WO2021139761A1 - Pd-1亲和肽及其应用 - Google Patents
Pd-1亲和肽及其应用 Download PDFInfo
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- WO2021139761A1 WO2021139761A1 PCT/CN2021/070838 CN2021070838W WO2021139761A1 WO 2021139761 A1 WO2021139761 A1 WO 2021139761A1 CN 2021070838 W CN2021070838 W CN 2021070838W WO 2021139761 A1 WO2021139761 A1 WO 2021139761A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the invention belongs to the technical field of biopharmaceuticals.
- PD-1 Programmed death-1
- TasukuHonj was originally discovered and cloned by TasukuHonj from apoptotic hybridomas. It is also called CD279 and belongs to the CD28 superfamily of immunoglobulins. PD-1 is mainly expressed on the surface of activated T cells, B cells and NK cells, and is a surface molecule of activated T cells. The ligands of PD-1, PD-L1 (CD274) and PD-L2 (CD273), belong to the CD28/B7 family. Blocking the PD-1/PD-L1 signal pathway can block negative regulatory signals, restore the function of T cells to a certain extent, kill tumor cells, and achieve the goal of anti-tumor.
- the current marketed drugs targeting PD-1 are all monoclonal antibodies. Although they have achieved very good curative effects, antibody drugs are expensive, immunogenic, poor in tumor invasion, long half-life, and cannot be used when adverse immune reactions occur. Timely termination of removal and other shortcomings, so the development of other types of blocking agents has become the current research focus.
- Polypeptide drugs generally only have primary and secondary structures, not more complex three-dimensional structures. Compared with traditional chemotherapeutics and antibodies, peptide drugs have the advantages of small molecular weight, strong permeability, low immunogenicity, and low price, which makes peptide drugs receive great attention in anti-tumor research. Therefore, the development of safer and more effective peptide blockers has far-reaching clinical research value.
- the present invention relates to an affinity peptide that has affinity for PD-1, which is a modified peptide of peptide P1 or P1 peptide.
- the amino acid sequence of peptide P1 is shown in SEQ ID NO. 1, and the modified peptide is selected from the group consisting of The peptide defined by the peptide a, b or c or a combination thereof:
- Peptide a Alanine scanning peptide of peptide P1, the amino acid sequence is independently selected from SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (NOs are represented in the art Parallel listing of sequence numbers, that is, the amino acid sequences are SEQ ID NO.2, SEQ ID NO.3...);
- Peptide b a point mutation peptide of peptide P1, which has point mutations at the 1, 4, 5, 8, 9 and/or 10 amino acids of peptide P1;
- Peptide c C-terminal truncated peptide of peptide P14, peptide P14 is a point mutant peptide in which amino acid R at position 4 of peptide P1 is mutated to Y, and the number of amino acids of the truncated peptide is 3, 4, 5, 6, 7 , 8, 9, 11 or 12;
- each amino acid of the affinity peptide is independently selected from D-type or L-type, for example, each amino acid is D-type or L-type.
- C-terminal truncated peptides in the art refer to a series of shorter peptides obtained by truncating 1 to more amino acids from the C-terminus of the protein parent, as shown in SEQ ID NO.21.
- the point mutations in peptide b are independently selected from the following mutations: D mutation is Y or N; F mutation is R; R mutation is Y; V mutation is Y or W; P mutation is W; N mutation is D Or W.
- peptide b is a peptide obtained by point mutation of one amino acid of peptide P1.
- the amino acid sequence of the affinity peptide is independently selected from SEQ ID NOs: 1-28 (NOs: 1-28 is the abbreviated form of the NOs listed side by side, and the sequence numbers included are between 1-28. Any integer).
- 1, 2, or 3 amino acids at the N-terminus of the affinity peptide are D-type, and/or 1 or 2 amino acids at the C-terminus are D-type;
- other amino acids are defaulted to be L-shaped.
- the sequence is as follows: dDFYVWWPNFPR, ddFYVWWPNFPR, ddfYVWWPNFPR, dDFYVWWPNFPr, ddFYVWWPNFPr, dDFYVWWPNFpr, ddFrYVWWPNFVWWFP or ddf
- D form of the corresponding amino acid such as Asp D -Asp D -Phe D -Tyr-Val-Trp-Trp-Pro-Asn-Phe-Pro D -Arg D , abbreviated as ddfYVWWPNFpr.
- the present invention provides a pharmaceutical composition or kit containing the affinity peptide described in the first aspect.
- the present invention provides the application of the affinity peptide described in the first aspect in the preparation of a drug/pharmaceutical composition or kit.
- the affinity peptide can exist in free form or in the form of a pharmaceutically acceptable salt, the affinity peptide can also be chemically modified to extend the half-life, such as cyclization modification, acetylation modification, PAS modification, PEG modification, fatty acid modification, albumin modification, albumin Affinity peptide coupling, tumor homing peptide coupling, membrane penetrating peptide coupling, nanocarrier coupling; simple modifications based on the idea of this patent, all constitute equivalent infringement of this patent.
- composition of the second aspect or the third aspect may include a pharmaceutically acceptable excipient, and may be used for at least one of the following purposes:
- Anti-tumor such as solid tumor-colon cancer
- the PD-L1 of the present invention refers to the ligand of mammalian PD-1 protein, such as human PD-L1 (hPD-L1) or mouse.
- PD-1 and PD-L1 can be wild-type or mutant proteins that still retain their activity, such as wild-type or mutant PD-1 in the applicant's patent CN108794619.
- affinity peptide of this patent can exist in free form or in the form of its pharmaceutically acceptable salt.
- a simple modification based on the idea of this patent constitutes an equivalent infringement of this patent.
- the present invention screens and obtains PD-1 affinity peptides, which can block the combination of PD-1/PD-L1.
- High blocking rate peptides can significantly inhibit CT26 colon cancer in mice, and have good application prospects in tumor treatment, autoimmune diseases, inflammatory diseases, and so on.
- Figure 1 is a graph showing the experimental results of blocking the PD-1/PD-L1 protein binding by peptide P2-P13 at 200 ⁇ M and the relative blocking rate of 200 ⁇ M peptide P1;
- Figure 2 is a graph showing the experimental results of the relative blocking rate of peptides P1 and P14-P20;
- Figure 3 is a graph showing the experimental results of the relative blocking rates of peptides P14 and P21-P28;
- Figure 4 is a graph showing the experimental results of the relative blocking rate of P14, peptide P14-1 to peptide P14-9;
- Figure 5 is a graph of mouse tumor volume after administration of modified peptide P14-8 0.5 mg/kg. Significance analysis mark ** means P ⁇ 0.01.
- the phage display peptide library is screened in liquid phase to obtain the parent peptide P1.
- the screening process is as follows:
- the phage display dodecapeptide library was screened by the liquid phase differential screening method
- phage monoclonal with affinity to the target protein rhPD-1/Fc are enriched round by round;
- Micro thermal surge detects the affinity between the affinity peptide of the present invention and hPD-L1, and the detection process is as follows:
- wash the column Put the protein separation column into a 15mL centrifuge tube and wash with MST Buffer (usually wash 10 column volumes, the column volume is 500 ⁇ L);
- step 3 Separate and purify the protein: add the protein dye mixture (200 ⁇ L) in step 1 to the protein separation column in the air, and then add 300 ⁇ L of MST Buffer. When the column no longer drips liquid, add 500 ⁇ L of MST Buffer. Start collecting the first drop of liquid. After the collection is complete, test the fluorescence on the computer, and dilute the protein fluorescence value to about 600, and store it at -80°C after aliquoting;
- Sample preparation Dissolve the peptide to an appropriate concentration (10-200 ⁇ M), perform 15 times ratio dilutions to obtain 16 concentration gradient samples, and the system is 10 ⁇ L. Add 10 ⁇ L of labeled protein sample to each tube, mix well and centrifuge to remove air bubbles. After incubating on ice for 5 minutes, use MST special capillary to suck the incubated liquid and place it in the instrument holder;
- peptide P1K D 5.30 ⁇ 2.84, peptide P14-P20K D values are 0.19 ⁇ 0.14, 0.028 ⁇ 0.014, 0.80 ⁇ 0.37, 1.27 ⁇ 0.57, 0.38 ⁇ 0.27, 5.07 ⁇ 3.28, 4.23 ⁇ 2.93, peptide P14- The D values of 1 to peptide P14-9K are 0.017 ⁇ 0.009, 0.14 ⁇ 0.09, 0.024 ⁇ 0.016, 0.22 ⁇ 0.10, 0.001 ⁇ 0.001, 0.083 ⁇ 0.048, 0.53 ⁇ 0.25, 0.019 ⁇ 0.009, 0.47 ⁇ 0.16, respectively.
- the blocking experiment detects the ability of P1 and its series of peptides to block the binding of hPD-1/hPD-L1.
- the specific methods are as follows:
- Collect cells Collect CHO-K1-hPD-1 cells in good condition, centrifuge at 4000 rpm and 4°C, wash twice, and place on ice for later use;
- Flow cytometry Add 200 ⁇ L FACS buffer to each tube to resuspend the cells, and transfer to the flow tube in the dark to prepare for flow cytometry.
- each peptide can block hPD-1/hPD-L1 binding.
- the blocking rate of peptide P1 is 45%.
- the blocking rate is defined as 100%, which is the same
- the relative blocking rate measured by batch experiment testing P2-P13 is shown in Figure 1.
- the blocking rate of peptide P1 is still defined as 100%, which is the same batch
- the relative blocking rate measured by the experimental detection of peptides P14-P20 is shown in Figure 2; when the concentration of peptides P14 and P21-P28 is set to 100 ⁇ M, the relative blocking rate measured by the same batch of experimental detection of peptides P14 and P21-P28 is as follows: As shown in Figure 3; when the concentration of peptide P14, peptide P14-1 to peptide P14-9 is set to 100 ⁇ M, the relative blocking rate measured by the same batch of experiments for peptide P14, peptide P14-1 to peptide P14-9 is shown in Figure 4 Shown.
- Tumor-bearing mice have a tumor volume of 50-100 mm 3 and are divided into 2 groups according to the "S" pattern.
- the specific grouping and administration are as follows:
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Abstract
提供了一种PD-1亲和肽或其修饰肽,修饰肽为丙氨酸扫描肽、点突变肽或截短肽。本发明的肽在亲和PD-1的同时还能够阻断PD-1/PD-L1之间的相互作用,能用于抗肿瘤或治疗其它疾病。
Description
本发明属于生物制药技术领域。
程序性死亡蛋白-1(Programmed death-1,PD-1)最初是由TasukuHonj从凋亡杂交瘤中发现并克隆得到的,也称为CD279,属于免疫球蛋白CD28超家族。PD-1主要表达在活化的T细胞、B细胞以及NK细胞表面,是一种活化型T细胞表面分子。PD-1的配体PD-L1(CD274)和PD-L2(CD273),都属于CD28/B7家族。阻断PD-1/PD-L1的信号通路可以阻断负性调控信号,在一定程度上恢复T细胞的功能,杀伤肿瘤细胞,达到抗肿瘤的目的。
目前靶向PD-1的上市药物都为单克隆抗体,虽然取得了非常好的疗效,但是抗体类药物存在着价格昂贵、免疫原性强、肿瘤浸润性差、半衰期长、免疫不良反应发生时不能及时终止去除等缺点,因此开发其它类型的阻断剂成为目前研究的热点。
多肽类药物一般只有一级和二级结构,没有更复杂的立体结构。与传统化疗药物和抗体相比,多肽类药物具有分子量小、渗透性强、免疫原性低、价格低廉等优点,使得多肽类药物在抗肿瘤研究方面受到了极大的关注。因此,开发更安全、更有效的多肽阻断剂具有深远的临床研究价值。
发明内容:
第一方面,本发明涉及一种亲和PD-1的亲和肽,其为肽P1或P1肽的修饰肽,肽P1氨基酸序列如SEQ ID NO.1所示,所述修饰肽选自下述肽a、b或c所定义的肽或其组合:
肽a:肽P1的丙氨酸扫描肽,氨基酸序列独立选自SEQ ID NOs:2、3、4、5、6、7、8、9、10、11、12或13(NOs在本领域表示序列号的并列列举,即氨基酸序列分别为SEQ ID NO.2、SEQ ID NO.3……);
肽b:肽P1的点突变肽,其在肽P1的第1、4、5、8、9和/或10位氨基酸发生点突变;
肽c:肽P14的C端截短肽,肽P14为肽P1第4位的氨基酸R突变为Y后的点突变肽,所述截短肽的氨基酸数目为3、4、5、6、7、8、9、11或12个;
所述亲和肽的各氨基酸的构型独立地选自D型或L型,如,各氨基酸均为D型或L型。
C端截短肽,在本领域指从蛋白母体的C端截去1至多个氨基酸后得到的一系列更短的肽,如SEQ ID NO.21所示肽。
任选地,肽b中所述点突变独立选自如下突变:D突变为Y或N;F突变为R;R突变为Y;V突变为Y或W;P突变为W;N突变为D或W。
任选地,肽b为肽P1的1个氨基酸发生点突变所得到的肽。
任选地,所述亲和肽的氨基酸序列独立选自SEQ ID NOs:1-28(NOs:1-28为上述表示并列列举的NOs形式的简略形式,包含的序列号为1-28之间的任一个 整数)。
任选地,所述亲和肽N端的1、2或3个氨基酸为D型,和/或C端的1或2个氨基酸为D型;需说明的是,本文中,当限定某氨基酸为D型而未指明其他氨基酸构型时,均默认其他氨基酸为L型,作为本发明的详细技术方案示例,序列如下:dDFYVWWPNFPR、ddFYVWWPNFPR、ddfYVWWPNFPR、dDFYVWWPNFPr、ddFYVWWPNFPr、dDFYVWWPNFpr、ddFYVWWPNFpr、ddfYVWWPNFPr或ddfYVWWPNFpr,小写字母代表对应氨基酸的D型,比如Asp
D-Asp
D-Phe
D-Tyr-Val-Trp-Trp-Pro-Asn-Phe-Pro
D-Arg
D,缩写为d-d-f-Y-V-W-W-P-N-F-p-r。
第二方面,本发明提供了含前述第一方面所述亲和肽的药物组合物或试剂盒。
第三方面,本发明提供了前述第一方面所述亲和肽在制备药物/药物组合物或试剂盒中的应用,在制备成药物的过程中,亲和肽,可以游离形式存在,也可以其药学上可接受的盐的形式存在,也可对所述亲和肽进行化学修饰以延长半衰期,比如环化修饰、乙酰化修饰、PAS修饰、PEG修饰、脂肪酸修饰、白蛋白修饰、白蛋白亲和肽偶联、肿瘤归巢肽偶联、穿膜肽偶联、纳米载体偶联;基于本专利思路的简单变形,均构成对本专利的等同侵权。
第二方面或第三方面所述的药物组合物可包含药学上可接受的赋形剂,可用于下述至少一种用途:
1)抗肿瘤,如实体瘤-结肠癌
2)治疗细菌、病毒或真菌引起的感染
3)治疗自身免疫性疾病
4)阻断PD-1蛋白与PD-L1配体结合。
本发明的PD-L1是指哺乳动物PD-1蛋白的配体,如人PD-L1(hPD-L1)或小鼠的。PD-1与PD-L1可为野生型或仍保留其活性的突变型蛋白,比如本申请人专利CN108794619中的野生型或突变型PD-1。
另需注意的是,本专利的亲和肽,可以游离形式存在,也可以其药学上可接受的盐的形式存在,基于本专利思路的简单变形,均构成对本专利的等同侵权。
本发明筛选获得了PD-1亲和肽,这些肽均可阻断PD-1/PD-L1的结合。高阻断率肽可显著抑制小鼠CT26结肠癌,在肿瘤治疗及自身免疫性疾病、炎性疾病等方面均具有良好的应用前景。
图1为肽P2-P13在200μM阻断PD-1/PD-L1蛋白结合与200μM肽P1的相对阻断率的实验结果图;
图2为肽P1、P14-P20的相对阻断率的实验结果图;
图3为肽P14、P21-P28的相对阻断率的实验结果图;
图4为P14、肽P14-1至肽P14-9的相对阻断率的实验结果图;
图5为修饰肽P14-8 0.5mg/kg给药后小鼠瘤体积图,显著性分析标识**表示P<0.01。
对PD-1的亲和肽的制备过程简要说明如下:
噬菌体展示肽库液相筛选得到母体肽P1,筛选过程如下:
1.以真核蛋白rhPD-1/Fc为靶标,采用液相差相筛选法进行噬菌体展示十二肽库的筛选工作;
2.经过几轮的筛选后,与靶蛋白rhPD-1/Fc有亲和力的噬菌体单克隆逐轮得到富集;
3.从中挑选阳性克隆进行测序,共得到多个***十二肽序列,其中多个具有重复克隆,母体肽P1为阳性克隆之一,序列为DDFRVWWPNFPR,氨基酸均为L型。
基于上述筛选获得的多肽P1,进行标准的Fmoc固相合成得到其修饰肽P2-P28、P14-1至P14-8,其中P2-P28的序列依次如SEQ ID NO.2-SEQ ID NO.28所示,且各氨基酸均为L型;P14-1至P14-9的序列均如SEQ ID NO.14所示,其中部分氨基酸为D型,具体依次为:dDFYVWWPNFPR、ddFYVWWPNFPR、ddfYVWWPNFPR、dDFYVWWPNFPr、ddFYVWWPNFPr、dDFYVWWPNFpr、ddFYVWWPNFpr、ddfYVWWPNFpr、ddfYVWWPNFPr;字母小写代表对应氨基酸为D型。
质谱鉴定无误后,后续进行亲和实验、阻断实验检测多肽的亲和及阻断能力,并检测肽的稳定性、进一步确证其抗肿瘤效果。
1、微量热涌动(MST)检测本发明亲和肽与hPD-L1亲和力,检测过程如下:
1.标记蛋白:染料使用前用PBS(pH=7.4)稀释至被标记蛋白浓度的三倍,将染料与蛋白按体积比1:1混合(100μL浓度为10μM的蛋白,100μL浓度为30μM的染料),室温,避光孵育30min;
2.冲洗柱子:将蛋白分离柱子放入15mL离心管中,用MST Buffer冲洗(一般冲洗10个柱体积,柱体积为500μL);
3.分离纯化蛋白:将步骤1中的蛋白染料混合液(200μL)悬空滴加到蛋白分离柱中,然后加入300μL的MST Buffer,待柱子不再滴出液体时,再加入500μL MST Buffer,从第一滴液体开始收集,收集完成后,上机检测荧光,并将蛋白荧光值稀释至600左右,分装后-80℃保存;
4.冲洗柱子:用MST Buffer冲洗至少10个柱体积,最后将柱子保存至20%酒精中;
5.样品准备:将多肽溶解至适当浓度(10-200μM),进行15次倍比稀释,得到16个浓度梯度样品,体系为10μL。每管加入10μL标记好的蛋白样品,混合均匀后离心排除气泡。冰上孵育5min后用MST专用毛细管吸取孵育后的液体,放置于仪器托;
6.上机检测:打开电脑启动MO.Control软件,选择(Red)通道Binding Affinity模式进行检测;
7.分析结果:使用NanoTemper分析软件MO.Affinity Analysis v2.2.4计算结合解离常数(K
D值,单位μM):
经计算,肽P1K
D=5.30±2.84,肽P14-P20K
D值依次为0.19±0.14、0.028±0.014、0.80±0.37、1.27±0.57、0.38±0.27、5.07±3.28、4.23±2.93,肽P14-1至肽P14-9K
D值依次为0.017±0.009、0.14±0.09、0.024±0.016、0.22±0.10、0.001±0.001、0.083±0.048、0.53±0.25、0.019±0.009、0.47±0.16。
2、阻断实验检测P1及其同系列多肽阻断hPD-1/hPD-L1结合的能力,具体方法 如下:
1.收细胞:收集状态良好CHO-K1-hPD-1细胞,4000rpm 4℃离心洗涤两次,置于冰上备用;
2.孵肽:将步骤1中的细胞按5×10
5分至不同的1.5mL EP管中,实验组加入25μL多肽阻断剂(根据需要设置浓度,如肽P28、肽P14-1至肽P14-9浓度设为100μM)冰上孵育30min;设置蛋白管和阴性对照管,蛋白管和阴性对照管加入25μL PBS(pH=7.4);
3.孵蛋白:加入hPD-L1-Fc蛋白25μL(50ng)冰上孵育30min,阴性对照管加入25μL PBS(pH=7.4);
4.孵育二抗:加入10μL体系的Anti-Human IgG Fc PE荧光二抗,冰上避光孵育30min;
5.洗涤:1mL PBS(pH=7.4)4000rpm 4℃离心洗涤;
6.流式检测:每管中加200μL FACS buffer重悬细胞,避光转移到流式套管中,准备流式检测。
经测算,各肽均能阻断hPD-1/hPD-L1结合,肽P1-P13实验浓度设为200μM时,肽P1阻断率为45%,将其阻断率定义为100%,其同批次实验检测P2-P13测算得的相对阻断率如图1所示;肽P1、肽P14-P20实验浓度设为100μM时,仍将肽P1阻断率定义为100%,其同批次实验检测肽P14-P20测算得的相对阻断率如图2所示;肽P14、P21-P28浓度设为100μM时,同批次实验检测肽P14、P21-P28测算得的相对阻断率如图3所示;肽P14、肽P14-1至肽P14-9浓度设为100μM时,同批次实验检测肽P14、肽P14-1至肽P14-9测算得的相对阻断率如图4所示。
3、亲和肽酶降解稳定性实验
a)将代表性亲和肽用PBS(pH=7.4)溶解至100μM得母液,将100μL的10%的人血清溶液与900μL上述肽的母液迅速混匀,取出80μL混合液计时为0h,其余置于37℃培养箱进行酶解反应,并分别于之后的0.25h、0.5h、1h、2h、4h、8h、24h、48h取出80μL反应产物于EP管中,用于各个时间点酶解情况检测;
b)向上述不同时间点取出的样品中用移液枪加入90μL的乙腈,立即震荡混合均匀后,放置于冰上10min后移液枪加入90μL 0.5%的冰乙酸溶液以终止该酶解反应;
c)4℃预冷离心机,15000g离心15min后收集上清于新的EP管中,用于后续RP-HPLC分析。
酶降解稳定性实验表明,肽P14-2、肽P14-3、肽P14-7、肽P14-8等均比肽P14稳定性提高,其中肽P14-8在48h仍稳定存在,抗酶解能力显著。
4、为进一步展示亲和肽的抗癌等活性,在CT26结肠癌移植瘤模型以P14-8探究抗肿瘤效果,具体实施方法如下:
1.荷瘤
提前将小鼠的右侧背部剃毛等待荷瘤。收集生长状态良好的CT26单细胞悬液,调节细胞密度至1×10
6cells/mL,置于冰上。消毒荷瘤部位后,用1mL注射器吸取200μL CT26单细胞悬液(1×10
5cells)皮下荷瘤。
2.荷瘤小鼠分组及给药
荷瘤小鼠瘤体积至50-100mm
3,按“S”型方式分2组,具体分组及给药情况如下所示:
给药14天,期间小鼠自由进食,隔天称量小鼠体重,测量小鼠的瘤体积(V=1/2×a(长)×b(宽)×c(高)),记录并绘制小鼠瘤体积图,如图5所示。
Claims (10)
- PD-1蛋白的亲和肽,其为肽P1或其修饰肽,肽P1氨基酸序列如SEQ ID NO.1所示,所述修饰肽选自下述肽a、b或c所定义的肽或其组合:肽a:肽P1的丙氨酸扫描肽,氨基酸序列独立选自SEQ ID NOs:2-13;肽b:肽P1的点突变肽,其在肽P1的第1、4、5、8、9和/或10位氨基酸发生点突变;肽c:肽P14的C端截短肽,肽P14为肽P1第4位的氨基酸R突变为Y后的点突变肽,所述截短肽的氨基酸数目为3、4、5、6、7、8、9、11或12个;所述亲和肽的各氨基酸的构型独立地选自D型或L型,如,各氨基酸均为D型或L型。
- 如权利要求1所述的亲和肽,其特征是,肽b中所述点突变独立选自如下突变:D突变为Y或N;F突变为R;R突变为Y;V突变为Y或W;P突变为W;N突变为D或W。
- 如权利要求1或2所述的亲和肽,其特征是,肽b为肽P1的1个氨基酸发生点突变所得到的肽。
- 如权利要求1所述的亲和肽,其特征是,所述亲和肽的氨基酸序列独立选自SEQ ID NOs:1-28。
- 如任一在先权利要求所述的亲和肽,其特征是,所述亲和肽N端的1、2或3个氨基酸为D型。
- 如任一在先权利要求所述的亲和肽,其特征是,所述亲和肽C端的1或2个氨基酸为D型。
- 如权利要求1所述的亲和肽,其特征是,所述亲和肽的氨基酸序列为dDFYVWWPNFPR、ddFYVWWPNFPR、ddfYVWWPNFPR、dDFYVWWPNFPr、ddFYVWWPNFPr、dDFYVWWPNFpr、ddFYVWWPNFpr、ddfYVWWPNFPr或ddfYVWWPNFpr,字母小写代表对应氨基酸为D型。
- 含任一在先权利要求所述亲和肽的药物组合物或试剂盒。
- 任一在先权利要求所述亲和肽在制备药物或试剂盒中的应用。
- 如权利要求9所述的应用,其特征是,所述药物用于下述至少一种用途:1)抗肿瘤,如实体瘤,优选结肠癌2)治疗细菌、病毒或真菌引起的感染3)治疗自身免疫性疾病4)阻断PD-1蛋白与PD-L1配体结合;所述PD-1可为人源的野生型或仍保留其活性的突变型蛋白。
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Publication number | Priority date | Publication date | Assignee | Title |
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CN103897036A (zh) * | 2014-03-24 | 2014-07-02 | 郑州大学 | 一种pd-1蛋白胞外段亲和肽l8及其应用 |
CN104086627A (zh) * | 2014-05-29 | 2014-10-08 | 郑州大学 | 具有抗肿瘤活性的PD-L1 IgV亲和肽S10 |
CN105504018A (zh) * | 2016-01-13 | 2016-04-20 | 郑州大学 | Lag-3亲和肽n13、制备方法及其应用 |
CN108727470A (zh) * | 2017-04-17 | 2018-11-02 | 上海大学 | 一种多肽及其应用 |
CN110087672A (zh) * | 2016-07-29 | 2019-08-02 | 朱诺治疗学股份有限公司 | 免疫调节多肽及相关组合物和方法 |
CN110330550A (zh) * | 2019-08-02 | 2019-10-15 | 郑州大学 | PD-L1-IgV的亲和肽及其应用 |
CN111153961A (zh) * | 2020-01-08 | 2020-05-15 | 郑州大学 | 一种亲和pd-1的肽及其应用 |
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Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103897036A (zh) * | 2014-03-24 | 2014-07-02 | 郑州大学 | 一种pd-1蛋白胞外段亲和肽l8及其应用 |
CN104086627A (zh) * | 2014-05-29 | 2014-10-08 | 郑州大学 | 具有抗肿瘤活性的PD-L1 IgV亲和肽S10 |
CN105504018A (zh) * | 2016-01-13 | 2016-04-20 | 郑州大学 | Lag-3亲和肽n13、制备方法及其应用 |
CN110087672A (zh) * | 2016-07-29 | 2019-08-02 | 朱诺治疗学股份有限公司 | 免疫调节多肽及相关组合物和方法 |
CN108727470A (zh) * | 2017-04-17 | 2018-11-02 | 上海大学 | 一种多肽及其应用 |
CN110330550A (zh) * | 2019-08-02 | 2019-10-15 | 郑州大学 | PD-L1-IgV的亲和肽及其应用 |
CN111153961A (zh) * | 2020-01-08 | 2020-05-15 | 郑州大学 | 一种亲和pd-1的肽及其应用 |
Non-Patent Citations (1)
Title |
---|
HAO-NAN CHANG, LIU BEI-YUAN, QI YUN-KUN, ZHOU YANG, CHEN YAN-PING, PAN KAI-MAI, LI WEN-WEN, ZHOU XIU-MAN, MA WEI-WEI, FU CAI-YUN, : "Blocking of the PD-1/PD-L1 interaction by a D-peptide antagonist for cancer immunotherapy", ANGEWANDTE CHEMIE INTERNATIONAL EDITION, vol. 54, no. 40, 28 September 2015 (2015-09-28), pages 11760 - 11764, XP055247320, ISSN: 1433-7851, DOI: 10.1002/anie.201506225 * |
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