1蛋白的亲和。动物实验结果表明，经PPLC肽治疗后的小鼠肿瘤体积显著的小于对照组。以上结果说明PPLC可以打破肿瘤免疫耐受，通过提高T细胞对肿瘤细胞的杀伤，达到治疗肿瘤的目的。
四、具体应用
肿瘤免疫疗法是近年来兴起的一种治疗肿瘤的新的手段，以PD-1/PD-L1蛋白为代表的免疫检测点阻断治疗是其中重要的组成部分。目前已经有PD-1/PD-L1抗体类药物通过了美国食品与药品监督管理局的批准，在临床使用当中，并且在黑色素瘤等疾病当中表现出了较好的效果。但是于此同时，抗体治疗也面临免疫原性较高、费用昂贵、副作用大以及脱靶等较多的问题。开发一种既具有靶向、又具有较小免疫原性的抗肿瘤药物是当今免疫学发展的一个重要方向。本发明提供的人PD-L1蛋白高亲和性肽可阻断PD-1/PD-L1蛋白的结合，打破肿瘤的免疫耐受，显著的抑制肿瘤细胞的生长，因此可作为潜在的肿瘤靶向治疗的药物。另外经过修饰的PPLC肽，可以检测人PD-L1蛋白的表达，作为检测试剂使用。

Claims (7)

  1. 一种人PD-L1蛋白高亲和性肽，其特征在于，氨基酸序列为SEQ.ID.NO.1，SEQ.ID.NO.3和SEQ.ID.NO.4之一；或者为SEQ.ID.NO.1，SEQ.ID.NO.3和SEQ.ID.NO.4单次重复或多次重复的串联或分支的肽分子序列，其氨基酸序列为SEQ.ID.NO.5。
  2. 一种编码如权利要求1所述PD-L1蛋白高亲和性肽的编码基因，其特征在于，核苷酸序列为SEQ.ID.NO.2。
  3. 一种经过生物和化学基团修饰的肽，其特征在于，含有如权利要求1所述PD-L1蛋白高亲和性肽作为核心序列的C端，或N端，或侧链基团上加PEG修饰或其他分子基团共价修饰的结构特征分子。
  4. 一种经过修饰的多肽，其特征在于，如权利要求1所述PD-L1蛋白高亲和性肽的加FITC等荧光基团修饰、同位素标记、化学发光基团或酶标试剂修饰的分子，可用于检测PD-L1蛋白。
  5. 如权利要求1所述的人PD-L1蛋白高亲和性肽在制备PD-L1蛋白拮抗药物中的应用。
  6. 如权利要求1所述的人PD-L1蛋白高亲和性肽在制备PD-L1蛋白表达检测物和临床检测物中的应用。
  7. 如权利要求2所述的PD-L1蛋白高亲和性肽的编码基因在制备表达PD-L1蛋白拮抗药物和示踪检测物中的应用。
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