PDL1 targeting polypeptide, molecular imaging probe and application of PDL1 targeting polypeptide and molecular imaging probe

Abstract

The invention relates to a PDL1 targeting polypeptide, a molecular imaging probe and application of the PDL1 targeting polypeptide. The PDL1 targeting polypeptide comprises any one group of the following polypeptides: (1) a polypeptide as shown in at least one of SEQ ID NO: 1 to SEQ ID NO: 9; and (2) polypeptide which is obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence in (1), has the same function and is derived from the amino acid sequence in (1). The invention discloses a magnetic resonance molecular imaging probe based on PDL1 targeting polypeptide, which realizes early diagnosis and evaluation of tumor and immune myocardial injury.

Description

Technical Field

[0001] The present application relates to the field of biomedical technology, and in particular to a PDL1 targeting polypeptide, a molecular imaging probe and applications thereof. Background Art

[0002] Immune checkpoint inhibitors (ICIs) have attracted extensive attention and research in the comprehensive treatment of tumors by activating the immune system and specifically targeting, and have significantly improved the prognosis of tumor patients. PD-1 is an inhibitory receptor on the surface of T cells. When PD-1 binds to its ligand PD-L1, it inhibits the activity of T cells, thereby reducing the immune response. Many tumor cells escape the attack of the immune system by expressing PD-L1. PD-1 / PD-L1 inhibitors restore the anti-tumor function of T cells by blocking this pathway. With the increase in the application of immune checkpoint inhibitors, the incidence of immune-related adverse events (irAEs) has gradually increased. Related studies have shown that the incidence of severe irAEs in patients receiving combined immunotherapy is as high as 59%. In addition, the incidence of immunotherapy-related cardiovascular adverse events (cardiovascular death, cardiac arrest, and high-degree heart block) has increased by about 4 times. Among them, the mortality rate of immunotherapy-related myocarditis is as high as 40%-60%. Therefore, there is an urgent need for a technical means to monitor immune myocardial toxicity damage early in the clinic to guide treatment decisions. Summary of the invention

[0003] Based on this, one embodiment of the present application provides a PDL1 targeting polypeptide and constructs a magnetic resonance molecular imaging probe based on the PDL1 targeting polypeptide to achieve early diagnosis of immune myocardial injury and early monitoring of immune myocardial injury to guide treatment decisions.

[0004] The technical solutions include the following:

[0005] One embodiment of the present application provides a PDL1 targeting polypeptide, wherein the PDL1 targeting polypeptide includes any one of the following polypeptides:

[0006] (1) a polypeptide as shown in at least one of SEQ ID NO: 1 to SEQ ID NO: 9;

[0007] (2) A polypeptide derived from the amino acid sequence of (1) with the same function as that of the amino acid sequence of (1) after one or more amino acid residues are replaced and / or deleted and / or added.

[0008] In one preferred embodiment, the PDL1 targeting polypeptide comprises a polypeptide as shown in at least one of SEQ ID NO: 1 to SEQ ID NO: 4. The polypeptide has high affinity. In one preferred embodiment, the PDL1 targeting polypeptide comprises a polypeptide as shown in SEQ ID NO: 1.

[0009] One embodiment of the present application further provides a lipid carrier, wherein the lipid carrier comprises a targeting lipid containing the PDL1 targeting polypeptide.

[0010] In one embodiment, the targeting lipid further comprises DSPE-PEG2000-NHS.

[0011] In one embodiment, the PDL1 targeting polypeptide is linked to the DSPE-PEG2000-NHS via an amide bond.

[0012] In one embodiment, the molar ratio of the PDL1 targeting polypeptide to the DSPE-PEG2000-NHS is 1:2.8.

[0013] In one embodiment, the PDL1 targeting polypeptide is modified on the surface of the lipid carrier.

[0014] In one embodiment, the lipid carrier further comprises an excipient.

[0015] In one embodiment, the excipients include one or more of hydrogenated soy lecithin, cholesterol and DSPE-PEG2000.

[0016] In one embodiment, in the lipid carrier, the molar ratio of hydrogenated soybean lecithin, cholesterol, DSPE-PEG2000 and targeting lipid is 55:40:3:2.

[0017] In one embodiment, the lipid carrier comprises a lipid nanoparticle.

[0018] One embodiment of the present application also provides a molecular imaging probe, comprising the PDL1 targeting polypeptide and also comprising a signaling molecule.

[0019] This application uses phage display technology to screen PDL1 targeting peptides and construct molecular imaging probes based on PDL1 targeting peptides to provide a basis for the early diagnosis of immune myocardial injury.

[0020] Lipid carriers are nanovesicles composed of a phospholipid bilayer and have become a drug delivery carrier that has attracted much attention in recent years. Passive targeting of target tissues can be achieved by modifying the particle size of liposomes. In addition, lipid carriers can also be used to encapsulate signal molecules (such as magnetic resonance contrast agent Gd-DTPA) to achieve imaging.

[0021] Therefore, one embodiment of the present application also provides a molecular imaging probe, which includes the lipid carrier and also includes a signal molecule.

[0022] In one embodiment, the signal molecule is encapsulated inside the lipid carrier.

[0023] In one embodiment, the signal molecule has imaging function.

[0024] In one embodiment, the imaging function includes at least one of fluorescence imaging, magnetic resonance imaging, nuclear imaging, ultrasound imaging, photoacoustic imaging and electronic computer tomography imaging.

[0025] In one embodiment, the signal molecule having magnetic resonance imaging function includes at least one of a gadolinium-based contrast agent (such as Gd-DTPA), an iron-based contrast agent, and a manganese-based contrast agent.

[0026] In one embodiment, the average particle size of the molecular imaging probe is 138.71±11.96 nm, and the average charge is -10.60±1.51 mV.

[0027] An embodiment of the present application further provides a method for preparing the molecular imaging probe, comprising the following steps S1 to S3:

[0028] Step S1, reacting the DSPE-PEG2000-NHS with the PDL1 targeting polypeptide to prepare a targeting lipid.

[0029] In one embodiment, the DSPE-PEG2000-NHS and the PDL1 targeting polypeptide are dissolved in DMSO solution for reaction.

[0030] In one embodiment, the reaction is carried out at room temperature for 1 h to 2 h.

[0031] In one embodiment, the reaction further includes freeze-drying.

[0032] In one embodiment, DSPE-PEG2000-NHS and PDL1 targeting polypeptide were dissolved in DMSO, incubated at room temperature for 1 hour, and freeze-dried to obtain targeting lipids.

[0033] Step S2, dissolving the hydrogenated soybean lecithin, cholesterol, DSPE-PEG2000, targeting lipid and signal molecule with an organic solvent, mixing and removing the organic solvent to prepare a lipid film.

[0034] In one embodiment, the organic solvent comprises ethanol.

[0035] In one embodiment, the mixing comprises stirring.

[0036] In one embodiment, rotary vacuum is used to remove the ethanol.

[0037] In one embodiment, the mass and molar ratio of the targeting lipid to the signal molecule is (1-1.3) mg:2 mmol. Optionally, the mass and molar ratio of the targeting lipid to the signal molecule is 1 mg:2 mmol, 1.1 mg:2 mmol, 1.2 mg:2 mmol, 1.3 mg:2 mmol, or a range consisting of any of the above ratios.

[0038] Step S3, hydrating the lipid film to prepare a molecular imaging probe.

[0039] In one embodiment, a hydration medium is added to the lipid film, and the film is rotated and hydrated for 50 to 70 minutes, followed by ultrasonic treatment to prepare a molecular imaging probe.

[0040] In one embodiment, the hydration medium comprises PBS buffer.

[0041] In one embodiment, the ultrasonic time is 1 min to 2 min.

[0042] In one embodiment, the process further includes filtering and freeze-drying after sonication.

[0043] One embodiment of the present application also provides the use of the PDL1 targeting polypeptide, the lipid carrier or the molecular imaging probe in the preparation of a reagent for qualitatively and / or quantitatively detecting PDL1 expression, or expression location.

[0044] One embodiment of the present application also provides the use of the PDL1 targeting polypeptide, the lipid carrier or the molecular imaging probe in the preparation of reagents for diagnosis or tracing imaging of tumors or immune myocardial damage.

[0045] This tracer imaging reagent is used to monitor the development of diseases related to high PDL1 expression and provide a reference for clinical treatment decisions.

[0046] In one embodiment, the diagnosis includes early diagnosis of tumor or immune myocardial injury.

[0047] One embodiment of the present application also provides the use of the PDL1 targeting polypeptide, the lipid carrier or the molecular imaging probe in the preparation of a reagent for qualitatively and / or quantitatively detecting PDL1 expression or expression location.

[0048] One embodiment of the present application also provides the use of the PDL1 targeting polypeptide, the lipid carrier or the molecular imaging probe in the preparation of a drug for preventing and / or treating tumors.

[0049] One embodiment of the present application also provides the use of the PDL1 targeting polypeptide, the lipid carrier or the molecular imaging probe in the preparation of an evaluation product for diseases related to abnormal PDL1 expression.

[0050] In one embodiment, the diseases associated with abnormal PDL1 expression include immune myocardial injury and malignant tumors.

[0051] This assessment product can evaluate the extent of immune myocardial damage or the extent of tumor development and treatment, and guide clinical treatment decisions.

[0052] In one embodiment, abnormal expression of PDL1 includes abnormal increase or abnormal decrease.

[0053] In one embodiment, the malignant tumor includes but is not limited to melanoma, lung cancer, and renal cell carcinoma.

[0054] This application uses phage display technology to screen PDL1 targeting peptides, constructs a lipid carrier-coated magnetic resonance contrast agent (Gd-DTPA), and modifies the molecular imaging probe of the PDL1 targeting peptide on the surface of the lipid carrier. Through the targeting property of the PDL1 polypeptide, the molecular probe has the function of actively targeting to the inflammatory site, which can achieve early and accurate diagnosis of immune myocardial injury.

[0055] Therefore, one embodiment of the present application also provides a product for early diagnosis or evaluation of immune myocardial injury, including the PDL1 targeting polypeptide, the lipid carrier or the molecular imaging probe.

[0056] In one embodiment, the product comprises an early diagnosis or evaluation kit.

[0057] Compared with the traditional technology, this application has the following beneficial effects:

[0058] The present application uses phage display technology to screen and provide a circular and linear polypeptide and its sequence with specific targeting of PDL1 protein. After processing and modification, the polypeptide can be subsequently used in the research and development of PDL1 targeting probes and the development of specific targeted drugs, and has potential targeted diagnosis and treatment value for diseases with high expression of PDL1 protein.

[0059] Furthermore, the present application constructs a lipid carrier whose surface is modified with a PDL1 targeting polypeptide, and encapsulates a contrast agent with an imaging function, such as Gd-DTPA, to form a molecular imaging probe with a specific diagnostic function. The molecular imaging probe can actively target and deliver Gd-DTPA to the site of immune myocardial injury through the specific targeting of the PDL1 polypeptide, and then evaluate the immune myocardial injury through magnetic resonance imaging. The molecular imaging probe has good stability, high drug loading, simple and controllable preparation method, can realize mass production, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0061] Figure 1 Phage ELISA was used to evaluate the affinity of PDL1 targeting peptides to PDL1 protein.

[0062] Figure 2 The affinity of PDL1 targeting peptide to PDL1 protein was evaluated by SPR.

[0063] Figure 3A and Figure 3B It is the structural formula of PDL1 targeting peptide.

[0064] Figure 4 This is the AlphaFold3 predicted structure superposition of PDL1 targeting peptide and PDL1 protein.

[0065] Figure 5 .Binding mode of PDP#1 and Mouse PDL1 protein: In the figure, Mouse PDL1 protein is shown in green, PDP#1 is shown in blue, and the interacting residues are represented as Stick; the red dotted line is the hydrogen bond interaction, and the number represents the hydrogen bond length. The longer the bond length, the weaker the effect.

[0066] Figure 6 This is a TEM image of a targeted molecular probe, showing a circular structure with uniform particle size.

[0067] Figure 7 The particle size and charge of the targeted molecular probe.

[0068] Figure 8 To evaluate the efficacy of targeted molecular probes targeting PDL1 in vitro.

[0069] Fig. 9 Targeted molecular probes for in vivo evaluation of immune myocardial injury. DETAILED DESCRIPTION

[0070] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0072] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0073] Studies have shown that the expression of PDL1 in myocardial tissue is significantly increased in immune myocarditis. The inventors of this application found that the expression of PDL1 on the surface of myocardial cells and endothelial cells was significantly increased through immunohistochemical staining of myocardial samples from clinical patients with immune myocardial injury. By constructing a mouse model of immune myocarditis, it was found that the expression of PDL1 in endothelial cells and myocardial cells of myocardial tissue was increased in the early stage of immune myocardial injury. Therefore, by monitoring the expression of PDL1 in myocardial tissue, it is possible to achieve early identification of immune myocardial injury and provide a methodological basis for clinical decision-making.

[0074] Phage display technology is a powerful screening tool that is widely used to screen and identify polypeptides and proteins with specific binding properties. By cloning the coding gene of the polypeptide or protein into the structural gene of the phage coat protein, the exogenous polypeptide or protein is fused with the coat protein and expressed, and the fusion protein is expressed on the phage surface as the phage is reassembled. The displayed polypeptide or protein can maintain a relatively independent spatial structure and biological activity to facilitate the recognition and binding of the target molecule. Through multiple rounds of adsorption, elution, amplification and other screening processes, phages with specific affinity for the target are enriched. By sequencing the phage DNA, the polypeptide encoding nucleic acid sequence that binds to the target can be identified. The present application uses phage display technology to screen polypeptides with PDL1 specific targeting, and uses specific polypeptides to evaluate the expression of PDL1 in vivo.

[0075] Imaging examinations based on molecular probes can dynamically monitor changes in the molecular levels of diseased tissue cells, and are expected to achieve early diagnosis of immune myocardial injury. 68Ga-DOTATOC-PET / CT is a highly sensitive molecular probe for myocarditis, which can detect early myocardial injury lesions with elevated myocardial enzymes and normal MRI examinations. Due to the complex operation of PET / CT, low soft tissue resolution, and certain radiation damage, its application in the diagnosis of myocardial injury is limited. Cardiac magnetic resonance has the characteristics of no radiation and high soft tissue resolution. This application uses phage display technology to screen for PDL1-specific targeting peptide sequences, and constructs a magnetic resonance molecular imaging probe based on PDL1 targeting peptides to conduct early assessment of immune myocardial injury and provide a basis for clinical decision-making.

[0076] The present application provides a method for screening cyclic and linear polypeptides targeting programmed death ligand 1 (PDL1) using phage display technology, and application of the same in the diagnosis of immune myocardial injury.

[0077] PDL1 is an inhibitory immune checkpoint molecule that is widely expressed on the surface of myocardial cells and endothelial cells in immune myocardial injury. By constructing a peptide-based PDL1 targeted molecular imaging probe, the degree of myocardial injury in immune myocarditis can be assessed and clinical treatment decisions can be guided.

[0078] The present application provides a method for screening cyclic and linear peptides targeting programmed death ligand 1 (PDL1) by using phage display technology, comprising the following steps: incubating and panning the PDL1 protein with a phage display library for multiple rounds; recovering and amplifying the phages that bind to PDL1, and sequencing to obtain the coding sequence of the peptide; synthesizing the cyclic and linear peptides obtained by the screening, and performing in vitro binding experiments to verify the affinity with PDL1; performing in vitro cell experiments on the peptides obtained by the screening to evaluate their binding ability with PDL1; and evaluating the diagnostic efficacy of molecular imaging probes based on the PDL1 polypeptide in an animal model of myocardial injury.

[0079] Through the above method, the peptides screened in this application have high affinity and specificity, and can effectively bind to PDL1. In in vitro experiments, these peptides showed good affinity for PDL1, and the degree of myocardial damage in animal models of myocardial damage was evaluated by peptide-based molecular imaging probes, showing good imaging effects. Therefore, the polypeptides screened in this application have broad application prospects in the early assessment of immune myocardial damage. The present application provides a PDL1-targeting polypeptide sequence and its application in the diagnosis of immune myocardial damage, which is expected to provide an efficient and safe evaluation method for the evaluation of immune myocardial damage.

[0080] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0081] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0082] The phage display peptide library used in the following examples was commercially available from a biological company.

[0083] The PDL1-specific targeting polypeptides used in the following examples were synthesized by a biological company with a purity greater than 95%. The targeting polypeptides used were prepared into a mother solution of appropriate concentration using a solvent before the experiment.

[0084] The PDL1 targeting peptides in the following examples can all be prepared by standard Fmoc solid phase synthesis method.

[0085] Example 1 Screening of PDL1 targeting polypeptides using phage display technology

[0086] 1. Phage titer detection method

[0087] (1) After the ER2738 strain was inoculated into LB medium (containing 30 ug / mL Tet antibiotic) for activation, it was inoculated into 5 mL of fresh LB medium (containing 30 ug / mL Tet antibiotic) at a ratio of 1:100 and cultured until the OD600 was 0.4-0.6;

[0088] (2) Prepare agar plates: weigh 1.25 g IPTG and 1 g Xgal, dissolve in 25 mL DMF and store at -20°C until use; prepare agar plates by adding 15 g / L agar and 1 mL IPTG / Xgal storage solution to 1 L LB medium and store at 4°C in the dark until use;

[0089] (3) Dilute the phage with LB medium according to the 10-fold gradient dilution method; divide the expanded host bacteria equally according to the needs, add 10uL of the gradient phage diluted with LB to each tube, mix gently and incubate at room temperature for 30 minutes;

[0090] (4) Plating the infected host bacteria in step (3) on the plate prepared in step (2) and culturing at 37° C. overnight;

[0091] (5) Plaque counts.

[0092] 2. Phage expansion culture

[0093] (1) After the ER2738 strain was inoculated into LB medium (containing 30 μg / mL Tet antibiotic) for activation, it was inoculated into 5 mL of fresh LB medium (containing 30 μg / mL Tet antibiotic) at a ratio of 1:100 and cultured until the OD600 was 0.4-0.6;

[0094] (2) Take 10 μL of phage and inoculate it into the above-mentioned expanded host bacteria, shake and culture at 200 rpm and 37°C for 3-6 hours. Centrifuge at 8000 rpm to collect the supernatant, which is the phage culture.

[0095] 3. Phage display peptide library screening

[0096] (1) Antigen coating: Dilute the PDL1 protein standard to 5 μg / mL with PBS buffer. Select 2 duplicate wells of a 96-well ELISA plate and add 100 μL (400 ng / well) to each well. Coat overnight at 4°C. PBS is used as a negative control.

[0097] (2) Blocking: Discard the coating solution, add 200 μl of 2% skim milk powder to each well, and block at room temperature for 1 h;

[0098] (3) Incubation of phages: Wash with PBST three times, take the phage solution prepared in the first step, and dilute it to 5 × 10 11 pfu / mL, added to ELISA plate, 200 μl / well, incubated at room temperature for 2 h;

[0099] (4) Elution: discard the phage sample, wash three times with PBST, add 200 μL Tris-HCl (pH = 3.0) to each well, let stand at room temperature for 10 min, aspirate the eluate and quickly neutralize to about pH = 7.0 with 40 μL / well Tris-HCl (pH = 8.0) (the amount of pH 8.0 Tris-HCl added needs to be re-determined before each neutralization).

[0100] (5) Determination of phage titer in the eluate: Determine the titer of phage in the eluate as in step 1;

[0101] (6) Repeat the expansion culture and screening twice to complete the second and third rounds of selection.

[0102] (7) Sequencing the positive phage clones, using the sequencing primer -96gIII (5'-CCCTCATAGTTAG CGTAACG-3', SEQ ID NO: 10), and the sequencing direction was reverse sequencing;

[0103] (8) The 36 gene sequences between the two restriction sites were found and translated into amino acid sequences on the website https: / / web.expasy.org / translate / . The translation results of 3'-5' were the polypeptide sequences displayed by the phage, as shown in Table 1.

[0104] Table 1. Screening of PDL1 targeting peptide sequences using phage display technology

[0105]

[0106]

[0107] Example 2 PDL1 targeting peptide affinity detection

[0108] 1. Phage enzyme-linked immunosorbent assay (ELISA)

[0109] (1) Coat the PDL1 protein on the ELISA plate at 4°C overnight;

[0110] (2) Block with 0.5% (w / v) BSA solution for 1 hour;

[0111] (3) After washing three times with 0.1% TBST, monoclonal phage (100 μL; ~1.0×10 11 pfu / mL, diluted in TBS) was added to each well and incubated at room temperature for 2 hours with shaking; TBS was used as a control.

[0112] (4) HRP-labeled anti-M13 monoclonal antibody (1:5000) was added to the plate, incubated at room temperature for 1 hour, and then washed three times with 0.1% TBST;

[0113] (5) adding reaction buffer and stop buffer;

[0114] (6) Measure the absorbance at 405 nm using a UV spectrophotometer.

[0115] The peptides screened and synthesized and capable of binding to PDL1 were used to perform an in vitro binding test with the coated PDL1 protein. The results were as follows: Figure 1 As shown, the polypeptides were able to bind to the PDL1 protein in vitro, and the absorbance after the binding was detected at 405 nm.

[0116] 2. Surface-plasmon resonance (SPR)

[0117] 2.1 Protein coupling

[0118] (1) Place the running buffer (200 mL 1× PBS Buffer), water bottle, and waste liquid bottle in the left and right trays respectively, and insert the corresponding liquid inlet tubes.

[0119] (2) Hold the CM5 chip with the side with the words facing up. Gently push the chip into the card slot in the direction of the arrow on the chip, and then close the door of the chip compartment.

[0120] (3) Activate channel 2 of the chip with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, GE Healthcare) and N-hydroxysuccinimide (NHS, GE Healthcare) at a flow rate of 10 μL / min.

[0121] (4) The ligand protein was diluted to 50 μg / mL with sodium acetate at pH 4.0, and the protein was fixed on channel 2 of the chip at a flow rate of 10 μL / min. The coupling amount was set to 9000, and a coupling map was generated.

[0122] (5) The channel was blocked with ethanolamine at a flow rate of 10 μL / min.

[0123] (6) Repeat steps (3)-(5) for channel 1 as a reference, except that protein-free acetate buffer is used in step (4).

[0124] 2.2 Solution correction

[0125] The running buffer of the test object is 1.PBS-P+ containing 5% DMSO. Replace the original running buffer in the tray on the left side of the system with 1.PBS-P+ containing 5% DMSO, and insert the corresponding liquid inlet tube. According to Table 2, mix 4.5% and 5.8% stock solutions to prepare a 5% DMSO concentration calibration curve.

[0126] Table 2. Solvent correction solution configuration table

[0127]

[0128] 2.3 Determination of analytes

[0129] (1) Each analyte is diluted to several concentrations in a 96-well plate and coupled to the target protein from low concentration to high concentration through the chip. The flow rate is 30 μL / min and the duration is 150 s.

[0130] (2) After each concentration point has passed, the chip is regenerated with 10 mM glycine hydrochloride (pH 2.0) solution for 5 min. This process is repeated until all the corresponding concentrations of the analyte have been run.

[0131] (3) The data of the samples were collected using BIAcore T200 Control software (v.2.0, GE Healthcare) and the data of the reference channel were subtracted from them. The data were globally fitted to a 1:1 Langmuir binding model using BIAcore T200 Evaluation software to obtain the association and dissociation constants.

[0132] The affinity between the peptide and PDL1 protein was determined according to the dissolved concentration, and the affinity between the peptide and PDL1 protein was detected by SPR. The results are shown in Table 3. Figure 2 As shown, the targeting peptide has a high affinity to PDL1, among which pep1 has the highest affinity to PDL1. pep1 is a cyclic peptide with the structural formula Figure 3A and Figure 3B shown.

[0133] Table 3. SPR determination of peptide affinity to PDL1

[0134]

[0135] 3. Molecular docking

[0136] The sequence of Mouse PDL1 protein was downloaded from the Uniprot website, and the sequence of the interval corresponding to the PD-1 binding domain (6SRU: 19-134) was extracted as follows:

[0137] Mouse PDL1:

[0138] SEQ ID NO: 11.

[0139] Molecular docking of PDP#1:ACKHEWSGEC and Mouse PDL1.

[0140] This project uses AlphaFold3 Server for protein-peptide docking. Mouse PDL1 is docked with PDP#1 peptide, and the best conformation (model_0) is selected from the docking results for analysis.

[0141] The experimentally resolved 3D structure of Mouse PDL1, 6SRU ​​structure, is basically consistent with the structure predicted by AlphaFold3. The RMSD of the predicted structure and experimental structure after superposition is 0.299. Figure 4As shown. The docking mode analysis of PDP#1 peptide and Mouse PDL1 protein is shown in Figure 5 shown.

[0142] Example 3 Preparation and characterization of molecular imaging probe targeting PDL1 ligand

[0143] (1) Preparation of peptide lipids: PDL1 peptide was synthesized by solid phase synthesis (in the process of peptide synthesis, AC (acetyl) group was added first and NH2 (amino) group was added at the end to block the end of the peptide). The peptide sequence used to prepare the molecular imaging probe was: PDP#1:AC-ACKHEWSGEC-NH2. The molar ratio of DSPE-PEG2000-NHS to PDL1 peptide was 1:2.8. 6.37 mg of DSPE-PEG2000-NHS and 7.18 mg of PDP#1 were weighed and dissolved in DMSO, incubated at room temperature for 1 h, and freeze-dried for later use.

[0144] (2) Weigh 27.99 mg of hydrogenated soybean lecithin, 10.33 mg of cholesterol, 5.5 mg of DSPE-mPEG2000, 6.18 mg of lipid polypeptide prepared in step (1), and 1 mL (100 mM) of magnetic resonance contrast agent. Dissolve all materials in 5 mL of ethanol solution, stir for 30 min, rotate and evacuate to remove ethanol. Then add 5 mL of PBS, stir for 5 min to hydrate, rotate on a rotary evaporator for 60 min, and ultrasonicate in an ice water bath with a probe type ultrasound instrument for 1 min to obtain a liposome solution coated with a magnetic resonance contrast agent. Use an ultrafiltration tube with a pore size of 3.5KD to ultrafilter 4 times, each time for 40 min, to remove the uncoated magnetic resonance contrast agent in the solution, and obtain a targeted liposome solution coated with a magnetic resonance contrast agent. Freeze-dry for later use.

[0145] (3) The morphology of the molecular imaging probe prepared in this example was characterized by TEM. The molecular imaging probe formed nanoparticles with regular morphology and individual dispersion, such as Figure 6 shown.

[0146] (4) The particle size and concentration of nanoparticles and the zeta potential analyzer were used to characterize the particle size and potential of the molecular imaging probe prepared in this embodiment. The average particle size of the molecular imaging probe prepared in this embodiment was 138.71±11.96 nm, and the average charge was -10.60±1.51 mV. Figure 7 shown.

[0147] (5) In vitro molecular imaging probe targeting PDL1 protein experiment: To verify the specific binding of PDL1 targeting probe to PDL1 protein, the targeted and non-targeted molecular probes were co-cultured with LV-PDL1 infected HL-1 cells (PDL1 highly expressed). highHL-1 cells were cultured in 6-well plates at 10 5 The cells were inoculated at a density of 10 cells for 24 hours. Targeted molecular imaging probes labeled with DIO or non-targeted molecular imaging probes labeled with DIL were added to the cell culture plate and incubated at 37°C for 4 hours. Unbound liposomes were then washed three times with PBS to remove them. The cells were fixed with 4% PFA and the nuclei were stained with DAPI for 10 minutes. Fluorescence images were observed using a confocal laser scanning microscope. Confocal imaging showed that the molecular imaging probe co-localized with the PDL1 protein on the surface of HL-1 cells, indicating that the molecular imaging probe had good PDL1 targeting ability, such as Figure 8 shown.

[0148] (6) In vivo evaluation of the efficacy of molecular imaging probes in immune myocardial injury: Female BALB / c mice aged 6-8 weeks were used to construct an immune myocardial injury model by combined subcutaneous injection of MyHc peptide and intraperitoneal injection of PD1 monoclonal antibody. The control group was given an equal amount of complete Freund's adjuvant and IgG isotype control antibody. The model mice were divided into two groups, and 150 μL of different samples of targeted and non-targeted molecular imaging probes were injected into the tail vein (150uL per mouse), and cardiac magnetic resonance imaging was performed 10 minutes after administration. The results are shown in Fig. 9 As shown, the non-targeted probe has poor cardiac targeting ability, while the targeted probe has better myocardial injury imaging effect.

[0149] The PDL1 targeting peptide provided in the present application can have affinity to the PDL1 protein and can be used to evaluate diseases with high PDL1 expression by preparing targeted molecular imaging probes.

[0150] This application demonstrates, through in vitro cell level experiments and immune myocardial injury mouse experiments, that the PDL1 targeting peptide has significant in vitro and in vivo targeting of the PDL1 protein, and can be used to prepare molecular imaging probes targeting PDL1, and even tumor-suppressing drugs, with good prospects for clinical transformation.

[0151] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0152] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.

Claims

1. A PDL1 targeting polypeptide, characterized in that The PDL1 targeting polypeptide includes any one of the following polypeptides: (1) a polypeptide as shown in at least one of SEQ ID NO: 1 to SEQ ID NO: 9; (2) A polypeptide derived from the amino acid sequence of (1) with the same function as that of the amino acid sequence of (1) after one or more amino acid residues are replaced and / or deleted and / or added.

2. A lipid carrier, characterized in that The lipid carrier comprises a targeting lipid containing the PDL1 targeting polypeptide according to claim 1.

3. The lipid carrier according to claim 2, characterized in that The targeting lipid also includes DSPE-PEG2000-NHS; Optionally, the PDL1 targeting polypeptide is linked to the DSPE-PEG2000-NHS via an amide bond; Optionally, the molar ratio of the PDL1 targeting polypeptide to the DSPE-PEG2000-NHS is 1:2.8; Optionally, the PDL1 targeting polypeptide is modified on the surface of the lipid carrier.

4. The lipid carrier according to claim 3, characterized in that The lipid carrier also includes auxiliary materials; Optionally, the excipients include one or more of hydrogenated soybean lecithin, cholesterol and DSPE-PEG2000; Optionally, in the lipid carrier, the molar ratio of hydrogenated soybean lecithin, cholesterol, DSPE-PEG2000 and targeting lipid is 55:40:3:

2.

5. A molecular imaging probe, characterized in that: The method comprises the PDL1 targeting polypeptide according to claim 1 or the lipid carrier according to any one of claims 2 to 4, and further comprises a signal molecule.

6. The molecular imaging probe according to claim 5, characterized in that: The signal molecule is encapsulated inside the lipid carrier; Optionally, the signal molecule has an imaging function; Optionally, the imaging function includes at least one of fluorescence imaging, magnetic resonance imaging, nuclear imaging, ultrasound imaging, photoacoustic imaging and electronic computer tomography imaging; Optionally, the signal molecule having magnetic resonance imaging function includes at least one of a gadolinium-based contrast agent, an iron-based contrast agent, and a manganese-based contrast agent.

7. The method for preparing a molecular imaging probe according to claim 5 or 6, characterized in that: The preparation method comprises preparing the molecular imaging probe by a thin film dispersion method; Optionally, the following steps are included: reacting the DSPE-PEG2000-NHS with the PDL1 targeting polypeptide to prepare a targeting lipid; The hydrogenated soybean lecithin, cholesterol, DSPE-PEG2000, targeting lipid and signal molecule are dissolved in an organic solvent, and the organic solvent is removed after mixing to prepare a lipid film; and the lipid film is hydrated to prepare a molecular imaging probe.

8. The preparation method according to claim 7, characterized in that: The preparation method meets one or more of the following conditions: (1) The DSPE-PEG2000-NHS and the PDL1 targeting polypeptide are dissolved in a DMSO solution and reacted; the reaction time is 1 h to 2 h; (2) The mass and molar ratio of the targeting lipid to the signal molecule is (1-1.3) mg:2 mmol.

9. Use of the PDL1 targeting polypeptide according to claim 1, the lipid carrier according to any one of claims 2 to 4, or the molecular imaging probe according to any one of claims 5 to 6, the use comprising one or more of the following aspects: (1) Use in the preparation of a reagent for qualitatively and / or quantitatively detecting PDL1 expression or expression location; (2) Application in the preparation of reagents for diagnosis or tracing imaging of tumors or immune myocardial damage; (3) Use in the preparation of drugs for preventing and / or treating tumors; and (4) Application in the preparation of evaluation products for diseases related to abnormally high expression of PDL1; Optionally, the diseases associated with abnormally high expression of PDL1 include immune myocardial injury and tumors.

10. A product for early diagnosis or assessment of immune myocardial injury, characterized in that: The method comprises the PDL1 targeting polypeptide according to claim 1, the lipid carrier according to any one of claims 2 to 4, or the molecular imaging probe according to any one of claims 5 to 6.

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