CN114163496B

CN114163496B - PD-L1 targeting polypeptide and application thereof

Info

Publication number: CN114163496B
Application number: CN202111266397.5A
Authority: CN
Inventors: 胡志远; 钱怡霞
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: 2023-08-08
Anticipated expiration: 2039-06-04
Also published as: CN114163498A; CN112028969A; CN114163495B; CN112028969B; CN114163497B; CN114163495A; CN114163497A; CN114031670A; CN114163496A; CN114163498B; CN114031670B

Abstract

The invention relates to the technical fields of molecular biology and medicine, in particular to a polypeptide targeting PD-L1 and application thereof. The invention provides a polypeptide targeting PD-L1, which has an amino acid sequence of AHFQYTAREYRPAHE. The polypeptide targeting PD-L1 provided by the invention 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 tumor cells in practice or used as a targeting polypeptide to be conjugated or mixed with a preparation capable of killing cancer cells, is used for targeted treatment and imaging of various tumors, and has wide application prospects.

Description

PD-L1 targeting polypeptide and application thereof

Technical Field

The invention relates to a polypeptide targeting PD-L1, a preparation method and application thereof, which are divisional applications of patent application with the application number of 201910480489. X. The invention relates to the technical fields of molecular biology and medicine, in particular to a polypeptide targeting programmed death ligand-1 (PD-L1) and a preparation method and application thereof.

Background

With the rapid development and interpenetration of relevant subjects such as oncology, immunology and molecular biology, tumor immunotherapy has been developed as a new and important anti-tumor therapeutic approach. Unlike previous surgical, chemo, radiation and targeted therapies, tumor immunotherapy combat tumors by activating the human autoimmune system. Currently, inhibitors against immune checkpoints show better clinical effects in the treatment of various solid tumors, making it recognized that immunotherapy can truly become an important tool in the treatment of malignant tumors. Tumor immunotherapy has a variety of therapeutic strategies including nonspecific immunostimulants, tumor vaccines, adoptive immune cell therapies, and monoclonal antibody therapies. Because tumors have great heterogeneity and genetic instability, the pathogenesis of the tumors is complex, and the ideal anti-tumor effect is difficult to achieve by means of a certain treatment means alone, so that 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 among different treatment means.

At the heart of tumor immunotherapy is the activation of the anti-tumor response of T lymphocytes of tumor patients to increase their killing function against tumor cells. T cell mediated cellular immunity plays an important role in the process of recognizing and killing tumor cells, and the T cells have definite targeting and specificity on the tumor cells, can generate long-range immune response reaction by recognizing and aggregating to the tumor antigen expression part, and directly inhibit and kill the tumor cells. Dendritic Cells (DCs) play a vital role in T cell recognition. T cells recognize tumor cells by binding to the Major Histocompatibility Complex (MHC) with specific antigens on the surface of tumor cells via T Cell Receptors (TCRs). The interaction of TCR and MHC molecules is controlled by a series of immune checkpoints, which allow T cells to be activated or inhibited. Among them, the programmed death receptor 1 (PD-1) and programmed death ligand-1 (PD-L1) pathways are inhibitory immune checkpoints, and their binding can inhibit the immune activity of T cells, playing an important role in immune tolerance, which is also an important cause of tumor cell immune escape.

Programmed death ligand-1 (Programmed death receptor ligand-1, PD-L1, also known as B7-H1) is a ligand of Programmed death factor-1 (Programmed death-1, PD-1) which was first discovered and cloned as a gene in 1992, is a typical negative co-stimulatory molecule which transcriptionally induces Programmed death of T cells, and is one of the popular subjects in the field of immunology. PD-L1 is mainly expressed on antigen presenting cells, B cells, T cells, epithelial cells, muscle cells, endothelial cells and various tumor cells (PD-L1 is expressed on the surfaces of various tumor cells of human beings, 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 responses. PD-L1 belongs to the B7 family of type I transmembrane proteins, is composed of 290 amino acid subunits, and the extracellular segment is two immunoglobulin constant regions Ig C and Ig V-like domains. In tumor immune microenvironment, PD-L1 is taken as a representative negative co-stimulatory molecule, and through combining with the surface PD-1 of T lymphocytes, the polypeptide induces phosphorylation of an immunoreceptor tyrosine inhibition motif, can promote apoptosis of antigen-specific human T cell clones in vivo and in vitro, inhibit proliferation and differentiation of T cells and induce depletion of effector T cells. Tumor cells can abnormally up-regulate PD-L1 and the expression of PD-1, inhibit the immunocompetence of T cells, cause tumor immune escape, and cause tumor generation and development. Tumor patients with high expression of PD-L1 have poor prognosis and low survival rate. In conclusion, PD-L1 participates in the tumor generation and development process, and assists tumor cells to evade the killing effect of the immune system of the organism, so that the tumor can be further worsened. The PD-1/PD-L1 signal path plays a key role in tumor immunity, and simultaneously provides a novel molecular target for tumor immunotherapy. Therefore, the method blocks the PD-1/PD-L1 signal path, re-activates the killing effect of the organism immune system on the tumor, and reforms the 'soil' on which the tumor depends to survive into a new malignant tumor treatment hot spot.

In recent years, antibody drugs against PD-1/PD-L1 have been intensively studied, and some drugs have been marketed. Currently, immune checkpoint inhibitors that block the PD-1/PD-L1 pathway fall into two main categories: (1) Monoclonal antibodies directed against PD-1, nivolumab and pembrolizumab; (2) Monoclonal antibodies directed against PD-L1, BMS-936559, MPDL3280A, atezolizumab, avelumab and durvalumab. In clinical trials, at present, PD-1 or PD-L1 monoclonal antibodies have been clinically studied in various solid tumors such as melanoma, non-small cell lung cancer, renal cancer, prostate cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, liver cancer, gastric cancer, esophageal cancer, breast cancer, small cell lung cancer and the like, so that significant clinical effects are achieved, the progress of advanced metastatic tumors can be prevented, and substantial improvement of the total survival time of patients is expected. Study data show that Keytruda single drug treatment shows superiority at both the primary endpoint (progression free survival, PFS) and the secondary endpoint (total survival, OS) compared to standard chemotherapy in patients with advanced non-small cell lung cancer (NSCLC) with high tumor level expression of PD-L1 (tumor proportion score ∈50%).

The current consensus is that the PD-L1 expression is high, and the overall effect of PD1 or PDL1 immune medicines is relatively good. However, the results of several clinical trials suggest that only about 20% of NSCLC patients benefit from it, and that a large number of other tumor patients and malignant tumor patients do not respond to treatment, while there are drug-related adverse effects including skin itching, anorexia, fatigue, etc., dermatitis, colitis, hepatitis, hypophysitis, etc. The adverse reaction and the difference between the curative effects of the PD1 or PDL1 blocking agents need to be further discussed in clinical experiments, so that the medicine is used for guiding clinical medication, reducing the harm of the adverse reaction to special people and obtaining better clinical curative effects. Therefore, a proper curative effect detection marker is searched for, and the potential benefit group of the immunotherapy is accurately selected to become a research hot spot. The expression of PD-L1 in tumor cells or tumor stroma has been suggested as a potential efficacy predictive marker for the prediction of PD-1 or PD-L1 targeted immunotherapy responses. However, currently, the detection of PD-L1 expression has not yet achieved a uniform international standard. The expression condition of PD-L1 in different tumors and the correlation of the PD-L1 with clinical pathological parameters and prognosis of patients can be studied, so that clinical references and bases related to treatment and prognosis can be provided for the immunotherapy of different tumors; the close relationship between the two and the tumor determines that PD-L1 is one of hot molecular targets in the current tumor diagnosis and immunotherapy, and the expression level of the PD-L1 is also one of indexes of tumor prognosis.

To date, the extent of expression of PD-1 and PD-L1 has been detected by invasive techniques such as surgical removal of tumors or tissue biopsies, which can lead to inconsistent detection results due to differences in the brands used to detect antibodies, the detection technique, the environmental conditions at the time of detection, and the cut-off value at which PD-L1 is determined to be positive. Due to the associated risk of biopsy procedures and defects in Immunohistochemistry (IHC), factors such as insensitivity of tumor heterogeneous spatial expression leading to PD-L1 expression and incomplete tumor sampling may lead to false positives in PD-L1 detection. In addition, the baseline or other line treatment conditions that the patient may be in when the sample is taken are also one of the reasons for the differences in the test results. These factors affect not only the consistency of the detection of the PD-L1 expression level, but also the reliability and reproducibility of the detection. In addition, dynamic changes in the expression level of PD-L1 are also one of the factors that affect the determination of the exact expression state of PD-L1. Thus, conventional detection methods such as Immunohistochemical (IHC) methods, in situ hybridization (FISH) techniques, and the like, have certain one-sidedness and limitations in predicting the effect of anti-PD-1/anti-PD-L1 immunotherapy. With the continuous development of cancer immunotherapy, it is necessary to optimize the treatment method of individual patients by molecular typing and develop non-invasive molecular imaging tools to finally realize dynamic monitoring of clinical immune checkpoint blockade. Therefore, the method for rapidly, simply and dynamically and accurately identifying the expression level of the PD-L1 protein on the surface of the tumor cells has important significance for diagnosis, immunotherapy and prognosis evaluation of tumors.

Currently, noninvasive, reproducible and highly accurate detection of tumor PD-1 and PD-L1 expression levels and activities during or after clinical tumor therapy is difficult to achieve, and specific image detection techniques are urgently needed to guide tumor therapy. Molecular imaging plays an increasingly important role in immunotherapy and personalized medicine. Wherein, the preparation of molecular probes is the key of molecular imaging, and only high-sensitivity and specific molecular probes can be specifically combined with specific target molecules in cells after being introduced into the body to generate a certain signal, and the molecular probes can be processed by specific imaging equipment in vitro, such as: positron emission computed tomography (PET-CT), single Photon Emission Computed Tomography (SPECT), magnetic Resonance Imaging (MRI), and chemiluminescent devices, to achieve highly specific diagnosis.

The antibody drug has the problems of complicated preparation, poor in vitro stability, larger molecules, difficult labeling, weak penetrating power, post-translational modification, high cost and the like, and further application of the antibody drug is limited. Therefore, in order to improve the specificity and accuracy of cancer diagnosis and treatment, and to compensate for the defects of antibodies, there is an urgent need to search for a small molecular probe designed for tumor markers as an effective method for detecting and treating cancer. The polypeptide targeting small molecule drug and the diagnostic probe have the characteristics of low cost, small molecular weight, good biocompatibility, strong penetrability, no immunogenicity, higher 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, the high-specificity affinity polypeptide to cancer cells is reasonably designed and screened aiming at tumor markers in cancer research, and then the high-specificity affinity polypeptide is developed into diagnostic reagents and therapeutic drugs for tumors, which are effective ways for solving the problems. The use of non-invasive methods allows simultaneous imaging of the entire tumor and associated metastases, which may be different from the primary tumor in the PD-L1 expression state, with advantages that IHC is not comparable to, nor does it require any tissue excision. Based on the high affinity and specificity of the targeting polypeptide to PD-L1 and the enhanced tissue penetration thereof, the development of a polypeptide small molecule probe aiming at PD-L1 has important significance for 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 combined with the specificity and high efficiency of a marker PD-L1 protein related to various tumor immunotherapy; the invention also provides a product which is derived from the peptide and can be combined with PD-L1 protein, and the application of the polypeptide or the derived product thereof in preparing a preventive or therapeutic drug, a diagnostic reagent or an imaging preparation.

In order to achieve the above purpose, the technical scheme of the invention is as follows: the invention finds that some key amino acids of a CDR loop region play a main role in the binding process, such as PD-1 (Val 64, ile126, leu128, ala132, ile 134) and PD-L1 (Ile 54, tyr56, met115, ala121, tyr 123), through analysis and induction of crystal structure analysis of related PD-1-inhibitor-PD-L1 complexes in a PDB database. Three major hot spots were selected in the PD-L1/PD-1 interaction surface. The first hotspot was a classical hydrophobic pocket consisting of side chains of Tyr56, glu58, arg113, met115 and Tyr123 and sized to accommodate six-membered aromatic rings (this pocket is known as the Ile134 pocket). The second hotspot, located near Ile126, consists of Met115, ala121 and Tyr123, which can be anchored to the carbonyl-end donor group of Ala 121. The third hotspot is an extended slot containing Tyr68, gln75 and Thr 76. The invention designs and constructs the peptide library according to the hot spot amino acid locus 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 for carrying out mixing and equipartition to synthesize a single-bead one-peptide library. And (3) screening a high-flux one-bead one-object peptide library by utilizing 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 to PD-L1.

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

(1)NX ₅ X ₄ X ₃ EX ₂ X ₁ wherein X is ₁ Arginine, glutamine, aspartic acid, histidine, lysine or glutamic acid; x is X ₂ Glutamic acid, aspartic acid, leucine, tyrosine, threonine or glycine; x is X ₃ Is histidine, lysine, tyrosine, threonine, serine or glutamic acid; x is X ₄ Tryptophan, aspartic acid, tyrosine, arginine, histidine or lysine; x is X ₅ Is lysine, threonine, tyrosine, histidineProline or glutamic acid;

(2)X ₈ YX ₇ X ₆ TX ₅ X ₄ X ₃ X ₂ X ₁ wherein X is ₁ Arginine, glutamine, aspartic acid, histidine, lysine or glutamic acid; x is X ₂ Glutamic acid, aspartic acid, leucine, tyrosine, threonine or glycine; x is X ₃ Is histidine, lysine, tyrosine, threonine, serine or glutamic acid; x is X ₄ Serine, aspartic acid, tyrosine, arginine, histidine or lysine; x is X ₅ Lysine, threonine, tyrosine, histidine, tryptophan or glutamic acid; x is X ₆ Lysine, threonine, glutamic acid or tyrosine; x is X ₇ Methionine, asparagine or arginine; x is X ₈ Arginine, lysine, threonine or serine;

(3)X ₁₅ X ₁₄ X ₁₃ X ₁₂ X ₁₁ X ₁₀ X ₉ X ₈ X ₇ X ₆ X ₅ X ₄ X ₃ X ₂ X ₁ wherein X is ₁ Glutamic acid, glutamine or aspartic acid; x is X ₂ Glutamic acid, aspartic acid, histidine or serine; x is X ₃ Glutamic acid, serine or alanine; x is X ₄ Threonine, lysine, tyrosine, arginine or proline; x is X ₅ Tryptophan, tyrosine, arginine or histidine; x is X ₆ Lysine, threonine or tyrosine; x is X ₇ Is asparagine, threonine, glutamic acid, isoleucine or glutamine; x is X ₈ Methionine, asparagine or arginine; x is X ₉ Tyrosine, alanine or aspartic acid; x is X ₁₀ Arginine, lysine, threonine or serine; x is X ₁₁ Is threonine or proline; x is X ₁₂ Is glutamine or lysine; x is X ₁₃ Is phenylalanine or arginine; x is X ₁₄ Is histidine or tyrosine; x is X ₁₅ Is alanine or glutamine.

Preferably, the amino acid sequence of the polypeptide targeting PD-L1 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 to PD-L1.

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

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

Preferably, the nucleic acid comprises a nucleotide sequence encoding a polypeptide having an amino acid sequence of NKWTEDE, RYMETWKSDQ, KYNETWRSED, AHFQYTAREYRPAHE, QYFQTKDRIYHPAS or AHRKPSARQYRPASE.

The invention also provides biological materials comprising the nucleic acids.

The biological material includes an expression cassette, a vector, a transposon, a host cell or a transgenic cell line.

Such vectors include, but are not limited to, cloning vectors, expression vectors, plasmid vectors, all of which comprise at least one copy of the nucleic acid encoding the PD-L1-targeting polypeptide of the invention are within the scope of the invention.

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

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

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

The invention also provides derivatives of the polypeptides, which are bivalent or multivalent entities formed from the polypeptides, which are capable of targeting PD-L1.

Preferably, the bivalent or multivalent entity is formed by covalent or non-covalent attachment of a linker molecule or by non-covalent attachment by admixture with a multimer.

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

More preferably, the non-covalently linked linking 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 or a combination of at least two of polyethylene glycol (PEG), polyvinyl alcohol (PVA), cyclodextrin, polyamide-amine dendrimer (PAMAM), polylactic acid (PLA), and polylactic acid-ethanolamine (PLGA).

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 with a carrier in a covalent or non-covalent way.

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 polymer 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 comprises any one or more of nano gold, carbon material, calcium material, magnetic material, mesoporous silicon material and quantum dot.

The invention further provides the use of any one of said polypeptide or a nucleic acid encoding said polypeptide or a biological material comprising said nucleic acid or a derivative of said polypeptide or said conjugate:

(1) The application in preparing medicaments, diagnostic reagents, diagnostic kits or imaging preparations for preventing or treating PD-L1/PD1 mediated diseases;

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

(3) Use in the preparation of a reagent for detecting the expression level of PD-L1 in a cell;

(4) Use in detecting the level of cellular PD-L1 expression.

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, kidney cancer, prostate cancer, glioma, colorectal cancer, pancreatic cancer, cholangiocarcinoma, liver cancer, gastric cancer, esophagus cancer and breast cancer.

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

Preferably, the active ingredient of the medicament further comprises a formulation 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 photothermal therapy or a photodynamic therapy drug capable of killing tumor cells; or any one or more of alkylating agent, antimetabolite, natural antineoplastic agent, antitumor antibiotic, hormone, metal complex or tumor-emitting targeting marker.

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 use in the preparation of targeted drugs.

The carrier comprises any one of nano materials, liposome or oily compounds, or a mixture composed 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 transported to the target cell more stably in the body.

More preferably, the medicament may further comprise pharmaceutically acceptable excipients or adjuvants.

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

The invention also provides a diagnostic reagent or kit for a PD-L1/PD1 mediated disease comprising said polypeptide or a derivative of said polypeptide or said 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 label, magnetic resonance contrast agent or molecular imaging agent.

More preferably, in the imaging agent, the polypeptide or derivative of the polypeptide or the conjugate is coupled to or mixed with the imaging agent.

The invention has the beneficial effects that:

the PD-L1 targeting polypeptide provided by the invention 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 and reliability, can be prepared by adopting a chemical synthesis method, is simple and feasible in preparation method, low in cost and the like, and can be practically used for:

(1) The small molecular probe for detecting the PD-L1 expression condition in tumor cells is used for preparing a prediction and accompanying diagnostic reagent based on PD-L1 immunotherapy to monitor the curative effect of the immunotherapy in real time and screen patients suitable for the immunotherapy, provides a simple, convenient, quick, economical and accurate detection means for monitoring the recurrence and metastasis after the immunotherapy of the tumor in real time, so as to adjust the treatment scheme in time, perform clinical intervention as early as possible, prevent the progress of the illness state and improve the prognosis of the patients. The detection of the PD-L1 biomarker is beneficial to the establishment of a personalized treatment scheme to obtain an optimal treatment effect, and has good application prospect and clinical guidance significance.

(2) The targeting polypeptide is conjugated or mixed with a preparation capable of killing cancer cells and is used for targeted treatment and imaging of various tumors, or the targeting polypeptide is independently used as an active ingredient of a polypeptide inhibitor drug after being optimized, blocks PD-1/PD-L1 signal paths and activates immunotherapy. In the existing immunotherapy based on PD-1/PD-L1 blocker, how to apply to more malignant tumor treatments while reducing adverse reactions, provides important theory and reference significance for combined therapy with other immunotherapy, chemotherapy, radiotherapy and small molecule targeted drugs, and has wide application prospect.

The PD-L1 targeting polypeptide provided by the invention can provide important theoretical and clinical reference basis for early diagnosis of melanoma, non-small cell lung cancer, kidney cancer, prostate cancer, glioma, colorectal cancer, pancreatic cancer, cholangiocarcinoma, liver cancer, gastric cancer, esophagus cancer, breast cancer and other tumors, dynamic monitoring of check points in immunotherapy and the like, and has important significance and application value for optimizing the treatment scheme of individual patients. In view of the fact that the PD-1/PD-L1 pathway also plays an important role in regulating other multisystem diseases, the polypeptide provides a new thought for the immunotherapy of diseases such as rheumatoid arthritis, HCV virus infection, allergic purpura, aplastic anemia, atherosclerosis and coronary heart disease, screening and prognosis detection evaluation of suitable people and the like.

Drawings

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

FIG. 2 is a screening chart of PD-L1 positive polypeptides-magnetic beads in example 1 of the present invention.

FIG. 3 shows the detection of the affinity of PD-L1 positive polypeptides with human PD-L1 protein by Surface Plasmon Resonance (SPRi) method in example 2 of the present invention; wherein, (a) - (f) are the results of binding assays of polypeptides P1, P2, P3, P4, P5, P6, respectively, to human PD-L1 protein.

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

FIG. 5 shows the detection of specific affinities of polypeptides P3 and P4 of example 6 of the present invention with the cellular levels of the PD-L1 highly expressed cell lines MDA-MB-231, MC38 and negative cell A549, respectively; wherein, (a) is the detection of the cell level specific affinity of the polypeptide P3 and the cell lines MDA-MB-231, MC38 and negative cell A549 with high expression of PD-L1; (b) Is the detection of the specific affinity of the polypeptide P4 and the cell level of the cell lines MDA-MB-231, MC38 and negative cell A549 with high expression of PD-L1.

FIG. 6 shows the detection of specific affinities of polypeptides P5 and P6 of example 6 of the present invention with the cellular levels of the PD-L1 highly expressed cell lines MDA-MB-231, MC38 and negative cell A549, respectively; wherein, (a) is the detection of the cell level specific affinity of the polypeptide P5 and the cell lines MDA-MB-231, MC38 and negative cell A549 with high expression of PD-L1; (b) Is the detection of the specific affinity of the polypeptide P6 and the cell level of the cell lines MDA-MB-231, MC38 and negative cell A549 with high expression of PD-L1.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to 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 may be made by those skilled in the art without departing from the spirit and scope of this invention.

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

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

EXAMPLE 1 construction and screening of PD-L1 targeting polypeptide libraries

Experimental apparatus and materials

N-methylmorpholine (NMM), piperidine, trifluoroacetic acid (TFA), dichloromethane (DCM), ninhydrin, vitamin C, phenol, tetramethylurea Hexafluorophosphate (HBTU), piperidine, triisopropylsilane (TIS), ethylenediamine (EDT), N, N-Dimethylformamide (DMF), dehydrated ether, resins, methanol, various Fmoc protected amino acids, streptavidin magnetic beads (MB-strepitavidin), biotin labeling kits, polypeptide synthesis tubes, shaker, vacuum water pump, rotary evaporator, laser confocal microscope (ZEISS LSM 710), all of which are commercially available.

(II) solvent formulation

The deprotected solvent is prepared as piperidine: n, N dimethylformamide = 1:4, a step of;

the reaction solution was prepared as N-methylmorpholine N, N dimethylformamide=1: 24, a step of detecting the position of the base;

the lysate is prepared from 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 a (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 comprises the steps of coupling amino acids to solid-phase resin one by one randomly in a mixed splitting mode, removing side chain protecting groups under strong acid, and then screening. The specific method comprises the following steps:

(1) 400mg of Tentagel-NH was weighed out ₂ Resin, according to the solid-phase polypeptide synthesis program circulation, sequentially adding 200mg of Met and Gly to sequentially react;

(2) After the reaction is finished, dividing the resin into 3 parts, respectively adding 100mg of Glu, asp, gln and equal amount of HBTU into each tube for coupling, after the coupling is finished, deprotecting the 3 tubes of resin, mixing, dividing the resin into 4 parts, respectively adding 100mg of Glu, asp, his, ser and equal amount of HBTU into each tube for coupling;

(3) After the coupling is completed, the 4-tube resin is mixed after deprotection. The resin was then split equally into 3 parts, and 110mg of Glu, ser, ala was added to each tube separately to couple with equal amounts of HBTU;

(4) After the coupling is completed, the 4-tube resin is mixed after deprotection. The resin was then split equally into 5 parts, and 60mg of Thr, lys, tyr, arg, pro was added to each tube separately to couple with equal amounts of HBTU;

(5) After the coupling is completed, the 5-tube resin is mixed after deprotection. The resin was then split equally into 4 parts, and 80mg of Trp, tyr, arg, his was added to each tube separately to couple with equal amounts of HBTU;

(6) After the coupling is completed, the 4-tube resin is mixed after deprotection. The resin was then split equally into 3 parts, and 90mg of Lys, thr, tyr was added to each tube separately to couple with equal amounts of HBTU;

(7) After the coupling is completed, the 3-tube resin is mixed after deprotection. The resin was then split equally into 5 parts, and 70mg of Asn, glu, thr, gln, ile was added to each tube separately to couple with equal amounts of HBTU;

(8) After the coupling is completed, the 5-tube resin is mixed after deprotection. The resin was then split equally into 3 parts, and 120mg of Met, asn, arg was added to each tube separately to couple with equal amounts of HBTU;

(9) After the coupling is completed, the 3-tube resin is mixed after deprotection. The resin was then split equally into 3 parts, and 80mg of Tyr, ala, asp was added to each tube separately to couple with equal amounts of HBTU;

(10) After the coupling is completed, the 3-tube resin is mixed after deprotection. The resin was then split equally into 4 parts, and 80mg of Arg, lys, thr, ser was added to each tube separately to couple with equal amounts of HBTU;

(11) After the coupling is completed, the 4-tube resin is mixed after deprotection. Dividing the resin into 2 parts, and adding 120mg of Thr and Pro into each tube respectively to couple with the same amount of HBTU;

(12) After the coupling is completed, the 2 tubes of resin are mixed after deprotection. Then dividing the resin into 2 parts, and adding 180mg of Gln and Lys into each tube to couple with the same amount of HBTU;

(13) After the coupling is completed, the 2 tubes of resin are mixed after deprotection. Dividing the resin into 2 parts, and adding 120mg of Phe and Arg and equivalent HBTU into each tube for coupling;

(14) After the coupling is completed, the 2 tubes of resin are mixed after deprotection. Dividing the resin into 2 parts, and respectively adding 180mg of His and Tyr into each tube to couple with the same amount of HBTU;

(15) After the coupling is completed, the 2 tubes of resin are mixed after deprotection. The resin was then split into 2 parts, 180mg of Ala, gln and equal amounts of HBTU were added to each tube for coupling, and after coupling, the 2 tubes were deprotected and mixed. And (3) through methanol replacement and shrinkage steps, vacuum drying is carried out, and the dried resin loaded with the peptide library is obtained for standby.

(IV) screening of 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 peptide beads on a mixer at 37deg.C for 2h, and washing with 1 XPBS for 3 times;

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

(3) 100. Mu.L of MB-strepitavidine was then added to the peptide library and incubated for 2h on a mixer with mixing at 37℃in the dark. The incubated polypeptide library EP-containing tube was placed on a magnetic rack. The positive polypeptides are magnetically attracted to the EP tube side wall, while the negative polypeptides settle to the EP tube bottom due to gravity.

The principle of screening PD-L1 targeting polypeptide is schematically shown in figure 1, and when positive polypeptide beads are incubated with biotin-labeled receptor proteins, positive peptide beads specifically recognize proteins, and streptavidin-labeled magnetic beads recognize positive peptide beads by recognizing biotin. The PD-L1 positive polypeptide-magnetic bead screening result is shown in figure 2, wherein positive peptide beads are in a dotted line frame, and the surfaces of the positive peptide beads are magnetic due to the fact that the surfaces of the positive peptide beads are coated with a layer of magnetic beads, so that the positive peptide beads 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 are shown in a table 1, and 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 the table 1. The positive polypeptides were re-synthesized according to the amino acid sequences shown in Table 1 and fluorescence was labeled at the N-terminus of the polypeptides for cell imaging to verify the function of the polypeptides, which were used in subsequent experiments after MALDI-TOF identification and HPLC purification.

Table 1 amino acid sequence of PD-L1 Positive polypeptide

Sequence 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 affinity of Polypeptides P1, P2, P3, P4, P5, P6 with PD-L1 proteins

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

1mg/mL of the P1, P2, P3, P4, P5, P6 polypeptide and 1 XPBS were spotted onto the chip, incubated overnight at 4deg.C under wet conditions, then washed with 10 XPBS for 10min, then with 1 XPBS for 10min, finally with deionized water for 2 times, 10min each time, immersed in 1 XPBS containing 5% skimmed milk, incubated overnight at 4deg.C, then with 10 XPBS for 10min,1 XPBS for 10min, finally with deionized water for 2 times, 10min each time, blow-dried with nitrogen, and loaded onto the chip (PlexeraHT surface plasmon resonance imaging system).

The mobile phase was purified by passing through 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 in this order, and the SPRi signal was recorded and analyzed.

As shown in FIG. 3, the SPRi signals of P1, P2, P3, P4, P5 and P6 are gradually enhanced along with the increase of the protein concentration, which proves 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 ^-9 M, approaching the affinity of the antibody, can be used as a probe for targeting various tumor cells expressing PD-L1 for related research and application.

EXAMPLE 3 preparation of imaging Agents 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 methods:

300mg of wang-Glu resin is weighed, circulated according to a solid-phase polypeptide synthesis program, deprotected first, and then a certain amount of Glu, asp, glu, thr, trp, lys, asn and an equal amount of HTBU are sequentially added for reaction. And (3) deprotection and cleaning after the coupling reaction is finished. And adding epsilon-aminocaproic acid for reaction, and deprotecting and cleaning after coupling is finished. And adding FITC for light-shielding coupling reaction, and removing protection and cleaning after the reaction is finished. Finally, removing side chain protecting groups from the lysate of the resin, vacuum drying to obtain a crude polypeptide fluorescent conjugate, and performing HPLC purification to obtain imaging preparations of polypeptides P1, P2, P3, P4, P5 and P6 with purity of 95 percent, which can be used for fluorescent imaging.

Example 4 preparation of conjugates of 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 methods:

300mg of wang-Glu resin is weighed, circulated according to a solid-phase polypeptide synthesis program, deprotected first, and then a certain amount of Glu, asp, glu, thr, trp, lys, asn and an equal amount of HTBU are sequentially added for reaction. And (3) deprotection and cleaning after the coupling reaction is finished. Epsilon-aminocaproic acid was added to the reaction, and FITC was mixed with the peptide beads in a 1:5:7 pyridine/N, N-dimethylformamide/dichloromethane ratio to react overnight. After the reaction, cleaning. Finally, removing side chain protecting groups from the lysate in the resin, and vacuum drying to obtain the crude polypeptide fluorescent conjugate.

Example 5 preparation of kits based on polypeptides P1, P2, P3, P4, P5, P6

Polypeptide Fluorescein Isothiocyanate (FITC) conjugate is obtained by adopting a solid phase synthesis method, and epsilon-aminocaproic acid is continuously coupled on the synthesized P1, P2, P3, P4, P5 and P6 polypeptide microbeads respectively. FITC was mixed with peptide beads in a 1:5:7 ratio of pyridine/N, N dimethylformamide/dichloromethane solution overnight. After cleavage by the lysate, P1, P2, P3, P4, P5, P6 polypeptide FITC conjugates were obtained and purified by MALDI-TOF identification and HPLC for subsequent experiments.

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

This example analyzes the interaction of polypeptides P1, P2, P3, P4, P5, P6 with PD-L1 highly expressed cells MDA-MB-231 and MC38 and low expressing cell A549, wherein the breast cancer cell line MDA-MB-231 was cultured in DMEM medium containing 10% fetal bovine serum, the colon cancer cell line MC38 was cultured in MEM/EBSS medium containing 10% fetal bovine serum, and the non-small cell lung cancer cell line A549 was 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 1X 10 ⁵ Cell concentration/mL was seeded in round glass bottom dishes (35 mm), 37℃at 5% CO ₂ After culturing for 24 hours in a cell culture box, the culture solution is discarded, and after 1 mu mol/L Hoechst is added into three cells respectively and incubated for 15 minutes at 33342,4 ℃ in a dark place, the three cells are washed for 2 times by precooled 1 XPBS, 50 mu M of FITC labeled polypeptide P1 is added respectively, incubated for 20 minutes at 4 ℃ in a dark place, and the three cells are washed for 3 times by precooled 1 XPBS. Fluorescence distribution in cells was detected with a laser scanning confocal microscope (ZEISS LSM 710).

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

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

At 1X 10 ⁵ Cell concentration/mL was seeded in round glass bottom dishes (35 mm), 37℃at 5% CO ₂ After culturing for 24 hours in the cell culture box, the culture solution is discarded,after adding 1. Mu. Mol/L Hoechst 33342,4 ℃for 15min in the dark, the three cells were washed 2 times with pre-chilled 1 XPBS, 50. Mu.M FITC-labeled polypeptide P2 was added, and after incubation 20min at 4℃in the dark, the cells were washed 3 times with pre-chilled 1 XPBS. Fluorescence distribution in cells was detected with a laser scanning confocal microscope (ZEISS LSM 710).

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

(3) Interaction of polypeptide P3 with PD-L1 highly expressed cells MDA-MB-231 and MC38 and lowly expressed cell A549, respectively

At 1X 10 ⁵ Cell concentration/mL was seeded in round glass bottom dishes (35 mm), 37℃at 5% CO ₂ After culturing for 24 hours in a cell culture box, the culture solution is discarded, and after 1 mu mol/L Hoechst is added into three cells respectively and incubated for 15 minutes at 33342,4 ℃ in a dark place, the three cells are washed for 2 times by precooled 1 XPBS, 50 mu M of FITC labeled polypeptide P3 is added respectively, and after 20 minutes at 4 ℃ in a dark place, the three cells are washed for 3 times by precooled 1 XPBS. Fluorescence distribution in cells was detected with a laser scanning confocal microscope (ZEISS LSM 710).

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

(4) Interaction of polypeptide P4 with PD-L1 highly expressed cells MDA-MB-231 and MC38 and lowly expressed cell A549, respectively

At 1X 10 ⁵ Cell concentration/mL was seeded in round glass bottom dishes (35 mm), 37℃at 5% CO ₂ After culturing for 24 hours in a cell culture box, the culture solution is discarded, and after 1 mu mol/L Hoechst is added into three cells respectively and incubated for 15 minutes at 33342,4 ℃ in a dark place, the three cells are washed for 2 times by precooled 1 XPBS, 50 mu M of FITC labeled polypeptide P4 is added respectively, incubated for 20 minutes at 4 ℃ in a dark place, and the three cells are washed for 3 times by precooled 1 XPBS. Fluorescence distribution in cells was detected with a laser scanning confocal microscope (ZEISS LSM 710).

As a result, as shown in FIG. 5 b, strong green fluorescence was observed on the cell membranes of MDA-MB-231 and MC38 added to the polypeptide P4, whereas A549 cells did not. The result shows that the P4 polypeptide is combined on the cell membrane of the positive cell, has specificity with the identification of the human PD-L1 positive cell line, has positive correlation with the expression quantity of the target protein, and can be used as a targeting molecule for related diagnosis and detection. In contrast, no green fluorescent signal and no antibody signal were observed for negative cells a549 with low PD-L1 expression by the P4 polypeptide, further demonstrating that polypeptide P4 is able to 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 highly expressed cells MDA-MB-231 and MC38 and lowly expressed cell A549, respectively

At 1X 10 ⁵ Cell concentration/mL was seeded in round glass bottom dishes (35 mm), 37℃at 5% CO ₂ After culturing for 24 hours in a cell culture box, the culture solution is discarded, and after 1 mu mol/L Hoechst is added into three cells respectively and incubated for 15 minutes at 33342,4 ℃ in a dark place, the three cells are washed for 2 times by precooled 1 XPBS, 50 mu M of FITC labeled polypeptide P5 is added respectively, incubated for 20 minutes at 4 ℃ in a dark place, and the three cells are washed for 3 times by precooled 1 XPBS. Fluorescence distribution in cells was detected with a laser scanning confocal microscope (ZEISS LSM 710).

As a result, as shown in FIG. 6 a, strong green fluorescence was observed on the cell membranes of MDA-MB-231 and MC38 added to the polypeptide P5, whereas A549 cells did not. The result shows that the P5 polypeptide is combined on the cell membrane of the positive cell, has specificity with the identification of the human PD-L1 positive cell line, has positive correlation with the expression quantity of the target protein, and can be used as a targeting molecule for related diagnosis and detection. In contrast, no green fluorescent signal and no antibody signal were observed for negative cells a549 with low PD-L1 expression by the P5 polypeptide, further demonstrating that polypeptide P5 was able to 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 highly expressed cells MDA-MB-231 and MC38 and lowly expressed cell A549, respectively

At 1X 10 ⁵ Cell concentration/mL was seeded in round glass bottom dishes (35 mm), 37℃at 5% CO ₂ After culturing for 24 hours in a cell culture box, the culture solution is discarded, and after 1 mu mol/L Hoechst is added into three cells respectively and incubated for 15 minutes at 33342,4 ℃ in a dark place, the three cells are washed for 2 times by precooled 1 XPBS, 50 mu M of FITC labeled polypeptide P6 is added respectively, incubated for 20 minutes at 4 ℃ in a dark place, and the three cells are washed for 3 times by precooled 1 XPBS. Fluorescence distribution in cells was detected with a laser scanning confocal microscope (ZEISS LSM 710).

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

In summary, the polypeptides P1, P2, P3, P4, P5, P6 of the present invention have the property of targeting tumor cells expressing PD-L1 positive, so that in practical application, the polypeptides P1, P2, P3, P4, P5, P6 of the present invention can be conjugated or mixed with agents capable of killing cancer cells as targeting polypeptides for targeted treatment and imaging of tumors.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Sequence listing

<110> national center of nanoscience

<120> a PD-L1 targeting polypeptide and use thereof

<130> KHP211123099.8D

<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 PD-L1-targeting polypeptide, which has the amino acid sequence AHFQYTAREYRPAHE.

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

3. A biological material comprising the nucleic acid of claim 2, wherein the biological material comprises an expression cassette, a vector, a transposon, a host cell, or a transgenic cell line.

4. A conjugate of a polypeptide according to claim 1, wherein the conjugate is obtained by covalent or non-covalent attachment or interaction of the polypeptide according to claim 1 with a carrier;

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

5. Use of a polypeptide according to claim 1 or a nucleic acid according to claim 2 or a biomaterial according to claim 3 or a conjugate according to claim 4, for any of the following:

(1) Use in the preparation of a diagnostic reagent, diagnostic kit or imaging formulation for PD-L1 mediated diseases;

(2) Use in the preparation of a reagent for detecting the expression level of cellular PD-L1.

6. A diagnostic reagent or kit for PD-L1 mediated diseases, comprising the polypeptide of claim 1 or the conjugate of claim 4.

7. An imaging agent comprising the polypeptide of claim 1 or the conjugate of claim 4.

8. The imaging agent of claim 7, further comprising an imaging agent, wherein the imaging agent is any one of a radionuclide, a radionuclide label, a magnetic resonance contrast agent, or a molecular imaging agent.

9. The imaging formulation of claim 8, wherein the polypeptide of claim 1 or the conjugate of claim 4 is conjugated or mixed with the imaging agent in the imaging formulation.