CN110330550A - The affinity peptide of PD-L1-IgV and its application - Google Patents

The affinity peptide of PD-L1-IgV and its application Download PDF

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CN110330550A
CN110330550A CN201910711326.8A CN201910711326A CN110330550A CN 110330550 A CN110330550 A CN 110330550A CN 201910711326 A CN201910711326 A CN 201910711326A CN 110330550 A CN110330550 A CN 110330550A
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affinity
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artificial sequence
glu
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CN110330550B (en
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高艳锋
李琬琼
翟文杰
周秀曼
祁元明
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Zhengzhou University
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Abstract

The present invention relates to the affinity peptide of PD-L1-IgV a kind of and its applications.The affinity peptide is selected from peptide defined in following peptide a, b or c or combinations thereof: peptide a: amino acid sequence is selected from SEQ ID NOs:1,2,3 or 5；Polypeptide shown in peptide b:SEQ ID NO.4 or the mutant peptide of its 4th, the 5th, the 10th, and/or the 11st generation point mutation；Polypeptide shown in peptide c:SEQ ID NO.8, alanine scan peptide or its N-terminal or C-terminal truncated peptide, and the amino acid number of the truncated peptide is 4,5,6,7,8,9,10 or 11 or the alanine of the truncated peptide scans peptide.The present invention is off the beaten track, and the peptide obtained by repeated screening, optimization can preferably block the interaction between PD-1/PD-L1, to treat tumour or other disease types.

Description

The affinity peptide of PD-L1-IgV and its application
Technical field:
The invention belongs to biopharmaceutical technologies, and in particular to the peptide of a kind of affine PD-L1-IgV and its in tumour etc. Application in terms of related disease etc..
Background technique:
Tumour is to seriously threaten the major disease of human health, and tumour has heterogeneity, diversity and the changeability of height, Understanding tumor development mechanism and searching treatment oncology tools are still the huge challenge that scientists face.Operation, radiotherapy It is method more universal used by traditional tumour treatment with chemotherapy, but can not after excision naked eyes visual tumors of performing the operation Tumour is completely eliminated, radiation and chemotherapy then has non-specificity, i.e., cannot be targeted to tumor locus, is also easy to produce more tight The side effect of weight.Compared with above-mentioned three kinds of methods, the immunization therapy of tumour can activate well the immune system of body to make it Specific killing tumour, and make body immunocyte generate immunological memory, so immunotherapy can effectively prevent tumor recurrence and Transfer, and side effect is smaller." science " magazine in 2013 is classified as the immunization therapy of tumour first of the breakthrough of ten big sciences, and tumour is exempted from Epidemic disease cure gradually becomes the 4th kind of important therapy after three great tradition treatment methods.The immunization therapy of tumour is scientific circles A big hot spot, Nobel prize's soul in 2018 also authorizes two immunologists in this field, to commend them " it was found that contribution in terms of the therapy of negative immune adjustment for the treatment of cancer ".
T cell is the core of body immune system anti-tumor immune response, its activation needs dual signal: the first signal is The compound of surface receptor TCR the identification Antigenic Peptide and MHC molecule of T lymphocyte；Second signal is the corresponding receptor in T cell surface TCR is combined with costimulatory molecules on antigen presenting cell.Immunologic test point can be believed by balance costimulation and co-suppression Number T cell immune response strength is controlled, immunocyte is expressed in tumor microenvironment inhibition molecule such as PD-1, CTLA-4, TIGIT, LAG-3, TIM-3 etc., by interacting with T cell or antigen presenting cell surface ligand, to make T cell Function is exhausted, is inhibited its antitumor action, is caused the generation of immunologic escape.Block PD-1, CTLA-4, TIGIT, LAG-3, TIM- 3 equal inhibitions molecules can reactivate the tumor specific cytotoxic T lymphocyte in tumor microenvironment, and the tumour for breaking body foundation is exempted from Epidemic disease tolerance brings hope for oncotherapy.
PD-L1 is the major ligand of PD-1, and tumour cell can express PD-L1 by height and lymphocyte function is caused to exhaust And the immunologic escape of mediate tumor, but also go forward side by side at present some researches show that tumour cell also can secrete PD-L1 in the form of excretion body Enter lymph node and inhibit T cell function, clinical data shows that becoming for negative correlation is presented in the prognosis of PD-L1 expression and tumor patient Gesture.Also there is data to show that PD-L1 can be combined with B7-1, inhibit B7-1/CD28 access, fall into T lymphocyte and exempt from Epidemic disease holddown causes the reduced capability of its proliferative capacity and the lethal cell factor of secretion, it is believed that the suppression that PD-L1 is mediated Signal processed is two-way.Under normal condition, PD-1/PD-L1 is the excessive activation and autoimmune disease for avoiding immune system Occur, T lymphocyte proliferative capacity can be made to weaken, the cytokine secretions such as IL-2 and IFN-γ are reduced.Studies have found that individually It blocks PD-1/PD-L1 access to can be increased the ratio of tumor infiltrating T lymphocyte, improves the secretion level of its IFN-γ, together When, marrow source property inhibition percentage of lymphocyte is significantly reduced, and the function of T lymphocyte is restored and is promoted.
In addition, PD-1/PD-L1 usually participates in inducing T cell tolerance, except above-mentioned the answering in tumor area being widely noticed With, which is also constantly in progress in autoimmune disease, the research of virus and bacterium infection field and treatment use, therefore On the basis of PD-1/PD-L1 access, find rationally effective therapeutic strategy be also scientists facing with it is urgently to be resolved Problem.
It has been listed at present for the monoclonal antibody of PD-1/PD-L1 access, applied to the immunization therapy of tumour, but it is anti- Body class drug high production cost, tissue permeability be poor, long half time, cannot terminate immune adverse events quickly；Small point of polypeptide etc. Sub- pharmaceutical synthesis is convenient, tissue permeability is good, immunogenicity is relatively low, has preferable Development volue and application prospect.
Summary of the invention:
The present invention is off the beaten track, obtains the peptide of affine PD-L1-IgV a kind of by repeated screening, optimization, and by experiment The affinity peptide is demonstrated with affine PD-L1, the activity for blocking PD-1/PD-L1 to combine, tumour or other type diseases can be treated Disease.In a first aspect, the present invention provides the affinity peptide of PD-L1-IgV a kind of, it is selected from peptide or its group defined in following peptide a, b, c It closes:
Peptide a, amino acid sequence are selected from (arranged side by side column of the NOs in ability domain representation sequence number of SEQ ID NOs:1,2,3 or 5 It lifts, i.e., amino acid sequence is respectively SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.5)；
Polypeptide shown in peptide b, SEQ ID NO.4 or its 4th, the 5th, the 10th, and/or the 11st amino acids hair The mutant peptide of raw point mutation；
Polypeptide shown in peptide c, SEQ ID NO.8, alanine scan peptide or its N-terminal or C-terminal truncated peptide, the truncated peptide Amino acid number be that the alanine of 4,5,6,7,8,9,10 or 11 or the truncated peptide scans peptide.
As peptide c can be divided into:
Peptide c1 is that polypeptide shown in SEQ ID NO.8 or its alanine scan peptide；
Peptide c2 is polypeptide or its N-terminal or C-terminal truncated peptide shown in SEQ ID NO.8, the amino acid number of the truncated peptide It is 4,5,6,7,8,9,10 or 11；
Peptide c3 is that polypeptide shown in SEQ ID NO.32 or its alanine scan peptide.
The partial peptide of peptide c1 and peptide c2 can be combined into peptide c4:Arg-Val-Tyr-Ser-Phe and its N-terminal extends 7 amino 12 peptides of acid, 1 alanine is contained in this 7 amino acid of 12 peptides, and such as sequence is Gly-Gln-Ser-Glu-His-His- Ala-Arg-Val-Tyr-Ser-Phe。
Alanine scans peptide, refers to obtain any one amino acid substitution of albumen parent for alanine one in this field Serial single mutation peptide, such as a series of alanine of polypeptide shown in peptide c SEQ ID NO.8 scan peptide, and sequence is SEQ ID NO.15、SEQ ID NO.16…
Truncated peptide refers to that 1 is clipped from the N-terminal or C-terminal of albumen parent is a series of to obtaining after multiple amino acid in this field Shorter peptide, a series of N-terminal truncated peptides of the polypeptide as shown in peptide c SEQ ID NO.8, sequence are SEQ ID NO.26, SEQ ID NO.27……
Optionally, the point mutation is independently selected from following mutation:
1) Glu sports Gln, Asn, Asp；
2) His sports Gln, Glu；
3) Ala sports Trp, Phe, Tyr, His, Ile, Gln, Glu；
4) Ser sports Arg.
Optionally, the affinity peptide is the single-site mutant peptide of polypeptide shown in SEQ ID NO.4, i.e. above-mentioned the 1 of parent peptide Locate site (such as the 4th) and amino acid mutation occurs.
Optionally, the truncated peptide is the N-terminal truncated peptide of polypeptide shown in SEQ ID NO.8, amino acid number 5,6, 7,8,9,10 or 11.
Optionally, the sequence of the affinity peptide be selected from SEQ ID NOs:4,6,7,8,9,10,11,12,13,14,15,16, 17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36 or 37,38.
Optionally, the configuration of the amino acid of the affinity peptide can be D type or L-type, such as ammonia independently selected from D type or L-type Base acid is D configuration.
PD-L1 of the invention refers to the ligand of mammal PD-1 albumen, such as human PD-L 1 (hPD-L1) or mouse, PD- L1-IgV is the IgV spline structure domain of PD-L1.PD-1 and PD-L1 can retain for wild type or still its active mutein, than Such as the wild type or saltant type PD-1 in the first patent CN108794619 of the applicant.
It, can also be with its pharmaceutically acceptable salt separately it is noted that the affinity peptide of this patent, can exist in a free form Form exist, based on the simple deformation of this patent thinking, constitute the equivalent infringement to this patent.
Second aspect, the present invention provides pharmaceutical compositions or kit containing affinity peptide described in aforementioned first aspect.
The third aspect, the present invention provides affinity peptides described in aforementioned first aspect in preparation pharmaceutical composition or kit Application.
Pharmaceutical composition described in second aspect or the third aspect may include pharmaceutically acceptable excipient, under can be used for State at least one purposes:
1) antitumor, such as colon cancer or melanoma
2) bacterium, virus or fungus-caused infection are treated
3) autoimmune disease is treated
4) PD-1 albumen and PD-L1 ligand binding are blocked；The PD-1 and PD-L1 can be source of people or the wild type of small source of mouse Or still retain its active mutein.
Polypeptide of the invention can be made by synthesis in solid state, such as be prepared using Fmoc scheme.
The invention has the advantages that:
The present invention is off the beaten track, obtains PD-L1-IgV affinity peptide by repeated screening, optimization, affine blocking activity is real Verifying is real, these peptides can block the combination of PD-1/PD-L1.It is antitumor in further external affinity experiment and Mice Body Experimental verification, high blocking rate peptide can significantly inhibit the growth of mouse CT26 colon cancer and B16-OVA melanoma, and without obvious poison Side effect.As it can be seen that peptide of the invention oncotherapy and in terms of all have good application prospect.
Detailed description of the invention:
Fig. 1 is that peptide H5S, H7, H9, H12, H14 block the protein bound experimental result picture of PD-1/PD-L1；
Fig. 2 is that the mutant peptide of parent peptide H12 and H12 block the protein bound experimental result picture of PD-1/PD-L1；
Fig. 3 is the result of the horizontal affine experiment of polypeptide cells；
Fig. 4 is the influence result figure of BABL/c mouse weight variation of the peptide of the present invention to inoculation CT26；
Fig. 5 is influence of the peptide of the present invention to the BABL/c mice-transplanted tumor volume change of inoculation CT26；
Fig. 6 is knurl product influence diagram of the peptide of the present invention to the C57BL/6 mouse of inoculation B16-OVA；
The alanine that Fig. 7 is P8 scans peptide and blocks the protein bound experimental result picture of PD-1/PD-L1；
Fig. 8 is that truncated peptide blocks the protein bound experimental result picture of PD-1/PD-L1；
The alanine that Fig. 9 is peptide P32 scans peptide and blocks the protein bound experimental result picture of PD-1/PD-L1；
Significance analysis involved in each figure, which identifies *, indicates that P < 0.05, * * indicate P < 0.01.
Specific embodiment:
Embodiment of the present invention is described in detail below in conjunction with embodiment, but the following example is only used for The bright present invention, without that should be to limit the scope of the invention.
Unless otherwise instructed, reagent used below, biomaterial, culture medium and solution are commonly used in the art, Gong Zhongke To obtain or commercially available article.
Screening of the inventor to the affinity peptide of PD-L1-IgV, each description of test are as follows:
1. bacteriophage mirror image displayed polypeptide library screen in solution obtains parent peptide H12 etc., substantially screening process is as follows:
A) full chemistry synthesizes D-hPD-L1-IgV-biotin, and uses the SA magnetic capture D configuration albumen, using liquid phase The screening operation in screening method progress phage display dodecapeptide library；
B) after the screening excessively taken turns, the bacteriophage monoclonal of affinity obtains by wheel with target protein D-hPD-L1-IgV Enrichment；
C) it therefrom selects positive colony to be sequenced, multiple insertion dodecapeptide sequences (will wherein 1 polypeptide mutant) be obtained, Obtaining linear parent peptide H5S, H7, H9, H12, H14, (successively as shown in SEQ ID NO.1-5, each amino acid configuration closes sequence As D type).
2. blocking experiment:
A) culture CHO-K1-hPD-1 cell is long to logarithm to cell in 1640 culture medium of RMPI containing 10%FBS Phase pancreatin digests and collects cell in 1.5mL EP pipe, and every solencyte amount is 2 × 105, after 1mL PBS7.2 washes twice, set It is spare on ice.
B) antibody (Anti human PD-1 PE) the detection whether stable cell of hPD-1 is expressed in cell membrane first, is used in combination Isotype antibody compares (Mouse IgG1 κ iso control PE), to ensure that the cell can be used in subsequent blocking experiment.
C) polypeptide is incubated for altogether with hPD-L1-Fc: weighing the polypeptide of certain mass, PBS7.2 is dissolved to 100 μM.Take 200 μ L Low adsorption EP pipe, is incubated for 30min for the polypeptide vortex mixed of 100 μM of 50ng hPD-L1-Fc and 50 μ L jointly on ice.
D) said mixture is added in CHO-K1-hPD-1 cell, is vortexed after mixing and is incubated for 30min on ice.
E) it is added in the mixture of antibody (Human Fc γ specific PE) tri- kinds of substances of Yu Shangshu of 0.3 μ L, is vortexed It is protected from light on ice after mixing and is incubated for 30min.
F) wash antibody: 1mL ice-cold PBS7.2 is washed once, and after 1800rpm is centrifuged 5min, 200 μ L PBS7.2 weight is added It is outstanding, it is transferred to flow cytometer detection CHO-K1-hPD-1 cell and hPD-L1-Fc protein binding situation in streaming pipe.
Block the result shows that: polypeptide (H5S, H7, H9, H12, H14) of the invention can block hPD-L1 albumen with The combination of CHO-K1-hPD-1 cell, blocking rate are as shown in Figure 1.
3. carrying out subsequent Optimization Work using H12 as parent peptide, specific implementation method is as follows:
A) the 3D structure of PEPstrMOD on-line prediction H12 peptide: opening PEPstrMOD webpage, the polypeptide predicted needed for inputting Sequence clicks Submit and Go to Next Step.Since the polypeptide is D configuration, so Steriochemistry is changed It for Dextro (D), and clicks and submits after lower section inputs mailbox, polypeptide structure can be downloaded in mailbox after the completion of system prediction.
B) Z-DOCK molecular docking: the H12 peptide structure that above-mentioned prediction is obtained respectively with hPD-L1 (PDB ID:3BIK) into Row molecular docking, molecular docking are docked online using Z-DOCK, MOE analysis docking as a result, selection joint mode (comprehensive bond energy, Distance, interaction site account for), and 50ns molecular dynamics is run using MOE.
C) MOE single-site mutant: the joint mode after selecting above-mentioned molecular dynamics utilizes MOE run unit point amino Result after the completion of mutation is carried out affinity height and sorted by acid mutation, and selection marking higher first nine carry out subsequent mutation The synthesis of peptide, 9 mutant peptides obtained according to Fmoc solid-phase synthesis are respectively designated as P6-P14, and sequence is corresponding successively such as SEQ Shown in ID NO.6-SEQ ID NO.14, each amino acid is D type, after Mass Spectrometric Identification is errorless, is blocked according to the experiment 2-of front real It tests, detects mutant peptide blocking ability, the results showed that each peptide can block the knot of hPD-L1 albumen Yu CHO-K1-hPD-1 cell It closes, blocking rate is as shown in Figure 2.In addition, by taking peptide P8 as an example, the experiment that block mouse mPD-L1 referring to experiment 2 (by albumen and Cell correspondence replaces with source of mouse), the results showed that P8 can also block mPD-1/mPD-L1 to combine, and blocking rate is 30%.
4. micro heat springs up (MST) detection polypeptide P8 and PD-L1 affinity, detection process are as follows:
A) (theoretical: take 100 μ L concentration is 10 μM of albumen to NT647 dye marker hPD-L1-His albumen, and it is dense that 50 μ L are added Degree is in 20 μM of dyestuff).Testing hPD-L1-His protein concentration used is 7.14 μM.First be 20 μM by concentration, volume is After the NT647 dyestuff and albumen of 50 μ L is sufficiently mixed uniformly, room temperature is protected from light 30min.It is tried using NT.115TM protein labeling After agent box RED carries out the label of target protein hPD-L1-His, dyestuff albumen composition can be effectively formed.Label reaction is completed Later, unreacted dyestuff is removed using molecular sieve, in favor of going on smoothly for subsequent experimental.
B) column equilibration: carrying out the balance of chromatographic column during above-mentioned dye marker, and PBS7.2 balances 3 column volumes and is Can, pay attention to keeping pillar wet during column equilibration.
C) chromatographic column protein isolate: after albumen is protected from light with dyestuff and sufficiently to react 30min, said mixture is added pre- In the chromatographic column first balanced, the PBS7.2 of 400 μ L is added, when it is fully infiltrated into pillar when above two liquid, is added 600 μ L PBS7.2 simultaneously start to collect the liquid that pillar oozes, and every drop is collected into a new EP pipe (if molecular weight of albumen It is smaller, it may not be necessary to collect 3 drops for most starting to ooze), it collects to pillar and no longer oozes liquid.It collects and completes postcapillary absorption less Quantity of fluid, MST detection labeling effciency simultaneously rinse pillar well, and 4 DEG C are stored in 20% alcoholic solution.According to specific experiments The albumen marked suitably dilute by fluorescence intensity and protein concentration used.
D) sample to be tested prepares: electronic balance weighs appropriate polypeptide and dissolves, by polypeptide from 100 μM of 2 times of dilutions down, altogether After the dilution for carrying out 16 gradients, mixed that (10 μ L target proteins add 10 μ L more in equal volume with the fluorescin of above-mentioned label Peptide), it is placed on sample carrier after drawing 10 μ L said mixtures with capillary according to corresponding position and carries out next step analysis.
E) detect it is affine: on instrumentation interface, click start scan, detect every pipe fluorescence intensity whether one It causes, if consistency is preferable, clicks start MST measurement and start to measure.
F) experimental data is analyzed and organized.
To human PD-L 1 the experimental results showed that, linear D peptide P8 can be good at affine hPD-L1 albumen (in conjunction with PD-L1 Value=0.9 μM affinity Kd), and have with positive ligandin hPD-1 (value=0.4 μM Kd) affinity of peer-level. The affinity experiment (albumen and cell correspondence are replaced with into source of mouse) to mouse mPD-L1 is carried out referring to above-mentioned experiment, obtains similar knot Fruit: P8 also can affinity mPD-L1 (Kd=3.78 μM) very well, and have with mPD-1 affinity (the Kd=4.04 μ of peer-level M)。
5. the horizontal affine experiment of polypeptide cells
By high activity peptide P8 that fluorescent dye Cy5.5-NHS is synthesized with standard Fmoc solid phase, (C-terminal is connected to Rink tree Rouge, N-terminal exposure) after amino coupled, after purifying preparation, by fluorescent peptide P8 in PBS7.2 with 10 μM of its maxima solubility respectively with CHO-K1, CHO-K1-hPD-L1, CHO-K1-mPD-L1 are incubated for altogether, and can the flow cytomery mutant peptide specific affine HPD-L1 and mPD-L1, affinity rate statistical result are as shown in Figure 3.
6. probing into the antitumor of P8 respectively in CT26 colon cancer Transplanted tumor model and B16-OVA melanin Transplanted tumor model Effect, specific implementation method are as follows:
A) lotus knurl: the good CT26 colorectal cancer cell of growth conditions and B16-OVA melanoma cells are collected, respectively lotus Tumor BABL/c mouse (2 × 105Cells/ is only) and C57BL/6 mouse (5 × 105Cells/ is only).
B) to lotus knurl one week or so, mouse tumor volume is about grown to 40-80mm3When start S-shaped grouping and daily intraperitoneal administration, Successive administration 2 weeks, mouse tumor is measured using electronic balance weighing mouse weight, electronic digital indicator every other day during administration, is pressed It is calculated according to formula V=1/2 × a (length) × b (width) × c (height) and records mouse tumor volume change；
The product curve of knurl shown in Fig. 5 and Fig. 6 shows that P8 can be good at inhibiting under the dosage of 0.5mg/kg The growth of CT26 colorectal cancer xenografts and B16-OVA melanin transplantable tumor, and the result shows that P8 is 0.5mg/kg's shown in Fig. 4 The weight of BABL/c mouse is without substantially reduced phenomenon under dosage.The mouse state of mind is all during the administration of two kinds of Transplanted tumor models It is better.
7. affine peptase stability to degradation experiment
A) peptide P8 PBS7.2 is dissolved to 100 μM of mother liquors, the 10% human serum solution of 190 μ L and 10 μ L is above-mentioned The mother liquor of A10Y mixes rapidly, and 20 μ L mixed liquor timing of taking-up are 0h, remaining is placed in 37 DEG C of incubators and carries out enzyme digestion reaction, and divides Not in later 0.5h, 1h, 2h, 4h, 8h, 16h, for 24 hours, 48h take out 20 μ L reaction products in 1.5mL EP pipe, for each Time point digests situation detection；
B) acetonitrile of 90 μ L, immediately concussion mixing are carefully added into the sample that Xiang Shangshu different time points are taken out with liquid-transfering gun After uniformly, the glacial acetic acid solution of 90 μ L0.5% is added to terminate the enzyme digestion reaction in liquid-transfering gun after being placed in 10min on ice；
C) 4 DEG C of pre-cooling centrifuges, 10000g are centrifuged after 20min and collect supernatant in new EP pipe, are used for subsequent RP- HPLC analysis
The experiment of enzyme stability to degradation shows that polypeptide P8 is still stabilized in 48h, and resistance to enzymolysis ability is significant.
8. peptide P8 is carried out alanine scanning, blocking experiment studies its alanine scanning peptide blocking ability, specific embodiment party Method and above-mentioned experiment 2 are described essentially identical.
Using P8 to the numerical value of the blocking rate of hPD-L1 as 100%, opposite blocking rate of other each peptides with respect to the peptide is calculated, As shown in Figure 7: the alanine scanning Peptide P15-P25 of P8 can block PD-1/PD-L1 protein binding under 100 μM of concentration.
9. peptide P8 is truncated, blocking experiment studies its truncated peptide blocking ability, specific implementation method and above-mentioned experiment 2 It is described essentially identical.
Using P8 to the numerical value of the blocking rate of hPD-L1 as 100%, opposite blocking rate of other each peptides with respect to the peptide is calculated, As shown in Figure 8: the truncated peptide P26-P33 of P8 can block PD-1/PD-L1 protein binding under 100 μM of concentration.
10. peptide P32 is carried out alanine scanning, blocking experiment studies its alanine scanning peptide blocking ability, specific implementation Method and above-mentioned experiment 2 are described essentially identical.
Using P32 to the numerical value of the blocking rate of hPD-L1 as 100%, calculates other each peptides and blocked with respect to the opposite of the peptide Rate, as shown in Figure 9: the alanine scanning peptide P34-38 of P32 (8-12) can block PD-1/PD-L1 albumen under 100 μM of concentration In conjunction with.
Amino acid (amino acid configuration is D type) sequence table of 1 each peptide of the present invention of table
Sequence table
<110>Zhengzhou University
<120>affinity peptide of PD-L1-IgV and its application
<160> 38
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
His Pro Trp Ser Ser Gly Leu Arg Leu Asp Leu Arg
1 5 10
<210> 2
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Ser Ser His Leu Thr Asn Trp Trp Arg Asn Gly Ile
1 5 10
<210> 3
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Val Val Ser Pro Asp Met Asn Leu Leu Leu Thr Asn
1 5 10
<210> 4
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
Gly Gln Ser Glu His His Met Arg Val Ala Ser Phe
1 5 10
<210> 5
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 5
Ala Ser Ser Ala Tyr Leu Lys Ser Met Asp Pro Ala
1 5 10
<210> 6
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 6
Gly Gln Ser Glu His His Met Arg Val Trp Ser Phe
1 5 10
<210> 7
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 7
Gly Gln Ser Glu His His Met Arg Val Phe Ser Phe
1 5 10
<210> 8
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 8
Gly Gln Ser Glu His His Met Arg Val Tyr Ser Phe
1 5 10
<210> 9
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 9
Gly Gln Ser Glu His His Met Arg Val His Ser Phe
1 5 10
<210> 10
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 10
Gly Gln Ser Glu Glu His Met Arg Val Ala Ser Phe
1 5 10
<210> 11
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 11
Gly Gln Ser Glu His His Met Arg Val Ala Arg Phe
1 5 10
<210> 12
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 12
Gly Gln Ser Glu His His Met Arg Val Ile Ser Phe
1 5 10
<210> 13
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 13
Gly Gln Ser Glu His His Met Arg Val Glu Ser Phe
1 5 10
<210> 14
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 14
Gly Gln Ser Asn His His Met Arg Val Ala Ser Phe
1 5 10
<210> 15
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 15
Ala Gln Ser Glu His His Met Arg Val Tyr Ser Phe
1 5 10
<210> 16
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 16
Gly Ala Ser Glu His His Met Arg Val Tyr Ser Phe
1 5 10
<210> 17
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 17
Gly Gln Ala Glu His His Met Arg Val Tyr Ser Phe
1 5 10
<210> 18
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 18
Gly Gln Ser Ala His His Met Arg Val Tyr Ser Phe
1 5 10
<210> 19
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 19
Gly Gln Ser Glu Ala His Met Arg Val Tyr Ser Phe
1 5 10
<210> 20
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 20
Gly Gln Ser Glu His Ala Met Arg Val Tyr Ser Phe
1 5 10
<210> 21
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 21
Gly Gln Ser Glu His His Ala Arg Val Tyr Ser Phe
1 5 10
<210> 22
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 22
Gly Gln Ser Glu His His Met Ala Val Tyr Ser Phe
1 5 10
<210> 23
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 23
Gly Gln Ser Glu His His Met Arg Ala Tyr Ser Phe
1 5 10
<210> 24
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 24
Gly Gln Ser Glu His His Met Arg Val Tyr Ala Phe
1 5 10
<210> 25
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 25
Gly Gln Ser Glu His His Met Arg Val Tyr Ser Ala
1 5 10
<210> 26
<211> 11
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 26
Gln Ser Glu His His Met Arg Val Tyr Ser Phe
1 5 10
<210> 27
<211> 10
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 27
Ser Glu His His Met Arg Val Tyr Ser Phe
1 5 10
<210> 28
<211> 9
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 28
Glu His His Met Arg Val Tyr Ser Phe
1 5
<210> 29
<211> 8
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 29
His His Met Arg Val Tyr Ser Phe
1 5
<210> 30
<211> 7
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 30
His Met Arg Val Tyr Ser Phe
1 5
<210> 31
<211> 6
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 31
Met Arg Val Tyr Ser Phe
1 5
<210> 32
<211> 5
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 32
Arg Val Tyr Ser Phe
1 5
<210> 33
<211> 4
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 33
Val Tyr Ser Phe
1
<210> 34
<211> 5
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 34
Ala Val Tyr Ser Phe
1 5
<210> 35
<211> 5
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 35
Arg Ala Tyr Ser Phe
1 5
<210> 36
<211> 5
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 36
Arg Val Ala Ser Phe
1 5
<210> 37
<211> 5
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 37
Arg Val Tyr Ala Phe
1 5
<210> 38
<211> 5
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 38
Arg Val Tyr Ser Ala
1 5

Claims (10)

  1. The affinity peptide of 1.PD-L1-IgV is selected from peptide defined in following peptide a, b or c or combinations thereof:
    Peptide a: amino acid sequence is selected from SEQ ID NOs:1,2,3 or 5；
    It is prominent that point occurs for polypeptide shown in peptide b:SEQ ID NO.4 or its 4th, the 5th, the 10th, and/or the 11st amino acids The mutant peptide of change；
    Polypeptide shown in peptide c:SEQ ID NO.8, alanine scan peptide or its N-terminal or C-terminal truncated peptide, the ammonia of the truncated peptide Base acid number is 4,5,6,7,8,9,10 or 11 or the alanine of the truncated peptide scans peptide.
  2. 2. affinity peptide as described in claim 1, characterized in that the point mutation is independently selected from following mutation:
    1) Glu sports Gln, Asn or Asp；
    2) His sports Gln or Glu；
    3) Ala sports Trp, Phe, Tyr, His, Ile, Glu or Gln；
    4) Ser sports Arg.
  3. 3. affinity peptide as described in claim 1, characterized in that the point mutation is independently selected from following mutation:
    1) Glu sports Asn；
    2) His sports Glu；
    3) Ala sports Trp, Phe, Tyr, His, Ile or Glu；
    4) Ser sports Arg.
  4. 4. the affinity peptide as described in any first claim, characterized in that the affinity peptide is polypeptide shown in SEQ ID NO.4 Single-site mutant peptide.
  5. 5. affinity peptide as described in claim 1, characterized in that the truncated peptide is that the N-terminal of polypeptide shown in SEQ ID NO.8 is cut Small peptide, amino acid number 5,6,7,8,9,10 or 11.
  6. 6. affinity peptide as described in claim 1, characterized in that the sequence of the affinity peptide be selected from SEQ ID NOs:4,6,7, 8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、 34,35,36,37 or 38.
  7. 7. the affinity peptide as described in any first claim, characterized in that the configuration of each amino acid of the affinity peptide is independent Ground is selected from D type or L-type, and each amino acid can be D type or L-type, and such as each amino acid is D configuration.
  8. 8. pharmaceutical composition or kit containing affinity peptide described in any first claim.
  9. 9. application of the affinity peptide described in any first claim in preparation pharmaceutical composition or kit.
  10. 10. application as claimed in claim 9, characterized in that described pharmaceutical composition is for following at least one purposes:
    1) antitumor, tumour may include colon cancer or melanoma
    2) bacterium, virus or fungus-caused infection are treated
    3) autoimmune disease is treated
    4) PD-1 albumen and PD-L1 ligand binding are blocked；The PD-1 and PD-L1 can be the wild type or still of source of people or small source of mouse Retain its active mutein.
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CN112409450A (en) * 2020-03-29 2021-02-26 郑州大学 Affinity agent of TIGIT-IgV and application thereof
CN112724199A (en) * 2020-12-30 2021-04-30 郑州大学 Polypeptide with affinity Clec9a and application thereof
CN112812174A (en) * 2021-01-15 2021-05-18 新乡学院 Pig PD-L14QN-AF epitope polypeptide and application thereof
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WO2021139761A1 (en) * 2020-01-08 2021-07-15 郑州大学 Peptide having affinity to pd-1 and application thereof
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Publication number Priority date Publication date Assignee Title
WO2021023140A1 (en) * 2019-08-02 2021-02-11 郑州大学 Affinity peptide of pd-l1-igv and application thereof
WO2021116079A1 (en) 2019-12-10 2021-06-17 Université de Mons Peptides binding to ldl-receptor as carriers for crossing the blood brain barrier
WO2021139761A1 (en) * 2020-01-08 2021-07-15 郑州大学 Peptide having affinity to pd-1 and application thereof
CN112409450A (en) * 2020-03-29 2021-02-26 郑州大学 Affinity agent of TIGIT-IgV and application thereof
CN112409450B (en) * 2020-03-29 2023-01-24 郑州大学 Affinity agent of TIGIT-IgV and application thereof
CN112724199A (en) * 2020-12-30 2021-04-30 郑州大学 Polypeptide with affinity Clec9a and application thereof
CN112812174A (en) * 2021-01-15 2021-05-18 新乡学院 Pig PD-L14QN-AF epitope polypeptide and application thereof
CN114044808A (en) * 2021-11-19 2022-02-15 郑州大学 Albumin affinity agent and application thereof
CN114044808B (en) * 2021-11-19 2024-01-30 郑州大学 Albumin affinity agent and application thereof

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