CN114163495A

CN114163495A - Polypeptide targeting PD-L1 and application thereof

Info

Publication number: CN114163495A
Application number: CN202111265100.3A
Authority: CN
Inventors: 胡志远
Original assignee: National Center for Nanosccience and Technology China
Current assignee: National Center for Nanosccience and Technology China
Priority date: 2019-06-04
Filing date: 2019-06-04
Publication date: 2022-03-11
Anticipated expiration: 2039-06-04
Also published as: CN112028969A; CN114163496A; CN114163497A; CN114163498A; CN112028969B; CN114163497B; CN114031670B; CN114031670A; CN114163498B; CN114163495B; CN114163496B

Abstract

The invention relates to the technical field of molecular biology and medicines, in particular to a polypeptide targeting PD-L1 and application thereof. The invention provides a polypeptide targeting PD-L1, and the amino acid sequence of the polypeptide is RYMETWKSDQ. The polypeptide of the targeted PD-L1 has higher affinity and specificity to PD-L1, can specifically and efficiently target PD-L1 positive tumor cells, can be used as a small molecular probe for detecting the expression condition of PD-L1 in the tumor cells in practice or as a targeted polypeptide to be conjugated or mixed with a preparation capable of killing cancer cells, is used for targeted therapy and imaging of various tumors, and has wide application prospect.

Description

Polypeptide targeting PD-L1 and application thereof

Technical Field

The invention is a divisional application of a patent application with the application number of 201910480489.X and the name of 'a polypeptide targeting PD-L1 and a preparation method and application thereof'. The invention relates to the technical field of molecular biology and medicines, in particular to a polypeptide of a targeted programmed death ligand-1 (PD-L1) and a preparation method and application thereof.

Background

With the rapid development and interpenetration of related disciplines such as oncology, immunology, molecular biology and the like, tumor immunotherapy has developed into a new and important anti-tumor therapeutic approach. Unlike previous surgical, chemical, radiation and targeted therapies, tumor immunotherapy combats tumors by activating the body's own immune system. At present, inhibitors against immune checkpoints show better clinical effects in the treatment of various solid tumors, leading people to realize that immunotherapy can really become an important tool for the treatment of malignant tumors. There are a variety of treatment strategies for tumor immunotherapy, including non-specific immunostimulants, tumor vaccines, adoptive immune cell therapy, and monoclonal antibody therapy. Because tumors have great heterogeneity and genetic instability, the pathogenesis of the tumors is complex, and the ideal anti-tumor effect cannot be achieved by depending on one treatment means alone, the anti-tumor strategy combining tumor targeted therapy and different types of immunotherapy is likely to be the development direction of future tumor immunotherapy on the basis of deeply researching the interaction mechanism between different treatment means.

The core of tumor immunotherapy is to activate the anti-tumor reaction of T lymphocytes of tumor patients so as to improve the killing function of the T lymphocytes on tumor cells. T cell mediated cell immunity plays an important role in the process of identifying and killing tumor cells, the T cells have definite targeting and specificity to the tumor cells, and can generate long-range immune response reaction by identifying and gathering to the tumor antigen expression part to directly inhibit and kill the tumor cells. Dendritic Cells (DCs) play a crucial role in T cell recognition. T cells recognize tumor cells by binding to the Major Histocompatibility Complex (MHC) with a specific antigen on the surface of the tumor cells via a T Cell Receptor (TCR). The interaction of TCR and MHC molecules is controlled by a series of immune checkpoints that can activate or inhibit T cells. Among them, the programmed death receptor 1(PD-1) and programmed death ligand-1 (PD-L1) pathways are inhibitory immune checkpoints, and their combination can inhibit the immune activity of T cells, playing an important role in immune tolerance, which is also an important reason for immune escape of tumor cells.

Programmed death ligand-1 (PD-L1, also known as B7-H1) is a ligand of Programmed death factor-1 (PD-1), is firstly found and cloned as a gene in 1992, is a typical negative co-stimulatory molecule, can transcribe and induce Programmed death of T cells, and is one of hot targets in the field of current immunological research. PD-L1 is mainly expressed in antigen presenting cells, B cells, T cells, epithelial cells, muscle cells, endothelial cells and various tumor cells (PD-L1 is expressed on the surface of various human tumor cells, such as lung cancer, melanoma, non-small cell lung cancer, liver cancer, ovarian cancer, esophageal cancer, breast cancer and the like), and participates in tumor-related immune response. PD-L1 belongs to type I transmembrane protein of B7 family, is transmembrane protein composed of 290 amino acid subunits, and the extracellular segment is two immunoglobulin constant regions Ig C and Ig V-like structural domains. In a tumor immune microenvironment, PD-L1 is taken as a representative negative co-stimulatory molecule, and can be combined with PD-1 on the surface of a T lymphocyte to induce phosphorylation of an immunoreceptor tyrosine inhibition motif, promote apoptosis of antigen-specific human T cell clones in vitro and in vivo, inhibit proliferation and differentiation of T cells and induce exhaustion of effector T cells. The tumor cells can abnormally up-regulate the expression of PD-L1 and PD-1, inhibit the immune activity of T cells, cause the immune escape of tumors and cause the occurrence and development of tumors. The tumor patient with high expression of PD-L1 has poor prognosis and low survival rate. In conclusion, PD-L1 participates in the process of generation and development of tumor, and assists tumor cells to evade the killing function of the immune system of the body, so that the tumor can be further worsened. The PD-1/PD-L1 signal channel plays a key role in tumor immunity and provides a new molecular target for tumor immunotherapy. Therefore, blocking the PD-1/PD-L1 signal path, reactivating the killing effect of the immune system of the organism on the tumor, and modifying the 'soil' on which the tumor lives becomes a new malignant tumor treatment hotspot.

In recent years, antibody drugs against PD-1/PD-L1 have been studied more intensively, and some of the drugs have been marketed. Currently, immune checkpoint inhibitors that block the PD-1/PD-L1 pathway fall into two major categories: (1) monoclonal antibodies to PD-1, nivolumab and pembrolizumab; (2) monoclonal antibodies against PD-L1, BMS-936559, MPDL3280A, atezolizumab, avelumab and durvalumab. In clinical tests, at present, the PD-1 or PD-L1 monoclonal antibody has been clinically researched in various solid tumors such as melanoma, non-small cell lung cancer, kidney cancer, prostate cancer, colorectal cancer, pancreatic cancer, bile duct cancer, liver cancer, gastric and esophageal cancer, breast cancer, small cell lung cancer and other diseases, and has a remarkable clinical effect, so that the process of late metastatic tumors can be prevented, and the overall survival period of patients can be substantially improved. The data from the study show that Keytruda monotherapy shows superiority in both primary (progression free survival, PFS) and secondary (overall survival, OS) endpoints compared to standard chemotherapy in patients with advanced non-small cell lung cancer (NSCLC) who express PD-L1 (tumor proportion score > 50%) at high tumor levels.

The current consensus is that the expression of PD-L1 is high, and the overall effect of PD1 or PDL1 immune drugs is better. However, the results of several clinical trials suggest that only about 20% of patients with NSCLC benefit from this therapy, and that a large number of patients with other tumors and patients with malignant tumors do not respond to the therapy, and that there are adverse drug-related reactions, including skin itching, anorexia, fatigue, etc., dermatitis, colitis, hepatitis, ptosis, etc. Adverse reactions and differences between therapeutic effects of two blocking agents, PD1 or PDL1, need to be further discussed in clinical trials to guide clinical medication, reduce the harm of adverse reactions to specific populations, and achieve better clinical efficacy. Therefore, it becomes a hot point of research to search for appropriate therapeutic effect detection markers and then accurately select people who have potential benefits in immunotherapy. The expression of PD-L1 in tumor cells or tumor stroma has been suggested as a potential therapeutic predictive marker for the prediction of PD-1 or PD-L1 directed immunotherapy responses. However, the detection of PD-L1 expression has not been achieved by a unified international standard at present. The expression condition of PD-L1 in different tumors and the correlation between the expression condition and clinical pathological parameters and prognosis of patients are researched, so that clinical references and bases related to treatment and prognosis can be provided for immunotherapy of different tumors; the close relationship between the two genes and the tumor determines that PD-L1 is one of the current hot molecular targets for tumor diagnosis and immunotherapy, and the expression level is also one of the indexes of tumor prognosis.

Up to now, detection of the degree of expression of PD-1 and PD-L1 by invasive techniques such as surgical removal of tumor or tissue biopsy may lead to inconsistency in detection results due to differences in brands used for detection of antibodies, detection techniques, environmental conditions at the time of detection, and cut-off values at which PD-L1 is judged to be positive. Factors such as insensitivity of PD-L1 expression due to spatial expression of tumor heterogeneity and incomplete tumor sampling may lead to false positives in PD-L1 detection due to the associated risks of biopsy procedures and Immunohistochemistry (IHC) deficiencies. In addition, the possibility that the patient may be in a baseline or other line-treatment state at the time the sample is taken is also one of the reasons for differences in the test results. These factors affect not only the consistency of the detection of the expression level of PD-L1, but also the reliability and repeatability of the detection. In addition, dynamic changes in the expression level of PD-L1 are also one of the factors that influence the determination of the exact expression state of PD-L1. Therefore, conventional detection methods such as Immunohistochemistry (IHC) method, in situ immunosterification technique (FISH), etc. exist to predict the effect of anti-PD-1/anti-PD-L1 immunotherapy in a certain way and limitation. With the ongoing development of cancer immunotherapy, there is a need to optimize the treatment of individual patients by molecular typing and to develop non-invasive molecular imaging tools, ultimately enabling dynamic monitoring of clinical immune checkpoint blockages. Therefore, the method for quickly, simply, conveniently and dynamically and accurately identifying the expression level of the PD-L1 protein on the surface of the tumor cell has important significance for the diagnosis, the immunotherapy and the prognosis evaluation of the tumor.

At present, in the clinical treatment process or after treatment of tumors, the noninvasive, repeatable and high-accuracy detection of the expression level and activity of tumor PD-1 and PD-L1 is difficult to realize, so that a specific image detection technology is urgently needed to guide the tumor treatment. Molecular imaging plays an increasingly important role in immunotherapy and personalized medicine. Wherein, the preparation of molecular probe is the key of molecular image, only the high sensitivity and specificity molecular probe can be combined with the specific target molecule in the cell and generate a certain signal after being introduced into the body, and the signal is generated by passing through a specific imaging device in vitro, such as: positron emission tomography (PET-CT), Single Photon Emission Computed Tomography (SPECT), Magnetic Resonance Imaging (MRI), and chemiluminescence devices, etc., to achieve highly specific diagnosis.

The antibody medicine has the problems of complicated preparation, poor in vitro stability, large molecules, difficult labeling, weak penetrating power, post-translational modification, high cost and the like, and further application of the antibody medicine is limited. Therefore, in order to improve the specificity and accuracy of cancer diagnosis and treatment and compensate for the defects of antibodies, the design of small molecule probes aiming at tumor markers as effective methods for detecting and treating cancer is urgently required. The polypeptide targeting small molecule drug and the diagnosis probe have the characteristics of low cost, small molecular weight, good biocompatibility, strong penetrability, no immunogenicity, high blood clearance rate, simple preparation and the like, show strong superiority in the aspects of tumor targeting drug delivery, cancer diagnosis and the like, and even show the trend of replacing antibody diagnosis and treatment reagents. Therefore, in cancer research, high specific affinity polypeptides for cancer cells are reasonably designed and screened for tumor markers, and then the polypeptides are developed into diagnostic reagents and therapeutic drugs for tumors, which is an effective way for solving the problems. The whole tumor and associated metastases can be imaged simultaneously using non-invasive methods, which may be different from the primary tumor in the PD-L1 expression state, with the advantage of being incomparable with IHC, and without the need to resect any tissue. Based on the high affinity and specificity of the targeting polypeptide to PD-L1 and the enhanced tissue penetration, the development of polypeptide small molecular probes aiming at PD-L1 has important significance for the diagnosis and treatment of various tumors with high expression of PD-L1.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention aims to provide a polypeptide targeting programmed death ligand-1 (PD-L1), which has higher specificity and affinity for PD-L1 and can be specifically and efficiently combined with a plurality of tumor immunotherapy-related markers PD-L1; the invention also provides a product derived from the peptide and capable of being combined with PD-L1 protein and application of the polypeptide or the derived product thereof in preparing preventive or therapeutic drugs, diagnostic reagents or imaging preparations.

In order to achieve the purpose, the technical scheme of the invention is as follows: according to the invention, through the analytic search and conclusion of crystal structures of compounds related to PD-1-inhibitor-PD-L1 in a PDB database, some key amino acids in a CDR loop region play a main role in a binding process, such as PD-1(Val64, Ile126, Leu128, Ala132 and Ile134) and PD-L1(Ile54, Tyr56, Met115, Ala121 and Tyr 123). Three major hot spots were selected in the PD-L1/PD-1 interaction surface. The first hotspot is a classical hydrophobic pocket consisting of the side chains of Tyr56, Glu58, Arg113, Met115 and Tyr123 and sized to accommodate a six-membered aromatic ring (this pocket is known as the Ile134 pocket). The second hot spot is located near Ile126 and consists of Met115, Ala121 and Tyr123, which can be anchored to the carbonyl-terminal donor group of Ala 121. The third hot spot was an elongated slot containing Tyr68, Gln75, and Thr 76. The invention designs and constructs the peptide library according to the hot spot amino acid sites of PD-1/PD-L1 interaction and the molecular recognition theory. Amino-modified TentaGel resin is used as a solid phase carrier, and Fmoc synthesis strategy is utilized to mix and equally divide to synthesize a bead-by-bead peptide library. Screening a high-throughput one-bead one-object peptide library by using a magnetic bead and magnetic field interaction method, and identifying positive peptide beads by mass spectrometry to obtain a series of active polypeptides capable of specifically binding PD-L1.

Specifically, the invention provides a polypeptide targeting PD-L1, which has a general formula as any one of the following (1), (2) and (3):

(1)NX₅X₄X₃EX₂X₁wherein X is₁Is arginine, glutamine, aspartic acid, histidine, lysine or glutamic acid; x₂Is glutamic acid, aspartic acid, leucine, tyrosine, threonine or glycine; x₃Is histidine, lysine, tyrosine, threonine, serine or glutamic acid; x₄Is tryptophan, aspartic acid, tyrosine, arginine, histidine or lysine; x₅Is lysine, threonine, tyrosine, histidine, proline or glutamic acid;

(2)X₈YX₇X₆TX₅X₄X₃X₂X₁wherein X is₁Is arginine, glutamine, aspartic acid, histidine, lysine or glutamic acid; x₂Is glutamic acid, aspartic acid, leucine, tyrosine, threonine or glycine; x₃Is histidine, lysine, tyrosine, threonine, serine or glutamic acid; x₄Is serine, aspartic acid, tyrosine, arginine, histidine or lysine; x₅Is lysine, threonine, tyrosine, histidine, tryptophan or glutamic acid; x₆Is lysine, threonine, glutamic acid or tyrosine; x₇Is methionine, asparagine or arginine; x₈Is arginine, lysine, threonine or serine;

(3)X₁₅X₁₄X₁₃X₁₂X₁₁X₁₀X₉X₈X₇X₆X₅X₄X₃X₂X₁wherein X is₁Is glutamic acid, glutamine or aspartic acid; x₂Is glutamic acid, aspartic acidHistidine or serine; x₃Is glutamic acid, serine or alanine; x₄Is threonine, lysine, tyrosine, arginine or proline; x₅Is tryptophan, tyrosine, arginine or histidine; x₆Is lysine, threonine or tyrosine; x₇Is asparagine, threonine, glutamic acid, isoleucine or glutamine; x₈Is methionine, asparagine or arginine; x₉Is tyrosine, alanine or aspartic acid; x₁₀Is arginine, lysine, threonine or serine; x₁₁Is threonine or proline; x₁₂Is glutamine or lysine; x₁₃Is phenylalanine or arginine; x₁₄Is histidine or tyrosine; x₁₅Is alanine or glutamine.

Preferably, the amino acid sequence of the polypeptide targeting PD-L1 of the present invention is any one of the following:

(1)NKWTEDE；

(2)RYMETWKSDQ；

(3)KYNETWRSED；

(4)AHFQYTAREYRPAHE；

(5)QYFQTKDRIYHPASE；

(6)AHRKPSARQYR PASE。

the polypeptide targeting PD-L1 has high specificity and affinity for PD-L1.

In the present invention, the amino acid residue may be in the L-form, D-form or a mixture of the L-form and D-form.

The present invention provides a nucleic acid comprising a nucleotide sequence encoding the polypeptide.

Preferably, the nucleic acid comprises a nucleotide sequence encoding a polypeptide having the amino acid sequence NKWTEE, RYMETWKSDQ, KYNETWRSED, AHFQYTAREYRPHAHE, QYFQTKDRIYHPAS or AHRKPSARQYRPASE.

The invention also provides biological materials comprising the nucleic acids.

The biological material includes expression cassettes, vectors, transposons, host cells or transgenic cell lines.

The vector includes but is not limited to cloning vector, expression vector, plasmid vector, and all vectors containing at least one copy of the nucleic acid encoding the targeted PD-L1 polypeptide of the present invention are within the scope of the present invention.

The host cell or transgenic cell line may be a cell or cell line derived from a microorganism, a plant or an animal, and all host cells or transgenic cell lines containing at least one copy of the nucleic acid encoding the targeted PD-L1 polypeptide of the present invention or comprising a vector carrying at least one copy of the nucleic acid are within the scope of the present invention.

The polypeptide targeting PD-L1 can be prepared by adopting a conventional preparation method in the field.

As one embodiment of the invention, the polypeptide targeting PD-L1 is prepared by Fmoc solid-phase polypeptide synthesis.

The invention also provides derivatives of the polypeptides, which are di-or polyvalent bodies formed by the polypeptides, wherein the di-or polyvalent bodies can target PD-L1.

Preferably, the bivalent or multivalent body is formed by covalent or non-covalent linkage of linking molecules, or by non-covalent linkage of molecules mixed with multimers.

More preferably, the covalently linked linking molecules are Fluorescein Isothiocyanate (FITC), 6-tertbutyloxycarbonylhydrazinonicotinic acid (HYNIC), 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

More preferably, the non-covalently linked linker molecule is a lipophilic near-infrared dye. The lipophilic near-infrared dye includes, but is not limited to DiR.

More preferably, the polymer is any one of polyethylene glycol (PEG), polyvinyl alcohol (PVA), cyclodextrin, polyamidoamine dendrimer (PAMAM), polylactic acid (PLA), polylactic-co-ethanolamine (PLGA), or a combination of at least two thereof.

The invention also provides a conjugate of the polypeptide or the derivative of the polypeptide, wherein the conjugate is obtained by connecting or acting the polypeptide or the derivative of the polypeptide and a carrier in a covalent or non-covalent mode.

Preferably, the carrier comprises any one or more of fluorescein, antibody, polymer, high molecular material, nano material, liposome, oily compound and inorganic material.

Further preferably, the high molecular material comprises any one or more of polyester, polyanhydride, polyamide phospholipid polymer micelle, polylactic acid-glycolic acid copolymer, polyethylene glycol and chitosan.

Further preferably, the inorganic material includes any one or more of nano gold, a carbon material, a calcium material, a magnetic material, a mesoporous silicon material, and a quantum dot.

The invention further provides any one of the following applications of the polypeptide or the nucleic acid encoding the polypeptide or the biological material containing the nucleic acid or the derivative of the polypeptide or the conjugate:

(1) the use in the manufacture of a medicament, diagnostic agent, diagnostic kit or imaging formulation for the prophylaxis or treatment of a PD-L1/PD1 mediated disease;

(2) the use in the diagnosis, prevention or treatment of PD-L1/PD1 mediated diseases;

(3) the application of the polypeptide in preparing a reagent for detecting the expression level of PD-L1 of cells;

(4) the application of the polypeptide in detecting the expression level of PD-L1 in cells.

In the invention, the PD-L1/PD1 mediated disease comprises any one of tumor, rheumatoid arthritis, HCV infection, allergic purpura, aplastic anemia, atherosclerosis and coronary heart disease.

Preferably, the tumor is a tumor with high expression of PD-L1. The PD-L1 high-expression tumor comprises any one of melanoma, non-small cell lung cancer, renal cancer, prostate cancer, glioma, colorectal cancer, pancreatic cancer, bile duct cancer, liver cancer, gastric cancer, esophageal cancer and breast cancer.

The invention also provides a medicament comprising the polypeptide or a derivative of the polypeptide or the conjugate.

Preferably, the active ingredient of the medicament further comprises an agent capable of killing tumor cells.

More preferably, the agent capable of killing tumor cells is any one or more of a chemical drug, a biological drug, a nano-drug, a radioactive drug, a photo-thermal therapy or a photodynamic therapy drug capable of killing tumor cells; or any one or more of alkylating agents, antimetabolites, natural antineoplastic agents, antineoplastic antibiotics, hormones, metal complexes or tumor radiotargeting markers.

More preferably, the medicament further comprises a carrier conjugated or admixed with said polypeptide, said derivative or said conjugate.

Such carriers include, but are not limited to, carriers for the preparation of targeted drugs.

The carrier comprises any one of a nano material, a liposome or an oily compound, or a mixture consisting of a plurality of oily compounds.

The drug obtained by conjugating or mixing the polypeptide or the derivative of the polypeptide or the conjugate with the carrier can be more stably transported to target cells in vivo.

More preferably, the medicament may further comprise a pharmaceutically acceptable adjuvant or adjuvant.

The polypeptide of the targeting PD-L1 provided by the invention has the function of specifically and efficiently targeting PD-L1, and can be used as a target head to effectively improve the content of a medicament or a carrier (such as a nano material, a liposome and the like) carrying the medicament in PD-L1 positive cells. And further adding pharmaceutically acceptable auxiliary materials or adjuvants to prepare a more effective targeted anticancer drug.

The invention also provides a diagnostic reagent or a kit for PD-L1/PD1 mediated diseases, which comprises the polypeptide or the derivative of the polypeptide or the conjugate.

The invention also provides an imaging agent comprising said polypeptide or a derivative of said polypeptide or said conjugate.

Preferably, the imaging agent further comprises an imaging agent. The imaging agent is any one of radionuclide, radionuclide marker, magnetic resonance contrast agent or molecular imaging agent.

More preferably, in said phase forming agent, said polypeptide or a derivative of said polypeptide or said conjugate is conjugated or mixed with said imaging agent.

The invention has the beneficial effects that:

the polypeptide of the target PD-L1 has higher affinity and specificity to PD-L1, can specifically and efficiently target PD-L1 positive tumor cells, has the advantages of strong selectivity, high purity, small molecular weight, strong specificity, no immunogenicity, safety, reliability, capability of being prepared by adopting a chemical synthesis method, simple and feasible preparation method, low cost and the like, and can be used for:

(1) the small molecular probe for detecting the expression condition of PD-L1 in tumor cells is used for preparing prediction and accompanying diagnostic reagents based on PD-L1 immunotherapy, so as to monitor the curative effect of the immunotherapy in real time and screen patients suitable for the immunotherapy, and a simple, convenient, rapid, economical and accurate detection means is provided for monitoring the relapse and metastasis of the tumor after the immunotherapy in real time, so that the treatment scheme can be adjusted in time, clinical intervention can be performed as early as possible, the progress of the disease can be prevented, and a new means is provided for improving the prognosis of the patients. The detection of the PD-L1 biomarker is beneficial to the establishment of a personalized treatment scheme to obtain the optimal treatment effect, and has better application prospect and clinical guiding significance.

(2) The polypeptide is used as a targeting polypeptide and is conjugated or mixed with a preparation capable of killing cancer cells, and is used for the targeted therapy and imaging of various tumors, or is independently used as an active component of a polypeptide inhibitor drug after being optimized, so that a PD-1/PD-L1 signal channel is blocked, and immunotherapy is activated. The method provides important theoretical and reference significance for the combined treatment of other immunotherapy, chemotherapy, radiotherapy and small molecule targeted drugs on how to reduce adverse reactions and apply the method to more malignant tumors in the existing immunotherapy based on the PD-1/PD-L1 blocking agent, and has wide application prospect.

The PD-L1-targeted polypeptide provided by the invention can provide important theoretical and clinical reference basis for early diagnosis of various tumors such as melanoma, non-small cell lung cancer, kidney cancer, prostate cancer, glioma, colorectal cancer, pancreatic cancer, cholangiocarcinoma, liver cancer, stomach cancer, esophageal cancer, breast cancer and the like, dynamic monitoring of detection points in immunotherapy and the like, and has important significance and application value for optimizing treatment schemes of individual patients. In view of the important regulation effect of the PD-1/PD-L1 pathway in other multi-system diseases, the polypeptide provides a new idea for immunotherapy of diseases such as rheumatoid arthritis, HCV virus infection, allergic purpura, aplastic anemia, atherosclerosis, coronary heart disease and the like, screening of suitable people, prognosis detection and evaluation and the like.

Drawings

FIG. 1 is a schematic diagram of the principle of screening PD-L1 targeting polypeptide in example 1 of the present invention.

FIG. 2 is a diagram showing the screening of magnetic beads, which are positive PD-L1 polypeptides, in example 1 of the present invention.

FIG. 3 is a graph showing the detection of the respective affinities of PD-L1 positive polypeptides and human PD-L1 protein by the Surface Plasmon Resonance (SPRi) method in example 2 of the present invention; wherein (a) - (f) are the results of analysis of the binding effect of the polypeptides P1, P2, P3, P4, P5 and P6 with human PD-L1 protein respectively.

FIG. 4 is the specific affinity detection of the polypeptides P1 and P2 in example 6 of the present invention at the cell level with PD-L1 high expression cell lines MDA-MB-231 and MC38 and negative cell A549, respectively; wherein (a) is the cell level specific affinity detection of the polypeptide P1 and PD-L1 high expression cell lines MDA-MB-231, MC38 and negative cell A549; (b) the cell level specific affinity detection of the polypeptide P2 and PD-L1 high expression cell lines MDA-MB-231, MC38 and negative cell A549 is carried out.

FIG. 5 is the specific affinity detection of the polypeptides P3 and P4 in example 6 of the invention at the cell level with PD-L1 high expression cell lines MDA-MB-231 and MC38 and negative cell A549 respectively; wherein (a) is the cell level specific affinity detection of the polypeptide P3 and PD-L1 high expression cell lines MDA-MB-231, MC38 and negative cell A549; (b) the cell level specific affinity detection of the polypeptide P4 and PD-L1 high expression cell lines MDA-MB-231, MC38 and negative cell A549 is carried out.

FIG. 6 is the specific affinity detection of the polypeptides P5 and P6 in example 6 of the present invention at the cell level with PD-L1 high expression cell lines MDA-MB-231 and MC38 and negative cell A549 respectively; wherein (a) is the cell level specific affinity detection of the polypeptide P5 and PD-L1 high expression cell lines MDA-MB-231, MC38 and negative cell A549; (b) the cell level specific affinity detection of the polypeptide P6 and PD-L1 high expression cell lines MDA-MB-231, MC38 and negative cell A549 is carried out.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 construction and screening of PD-L1 Targeted polypeptide library

Experimental instruments and materials

N-methylmorpholine (NMM), piperidine, trifluoroacetic acid (TFA), Dichloromethane (DCM), ninhydrin, vitamin C, phenol, tetramethyluronium Hexafluorophosphate (HBTU), piperidine, Triisopropylsilane (TIS), Ethanedithiol (EDT), N Dimethylformamide (DMF), dehydrated ether, resin, methanol, various Fmoc-protected amino acids, Streptavidin magnetic beads (MB-Streptavidin), biotin labeling kits, polypeptide synthesis tubes, shaker, vacuum pump, rotary evaporator, laser confocal microscope (ZEISS LSM 710), the above reagents and materials are commercially available.

(II) preparation of solvent

The deprotected solvent is formulated as piperidine: n, N-dimethylformamide ═ 1: 4;

the reaction solution was prepared from N-methylmorpholine, N-dimethylformamide ═ 1: 24;

preparing the lysate into trifluoroacetic acid (92.5%), triisopropylsilane (2.5%), ethanedithiol (2.5%) and ultrapure water (2.5%);

the ninhydrin test solution is prepared from ninhydrin: vitamin C: phenol 1: 1: 1.

synthesis of (III) PD-L1 "one-bead-one-substance" polypeptide library

The Fmoc solid phase peptide synthesis method is adopted to synthesize a polypeptide library, and the specific method is that amino acids are randomly coupled to solid phase resin one by one in a mixed splitting mode, then side chain protecting groups are removed under strong acid, and then screening is carried out. The specific method comprises the following steps:

(1) weighing 400mg of Tentagel-NH₂Resin, which is circulated according to the solid phase polypeptide synthesis program, and 200mg of Met and Gly are sequentially added for reaction;

(2) after the reaction is finished, equally dividing 3 parts of resin, respectively adding 100mg of Glu, Asp and Gln and an equal amount of HBTU into each tube for coupling, after the coupling is finished, deprotecting the 3 tubes of resin, mixing, equally dividing the resin into 4 parts, and respectively adding 100mg of Glu, Asp, His and Ser and an equal amount of HBTU into each tube for coupling;

(3) after the coupling is finished, 4 tubes of resin are deprotected and mixed. Dividing the resin into 3 parts, respectively adding 110mg of Glu, Ser and Ala into each tube, and coupling with HBTU with the same quantity;

(4) after the coupling is finished, 4 tubes of resin are deprotected and mixed. Dividing the resin into 5 parts, respectively adding 60mg of Thr, Lys, Tyr, Arg and Pro into each tube, and coupling with the same amount of HBTU;

(5) after the coupling is finished, 5 tubes of resin are deprotected and mixed. Dividing the resin into 4 parts, respectively adding 80mg of Trp, Tyr, Arg and His into each tube, and coupling with an equal amount of HBTU;

(6) after the coupling is finished, 4 tubes of resin are deprotected and mixed. Dividing the resin into 3 parts, respectively adding 90mg of Lys, Thr and Tyr into each tube, and coupling with the same amount of HBTU;

(7) after the coupling is finished, 3 tubes of resin are deprotected and mixed. Dividing the resin into 5 parts, respectively adding 70mg of Asn, Glu, Thr, Gln and Ile and an equal amount of HBTU into each tube for coupling;

(8) after the coupling is finished, 5 tubes of resin are deprotected and mixed. Dividing the resin into 3 parts, respectively adding 120mg of Met, Asn and Arg into each tube, and coupling with the same amount of HBTU;

(9) after the coupling is finished, 3 tubes of resin are deprotected and mixed. Dividing the resin into 3 parts, respectively adding 80mg of Tyr, Ala and Asp and the same amount of HBTU into each tube for coupling;

(10) after the coupling is finished, 3 tubes of resin are deprotected and mixed. Dividing the resin into 4 parts, respectively adding 80mg of Arg, Lys, Thr and Ser into each tube, and coupling with HBTU with the same amount;

(11) after the coupling is finished, 4 tubes of resin are deprotected and mixed. Dividing the resin into 2 parts, respectively adding 120mg of Thr and Pro and the same amount of HBTU into each tube for coupling;

(12) after the coupling is finished, 2 tubes of resin are deprotected and mixed. Dividing the resin into 2 parts, respectively adding 180mg of Gln and Lys into each tube, and coupling with the same amount of HBTU;

(13) after the coupling is finished, 2 tubes of resin are deprotected and mixed. Dividing the resin into 2 parts, respectively adding 120mg of Phe and Arg into each tube, and coupling with an equal amount of HBTU;

(14) after the coupling is finished, 2 tubes of resin are deprotected and mixed. Dividing the resin into 2 parts, respectively adding 180mg of His and Tyr and the same amount of HBTU into each tube for coupling;

(15) after the coupling is finished, 2 tubes of resin are deprotected and mixed. Dividing the resin into 2 parts, adding 180mg of Ala and Gln into each tube and HBTU with the same quantity for coupling, and after coupling is finished, deprotecting the 2 tubes of resin and mixing. And (3) performing methanol replacement and shrinkage steps, and performing vacuum pumping to obtain the dry resin loaded with the peptide library for later use.

(IV) screening for PD-L1-Positive Polypeptides

(1) Swelling the dried peptide library with 1 XPBS overnight, washing with 1 XPBS for 3 times, adding 5% skimmed milk, sealing the surface of the peptide beads on a vortex mixer at 37 ℃ for 2h, and washing with 1 XPBS for 3 times;

(2) labeling PD-L1 protein according to a biotin labeling kit, mixing the PD-L1 protein labeled by biotin (biotin) with a polypeptide library, incubating at 37 ℃ for 2h, and washing with 1 × PBS for 3 times;

(3) then 100. mu.L of MB-Streptavidin was added to the peptide library and incubated for 2h on a vortex mixer at 37 ℃ in the dark. After incubation, the EP tube containing the polypeptide library was placed on a magnetic frame. The positive polypeptide is magnetically attracted to the side wall of the EP tube, while the negative polypeptide settles to the bottom of the EP tube due to gravity.

A schematic diagram of the principle of screening PD-L1 targeting polypeptide is shown in FIG. 1, when positive polypeptide beads are incubated with biotin-labeled receptor protein, the positive peptide beads specifically recognize the protein, and the magnetic beads labeled with streptavidin recognize the positive peptide beads by recognizing biotin. The result of the PD-L1 positive polypeptide-magnetic bead screening is shown in fig. 2, wherein the dotted line frame is a positive peptide bead, and the surface of the positive peptide bead is magnetic due to the coating of a layer of magnetic bead and thus can be captured by a magnetic field. 6 PD-L1 positive polypeptides P1, P2, P3, P4, P5 and P6 are obtained through screening, the amino acid sequences of the polypeptides are shown in table 1, and the 6 polypeptides P1, P2, P3, P4, P5 and P6(SEQ ID NO. 1-6) are obtained through chemical synthesis according to the amino acid sequences shown in table 1. And (3) synthesizing a positive polypeptide again according to the amino acid sequence shown in the table 1, marking fluorescence at the N end of the polypeptide, and using the positive polypeptide for cell imaging to verify the function of the polypeptide, and using the positive polypeptide for subsequent experiments after MALDI-TOF identification and HPLC purification.

TABLE 1 amino acid sequence of PD-L1 Positive polypeptide

Serial number	Polypeptide name	Amino acid sequence
			SEQ ID NO.1	P1	NKWTEDE
SEQ ID NO.2	P2	RYMETWKSDQ
			SEQ ID NO.3	P3	KYNETWRSED
SEQ ID NO.4	P4	AHFQYTAREYRPAHE
			SEQ ID NO.5	P5	QYFQTKDRIYHPASE
SEQ ID NO.6	P6	AHRKPSARQYRPASE

Example 2 detection of the affinity of the Polypeptides P1, P2, P3, P4, P5, P6 with the PD-L1 protein

The affinity effect of the polypeptides P1, P2, P3, P4, P5 and P6 and PD-L1 protein is detected by a Surface Plasmon Resonance (SPRi) method, and the specific method is as follows:

dropping 1mg/mL P1, P2, P3, P4, P5, P6 polypeptide and 1 XPBS on a chip, incubating overnight at 4 ℃ under a humid condition, washing for 10min with 10 XPBS, washing for 10min with 1 XPBS, washing for 2 times with deionized water, soaking for 10min each time in 1 XPBS containing 5% skimmed milk, incubating overnight at 4 ℃, washing for 10min with 10 XPBS, washing for 10min with 1 XPPBS for 10min, washing for 2 times with deionized water, washing for 10min each time, drying with nitrogen, loading on a chip (Plexa)

HT surface plasmon resonance imaging system).

The mobile phase was purified sequentially by 1 XPBS, 2 XPBS, 0.625. mu.g/mL, 1.25. mu.g/mL, 2.5. mu.g/mL, 5. mu.g/mL and 10. mu.g/mL of human PD-L1, and SPRi signals were recorded for analysis.

The results are shown in FIG. 3, and SPRi signals of P1, P2, P3, P4, P5 and P6 are gradually enhanced along with the increase of protein concentration, which indicates that the P1, P2, P3, P4, P5 and P6 polypeptides of the invention have stronger binding force to PD-L1 and the affinity reaches 10^-8～10^-9M, close to the affinity of the antibody, can be used as a probe to target various tumor cells expressing PD-L1 and is used for related research and application.

Example 3 preparation of imaging preparation of Polypeptides P1, P2, P3, P4, P5, P6

This example provides the preparation of imaging agents for polypeptides P1, P2, P3, P4, P5, P6 by the following specific methods:

weighing 300mg of wang-Glu resin, circulating according to a solid-phase polypeptide synthesis program, firstly carrying out deprotection, and then sequentially adding a certain amount of Glu, Asp, Glu, Thr, Trp, Lys, Asn and an equal amount of HTBU for sequential reaction. And (3) after the coupling reaction is finished, deprotection and cleaning. Adding epsilon-aminocaproic acid for reaction, and after coupling, removing protection and cleaning. Adding FITC for coupling reaction in dark place, deprotecting after reaction, and washing. Finally, separating out the side chain protecting group from the cracking liquid in the resin, carrying out vacuum pumping to obtain a crude polypeptide fluorescent conjugate, and carrying out HPLC purification to obtain developing preparations of the polypeptides P1, P2, P3, P4, P5 and P6 with the purity of 95%, wherein the developing preparations can be used for fluorescence imaging.

Example 4 preparation of conjugates of the polypeptides P1, P2, P3, P4, P5, P6

This example provides the preparation of conjugates of polypeptides P1, P2, P3, P4, P5, P6 by the following specific methods:

weighing 300mg of wang-Glu resin, circulating according to a solid-phase polypeptide synthesis program, firstly carrying out deprotection, and then sequentially adding a certain amount of Glu, Asp, Glu, Thr, Trp, Lys, Asn and an equal amount of HTBU for sequential reaction. And (3) after the coupling reaction is finished, deprotection and cleaning. Epsilon-aminocaproic acid was added to the reaction solution, and FITC and peptide beads were mixed and reacted overnight in a solution of pyridine/N, N-dimethylformamide/dichloromethane at a ratio of 1:5: 7. And (4) after the reaction is finished, cleaning. Finally, the side chain protecting group is removed from the cracking solution in the resin, and the crude polypeptide fluorescent conjugate is obtained after vacuum pumping.

Example 5 preparation of a kit based on the polypeptides P1, P2, P3, P4, P5, P6

The polypeptide Fluorescein Isothiocyanate (FITC) conjugate is obtained by a solid phase synthesis method, and epsilon-aminocaproic acid is continuously coupled on newly synthesized P1, P2, P3, P4, P5 and P6 polypeptide microbeads respectively. FITC was mixed with peptide beads and reacted overnight in a solution of pyridine/N, N-dimethylformamide/dichloromethane at a ratio of 1:5: 7. And (3) obtaining P1, P2, P3, P4, P5 and P6 polypeptide FITC conjugates after cracking by a cracking solution, and carrying out MALDI-TOF identification and HPLC purification for subsequent experiments.

Example 6 interaction of the polypeptides P1, P2, P3, P4, P5, P6 with PD-L1 high expressing cells MDA-MB-231 and MC38

This example analyzes the interaction of polypeptides P1, P2, P3, P4, P5, P6 with PD-L1 high expression cells MDA-MB-231 and MC38 and low expression cell A549, wherein the breast cancer cell line MDA-MB-231 is cultured in DMEM medium containing 10% fetal bovine serum, the colon cancer cell line MC38 is cultured in MEM/EBSS medium containing 10% fetal bovine serum, and the non-small cell lung cancer cell line A549 is cultured in RPMI 1640 medium containing 10% fetal bovine serum, as follows:

(1) interaction of polypeptide P1 with PD-L1 high expression cells MDA-MB-231 and MC38 and low expression cell A549 respectively

At 1 × 10⁵Cell concentration per mL in round glass-bottom Petri dishes (35mm), 37 ℃, 5% CO₂Culturing in a cell culture box for 24h, removing culture solution, adding 1 μmol/L Hoechst 33342 into each of the three cells, incubating at 4 deg.C in dark for 15min, washing with precooled 1 × PBS for 2 times, adding 50 μ M FITC-labeled polypeptide P1, incubating at 4 deg.C in dark for 20min, and washing with precooled 1 × PBS for 3 times. Fluorescence distribution in cells was examined with a laser scanning confocal microscope (ZEISS LSM 710).

As shown in a of FIG. 4, strong green fluorescence was observed on the cell membranes of MDA-MB-231 and MC38 added with polypeptide P1, while the A549 cells did not fluoresce. The result shows that the P1 polypeptide is combined on the cell membrane of a PD-L1 positive cell, has specificity with the recognition of a human PD-L1 positive cell line, has positive correlation with the specificity and the expression quantity of a target protein, and can be used as a targeting molecule for relevant diagnosis and detection. In contrast, the P1 polypeptide observed no green fluorescence signal and no antibody signal for the negative cell a549 with low PD-L1 expression, further indicating that P1 can specifically target PD-L1, and further verifying the reliability of the SPRi data in a of fig. 3.

(2) Interaction of polypeptide P2 with PD-L1 high expression cells MDA-MB-231 and MC38 and low expression cell A549 respectively

At 1 × 10⁵Cell concentration per mL in round glass-bottom Petri dishes (35mm), 37 ℃, 5% CO₂Culturing in a cell culture box for 24h, removing culture solution, adding 1 μmol/L Hoechst 33342 into each of the three cells, incubating at 4 deg.C in dark for 15min, washing with precooled 1 × PBS for 2 times, adding 50 μ M FITC-labeled polypeptide P2, incubating at 4 deg.C in dark for 20min, and washing with precooled 1 × PBS for 3 times. Fluorescence distribution in cells was examined with a laser scanning confocal microscope (ZEISS LSM 710).

As a result, as shown in b of FIG. 4, strong green fluorescence was observed on the cell membranes of MDA-MB-231 and MC38 to which polypeptide P2 was added, whereas the A549 cells did not fluoresce. The result shows that the P2 polypeptide is combined on the cell membrane of the positive cell, has specificity with the recognition of the positive cell line of human PD-L1, has positive correlation with the expression quantity of the target protein in specificity, and can be used as a targeting molecule for relevant diagnosis and detection. In contrast, the P2 polypeptide observed no green fluorescence signal and no antibody signal for the negative cell a549 with low PD-L1 expression, further indicating that the polypeptide P2 can specifically target PD-L1, and further verifying the reliability of the SPRi data in b of fig. 3.

(3) Interaction of polypeptide P3 with PD-L1 high expression cells MDA-MB-231 and MC38 and low expression cell A549 respectively

At 1 × 10⁵Cell concentration per mL implanted in circular glass-bottom Petri dishes (35 m)m)，37℃，5％CO₂Culturing in a cell culture box for 24h, removing culture solution, adding 1 μmol/L Hoechst 33342 into each of the three cells, incubating at 4 deg.C in dark for 15min, washing with precooled 1 × PBS for 2 times, adding 50 μ M FITC-labeled polypeptide P3, incubating at 4 deg.C in dark for 20min, and washing with precooled 1 × PBS for 3 times. Fluorescence distribution in cells was examined with a laser scanning confocal microscope (ZEISS LSM 710).

As shown in a of FIG. 5, strong green fluorescence was observed on the cell membranes of MDA-MB-231 and MC38 added with the polypeptide P2P3, while the A549 cells did not fluoresce. The result shows that the P3 polypeptide is combined on the cell membrane of the positive cell, has specificity with the recognition of the positive cell line of human PD-L1, has positive correlation with the expression quantity of the target protein in specificity, and can be used as a targeting molecule for relevant diagnosis and detection. In contrast, the P3 polypeptide observed no green fluorescence signal and no antibody signal for the negative cell a549 with low PD-L1 expression, further indicating that the polypeptide P3 can specifically target PD-L1, and further verifying the reliability of the SPRi data in c of fig. 3.

(4) Interaction of polypeptide P4 with PD-L1 high expression cells MDA-MB-231 and MC38 and low expression cell A549 respectively

At 1 × 10⁵Cell concentration per mL in round glass-bottom Petri dishes (35mm), 37 ℃, 5% CO₂Culturing in a cell culture box for 24h, removing culture solution, adding 1 μmol/L Hoechst 33342 into each of the three cells, incubating at 4 deg.C in dark for 15min, washing with precooled 1 × PBS for 2 times, adding 50 μ M FITC-labeled polypeptide P4, incubating at 4 deg.C in dark for 20min, and washing with precooled 1 × PBS for 3 times. Fluorescence distribution in cells was examined with a laser scanning confocal microscope (ZEISS LSM 710).

As a result, as shown in b of FIG. 5, strong green fluorescence was observed on the cell membranes of MDA-MB-231 and MC38 to which polypeptide P4 was added, whereas the A549 cells did not fluoresce. The result shows that the P4 polypeptide is combined on the cell membrane of the positive cell, has specificity with the recognition of the positive cell line of human PD-L1, has positive correlation with the expression quantity of the target protein in specificity, and can be used as a targeting molecule for relevant diagnosis and detection. In contrast, the P4 polypeptide observed no green fluorescence signal and no antibody signal for the negative cell a549 with low PD-L1 expression, further indicating that the polypeptide P4 can specifically target PD-L1, and further verifying the reliability of the SPRi data in d of fig. 3.

(5) Interaction of polypeptide P5 with PD-L1 high expression cells MDA-MB-231 and MC38 and low expression cell A549 respectively

At 1 × 10⁵Cell concentration per mL in round glass-bottom Petri dishes (35mm), 37 ℃, 5% CO₂Culturing in a cell culture box for 24h, removing culture solution, adding 1 μmol/L Hoechst 33342 into each of the three cells, incubating at 4 deg.C in dark for 15min, washing with precooled 1 × PBS for 2 times, adding 50 μ M FITC-labeled polypeptide P5, incubating at 4 deg.C in dark for 20min, and washing with precooled 1 × PBS for 3 times. Fluorescence distribution in cells was examined with a laser scanning confocal microscope (ZEISS LSM 710).

As shown in a of FIG. 6, strong green fluorescence was observed on the cell membranes of MDA-MB-231 and MC38 added with polypeptide P5, while the A549 cells did not fluoresce. The result shows that the P5 polypeptide is combined on the cell membrane of the positive cell, has specificity with the recognition of the positive cell line of human PD-L1, has positive correlation with the expression quantity of the target protein in specificity, and can be used as a targeting molecule for relevant diagnosis and detection. In contrast, the P5 polypeptide observed no green fluorescence signal and no antibody signal for the negative cell a549 with low PD-L1 expression, further indicating that the polypeptide P5 can specifically target PD-L1, and further verifying the reliability of the SPRi data in e of fig. 3.

(6) Interaction of polypeptide P6 with PD-L1 high expression cells MDA-MB-231 and MC38 and low expression cell A549 respectively

At 1 × 10⁵Cell concentration per mL in round glass-bottom Petri dishes (35mm), 37 ℃, 5% CO₂Culturing in a cell culture box for 24h, removing culture solution, adding 1 μmol/L Hoechst 33342 into each of the three cells, incubating at 4 deg.C in dark for 15min, washing with precooled 1 × PBS for 2 times, adding 50 μ M FITC-labeled polypeptide P6, incubating at 4 deg.C in dark for 20min, and washing with precooled 1 × PBS for 3 times. Fluorescence distribution in cells was examined with a laser scanning confocal microscope (ZEISS LSM 710).

As a result, as shown in b of FIG. 6, strong green fluorescence was observed on the cell membranes of MDA-MB-231 and MC38 to which polypeptide P6 was added, whereas the A549 cells did not fluoresce. The result shows that the P6 polypeptide is combined on the cell membrane of the positive cell, has specificity with the recognition of the positive cell line of human PD-L1, has positive correlation with the expression quantity of the target protein in specificity, and can be used as a targeting molecule for relevant diagnosis and detection. In contrast, the P6 polypeptide observed no green fluorescence signal and no antibody signal for the negative cell a549 with low PD-L1 expression, further indicating that the polypeptide P6 can specifically target PD-L1, and further verifying the reliability of the SPRi data in f of fig. 3.

In conclusion, the polypeptides P1, P2, P3, P4, P5 and P6 of the present invention have the characteristic of targeting expression of PD-L1 positive tumor cells, so in practical application, the polypeptides P1, P2, P3, P4, P5 and P6 of the present invention can be used as targeting polypeptides, and can be conjugated or mixed with a preparation capable of killing cancer cells, so as to be used for targeting treatment and imaging of tumors.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> national center for Nano science

<120> polypeptide targeting PD-L1 and application thereof

<130> KHP211123095.4D

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Asn Lys Trp Thr Glu Asp Glu

1 5

<210> 2

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Arg Tyr Met Glu Thr Trp Lys Ser Asp Gln

1 5 10

<210> 3

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Lys Tyr Asn Glu Thr Trp Arg Ser Glu Asp

1 5 10

<210> 4

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Ala His Phe Gln Tyr Thr Ala Arg Glu Tyr Arg Pro Ala His Glu

1 5 10 15

<210> 5

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Gln Tyr Phe Gln Thr Lys Asp Arg Ile Tyr His Pro Ala Ser Glu

1 5 10 15

<210> 6

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Ala His Arg Lys Pro Ser Ala Arg Gln Tyr Arg Pro Ala Ser Glu

1 5 10 15

Claims

1. A polypeptide targeting PD-L1, characterized in that its amino acid sequence is RYMETWKSDQ.

2. A nucleic acid encoding the polypeptide of claim 1.

3. Biological material comprising nucleic acids according to claim 2, wherein the biological material comprises an expression cassette, a vector, a transposon, a host cell or a transgenic cell line.

4. A derivative of the polypeptide of claim 1, which is a bivalent or multivalent body formed from the polypeptide of claim 1, said bivalent or multivalent body being capable of targeting PD-L1;

preferably, the bivalent or multivalent body is formed by covalent or non-covalent linkage of a linking molecule, or by non-covalent linkage of a polymer in admixture;

more preferably, the covalently linked linking molecule is fluorescein isothiocyanate, 6-tertbutyloxycarbonylhydrazinonicotinic acid, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide or N-hydroxysuccinimide;

and/or, the non-covalently linked linker molecule is a lipophilic near-infrared dye;

and/or the polymer is any one or the combination of at least two of polyethylene glycol, polyvinyl alcohol, cyclodextrin, polyamide-amine dendrimer, polylactic acid and polylactic acid-ethanolamine.

5. A conjugate of the polypeptide of claim 1, wherein the conjugate is obtained by linking or interacting the polypeptide of claim 1 with a carrier in a covalent or non-covalent manner;

6. Use of any one of the following polypeptides according to claim 1 or nucleic acids according to claim 2 or biological material according to claim 3 or derivatives according to claim 4 or conjugates according to claim 5:

(4) the application of detecting the expression level of PD-L1 in cells;

preferably, the PD-L1/PD1 mediated disease comprises any one of tumor, rheumatoid arthritis, HCV infection, allergic purpura, aplastic anemia, atherosclerosis and coronary heart disease.

7. The use of claim 6, wherein the tumor is a tumor with high expression of PD-L1; the PD-L1 high-expression tumor comprises any one of melanoma, non-small cell lung cancer, renal cancer, prostate cancer, colorectal cancer, pancreatic cancer, bile duct cancer, liver cancer, gastric cancer, esophageal cancer and breast cancer.

8. A medicament comprising a polypeptide according to claim 1 or a derivative according to claim 4 or a conjugate according to claim 5;

preferably, the active ingredient of the medicament further comprises an agent capable of killing tumor cells;

9. A diagnostic reagent or kit for PD-L1/PD1 mediated diseases, characterized in that it comprises the polypeptide of claim 1 or the derivative of claim 4 or the conjugate of claim 5.

10. An imaging agent comprising the polypeptide of claim 1 or the derivative of claim 4 or the conjugate of claim 5;

preferably, the imaging agent further comprises an imaging agent, the imaging agent being any one of a radionuclide, a radionuclide label, a magnetic resonance contrast agent, or a molecular imaging agent;

more preferably, the polypeptide of claim 1 or the derivative of claim 4 or the conjugate of claim 5 is conjugated or mixed with the imaging agent in the imaging agent.