PDL1-Targeted Polypeptide, Molecular Imaging Probe and Their Applications

Abstract

This application relates to PDL1-targeting polypeptides, molecular imaging probes and their applications. The PDL1-targeting polypeptides include any one of the following polypeptides: (1) polypeptides shown by at least one of SEQ ID NO: 1 to SEQ ID NO: 9; (2) polypeptides derived therefrom with the same function, in which the amino acid sequence of (1) is subjected to substitution and / or deletion and / or addition of one or several amino acid residues. The magnetic resonance molecular imaging probe based on the PDL1-targeting polypeptide realizes the early diagnosis and evaluation of tumors and immune myocardial injuries.

Description

Technical Field

[0001] This application relates to the field of biomedical technologies, and particularly relates to a PDL1-targeting polypeptide, a molecular imaging probe, and their applications. Background Art

[0002] Immune checkpoint inhibitors (ICIs) have attracted extensive attention and research in the comprehensive treatment of tumors due to their characteristics of activating the immune system and specific 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, the activity of T cells will be inhibited, thereby reducing the immune response. Many tumor cells evade 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 increasing application of immune checkpoint inhibitors, the incidence of immune-related adverse events (irAEs) has also gradually increased. Relevant studies have shown that in patients receiving combined immunotherapy, the incidence of severe irAEs is as high as 59%. In addition, the incidence of immune therapy-related cardiovascular adverse events (cardiovascular death, cardiac arrest, high-degree heart block) has increased by about 4 times. Among them, the mortality rate of immune therapy-related myocarditis is as high as 40%-60%. Therefore, there is an urgent clinical need for a technical means for early monitoring of immune myocardial toxicity injury to guide treatment decisions. Summary of the Invention

[0003] Based on this, an embodiment of this 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 solution is as follows:

[0005] An embodiment of this application provides a PDL1-targeting polypeptide, which includes any one of the following groups of polypeptides:

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

[0007] (2) A polypeptide derived therefrom by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence of (1) and having the same function.

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

[0009] One embodiment of the present application also provides a lipid carrier, and the lipid carrier comprises a targeting lipid containing the PDL1-targeting polypeptide described above.

[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 through 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 excipients.

[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 soy lecithin, cholesterol, DSPE-PEG2000, and the targeting lipid is 55:40:3:2.

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

[0018] One embodiment of the present application also provides a molecular imaging probe, which comprises the PDL1-targeting polypeptide described above and further comprises a signal molecule.

[0019] The present application uses phage display technology to screen PDL1-targeting polypeptides and constructs molecular imaging probes based on PDL1-targeting polypeptides, providing a basis for the early diagnosis of immune myocardial injury.

[0020] Lipid carriers are nano-vesicles composed of phospholipid bilayers and have become drug delivery carriers attracting much attention in recent years. Passive targeting of target tissues can be achieved by modifying the particle size of liposomes. In addition, the imaging function can be realized by encapsulating signal molecules (such as the magnetic resonance contrast agent Gd-DTPA) through lipid carriers.

[0021] Therefore, an embodiment of the present application also provides a molecular imaging probe, including the lipid carrier described above and also including a signal molecule.

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

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

[0024] In one embodiment, the imaging function includes at least one of fluorescence imaging, magnetic resonance imaging, radionuclide imaging, ultrasonic imaging, photoacoustic imaging, and computed tomography imaging.

[0025] In one embodiment, the signal molecules with magnetic resonance imaging function include at least one of gadolinium-based contrast agents (such as Gd-DTPA), iron-based contrast agents, and manganese-based contrast agents.

[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 also provides a preparation method of the molecular imaging probe described above, including 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 a DMSO solution for reaction.

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

[0031] In one embodiment, freeze-drying treatment is also included after the reaction.

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

[0033] Step S2: Dissolve the hydrogenated soy lecithin, cholesterol, DSPE-PEG2000, targeting lipid, and signal molecule in an organic solvent. After mixing evenly, remove the organic solvent to prepare a lipid film.

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

[0035] In one embodiment, the method of mixing evenly includes stirring.

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

[0037] In one embodiment, the mass-to-mole ratio of the targeting lipid to the signal molecule is (1 - 1.3) mg: 2 mmol. Optionally, the mass-to-mole 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 the range composed of any of the above ratios.

[0038] Step S3: Hydrate the lipid film to prepare a molecular imaging probe.

[0039] In one embodiment, a hydration medium is added to the lipid film. After rotary hydration for 50 min to 70 min, ultrasonic treatment is performed to prepare a molecular imaging probe.

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

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

[0042] In one embodiment, after ultrasonic treatment, filtration and freeze-drying treatments are further included.

[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 the expression position.

[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 a reagent for diagnosing tumors or immune-related myocardial injuries, or for tracer imaging.

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

[0046] In one embodiment, the diagnosis includes the early diagnosis of tumors or immune-related myocardial injuries.

[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 the expression or expression location of PDL1.

[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 related to abnormal PDL1 expression include immune myocardial injury and malignant tumors.

[0051] This evaluation product can evaluate the degree of injury of immune myocardial injury or the degree of tumor development and treatment, and guide clinical treatment decisions.

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

[0053] In one embodiment, the malignant tumors include but are not limited to melanoma, lung cancer, and renal cell carcinoma.

[0054] In the present application, a PDL1-targeting polypeptide is screened by phage display technology, a lipid carrier-coated magnetic resonance contrast agent (Gd-DTPA) is constructed, and a molecular imaging probe with a PDL1-targeting polypeptide modified on the surface of the lipid carrier is developed. Through the targeting property of the PDL1 polypeptide, the molecular probe has the function of actively targeting the inflammatory site, and 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 includes an early diagnosis or evaluation kit.

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

[0058] The present application screens and provides a cyclic and linear polypeptide with specific targeting to PDL1 protein and its sequence through phage display technology. This polypeptide can be processed and modified for subsequent research and development of PDL1-targeting probes and development of specific targeting drugs, and has potential targeting diagnosis and treatment value for diseases with high expression of PDL1 protein.

[0059] Furthermore, the present application constructs a lipid carrier modified with a PDL1-targeting polypeptide on the surface and encapsulates a contrast agent with imaging function, such as Gd-DTPA, inside to form a molecular imaging probe with 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 immune myocardial injury through magnetic resonance imaging. The molecular imaging probe has good stability, high drug loading capacity, a simple and controllable preparation method, can be mass-produced, and has broad clinical application prospects. Brief Description of the Drawings

[0060] To more clearly illustrate the technical solutions in the embodiments of the present application and more comprehensively understand the present application and its beneficial effects, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following described drawings are only some embodiments of the present application. For those skilled in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0061] Figure 1 For Phage ELISA to evaluate the affinity between the PDL1-targeting polypeptide and PDL1 protein.

[0062] Figure 2 For SPR to evaluate the affinity between the PDL1-targeting polypeptide and PDL1 protein.

[0063] Figure 3A And Figure 3B For the structural formula of the PDL1-targeting polypeptide.

[0064] Figure 4 For the superimposed map of the predicted structures of the PDL1-targeting polypeptide and PDL1 protein by AlphaFold3.

[0065] Figure 5 .Binding mode of PDP#1 to Mouse PDL1 protein: In the figure, Mouse PDL1 protein is shown in green, PDP#1 is shown in blue, and the interacting residues are shown 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 interaction.

[0066] Figure 6 For the TEM image of the targeting molecular probe, showing a circular structure with uniform particle size.

[0067] Figure 7 For the particle size and charge of the targeting molecular probe.

[0068] Figure 8 For the in vitro targeting efficacy evaluation of the targeting molecular probe to PDL1.

[0069] Figure 9 To evaluate immune myocardial injury in vivo with a targeted molecular probe. Specific embodiments

[0070] To make the above objects, features, and advantages of the present application more apparent and understandable, the specific embodiments of the present application will be described in detail below. Many specific details are set forth in the following description 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 departing from the connotation of the present application. Therefore, 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 commonly understood by those of ordinary skill in the technical field to which this application belongs. The terms used in the specification of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0072] In this article, the term "and / or" includes any and all combinations of one or more of the related listed items.

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

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

[0075] Imaging examination methods based on molecular probes can dynamically monitor changes at the molecular level 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 magnetic resonance examination. 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. In this application, phage display technology is used to screen for PDL1-specific targeting polypeptide sequences, and a magnetic resonance molecular imaging probe based on PDL1-targeting polypeptides is constructed to conduct early evaluation of immune myocardial injury and provide a basis for clinical decision-making.

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

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

[0078] This application provides a method for screening cyclic and linear polypeptides targeting programmed death ligand 1 (PDL1) using phage display technology, including the following steps: incubating and panning the PDL1 protein with the phage display library for multiple rounds; recovering and amplifying the phages that bind to PDL1, and sequencing to obtain the coding sequences of the peptides; synthesizing the screened cyclic and linear peptides and conducting in vitro binding experiments to verify the affinity with PDL1; conducting in vitro cell experiments on the screened peptides to evaluate their binding ability with PDL1; evaluating the diagnostic efficacy of the molecular imaging probe based on PDL1 polypeptides 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 PDL1 affinity, and good imaging effects were shown in evaluating the degree of myocardial injury in an animal model of myocardial injury through a molecular imaging probe based on polypeptides. Therefore, the polypeptides screened in this application have broad application prospects in the early evaluation of immune myocardial injury. This application provides PDL1-targeting polypeptide sequences and their application in the diagnosis of immune myocardial injury, and is expected to provide an efficient and safe evaluation method for the evaluation of immune myocardial injury.

[0080] The implementation solutions of the present application will be described in detail below in conjunction with embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specific conditions indicated in the following embodiments, the guidelines given in the present application shall be preferentially referred to, and it is also possible to follow the experimental manuals or conventional conditions in the art, or the conditions recommended by the manufacturer, or refer to the experimental methods known in the art.

[0081] In the following specific embodiments, for the measurement parameters of raw material components, if not otherwise specified, there may be slight deviations within the weighing accuracy range. For the 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 obtained from a biological company.

[0083] The PDL1-specific targeting polypeptide used in the following examples was synthesized by a biological company, with a purity greater than 95%. The targeting polypeptide used was formulated into a stock solution with an appropriate concentration using a solvent before the experiment.

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

[0085] Example 1 Screening of PDL1 Targeting Polypeptides by Phage Display Technology

[0086] 1. Phage Titer Detection Method

[0087] (1) After inoculating the ER2738 strain 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 OD600 was 0.4 - 0.6;

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

[0089] (3) Dilute the phage with LB medium according to the 10-fold serial dilution method; After equally dividing the expanded host bacteria as required, add 10 μL of the gradient phage diluted with LB to each tube, mix gently, and incubate at room temperature for 30 min;

[0090] (4) Spread the host bacteria infected in step (3) on the plates prepared in step (2) and culture them overnight at 37 °C;

[0091] (5) Count the number of plaques.

[0092] 2. Phage amplification culture

[0093] (1) After inoculating the ER2738 strain 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 OD600 reached 0.4 - 0.6;

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

[0095] 3. Screening of phage display peptide library

[0096] (1) Antigen coating: Dilute the PDL1 protein standard with PBS buffer to 5 μg / mL. Take a 96-well ELISA plate, select 2 replicates, add 100 μL (400 ng / well) to each well, and coat overnight at 4 °C. Use PBS as the 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) Incubate phage: Wash 3 times with PBST. Take the phage solution prepared in the first step, dilute it with 2% skim milk powder to 5×10 11 pfu / mL, add it to the ELISA plate, 200 μl / well, and incubate at room temperature for 2 h;

[0099] (4) Elution: Discard the phage sample, wash 3 times with PBST, add 200 μL of Tris-HCl (pH = 3.0) to each well, let it stand at room temperature for 10 min, and quickly neutralize the eluate with 40 μL / well of Tris-HCl (pH = 8.0) to about pH = 7.0 (the amount of Tris-HCl added at pH 8.0 needs to be re-determined before each neutralization).

[0100] (5) Determine the phage titer of the eluate: Refer to the method in step 1 to determine the phage titer in the eluate;

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

[0102] (7) Sequence the positive phage clones. The sequencing primer is -96gIII (5’-CCCTCATAGTTAG CGTAACG-3’, SEQ ID NO: 10), and the sequencing direction is reverse sequencing;

[0103] (8) Find the 36 gene sequences between the two restriction enzyme sites and translate them into amino acid sequences on the website https: / / web.expasy.org / translate / . The translation result in the 3'-5' direction is the polypeptide sequence for phage display, as shown in Table 1.

[0104] Table 1. Screening of PDL1-targeting polypeptide sequences by phage display technology

[0105]

[0106]

[0107] Example 2 Detection of the affinity of PDL1-targeting polypeptides

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

[0109] (1) Coat the enzyme-labeled plate with PDL1 protein and incubate overnight at 4°C;

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

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

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

[0113] (5) Add reaction buffer and termination buffer;

[0114] (6) Measure the absorbance at 405 nm using an ultraviolet spectrophotometer.

[0115] Perform an in vitro binding assay with the screened and synthesized polypeptide that can bind to PDL1 and the pre-coated PDL1 protein. The results are as Figure 1 shown. The polypeptides can all bind to the PDL1 protein in vitro, and the absorbance after their binding is detected at 405 nm.

[0116] 2. Surface plasmon resonance (SPR)

[0117] 2.1 Protein coupling

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

[0119] (2) Hold the CM5 chip with the side with words facing up. Gently push the chip into the card slot in the direction of the arrow on the chip, and finally 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) Dilute the ligand protein to 50 μg / mL with sodium acetate at pH 4.0, and fix the protein on channel 2 of the chip at a flow rate of 10 μL / min. Set the coupling amount to 9000 to generate a coupling map.

[0122] (5) Block the channel with ethanolamine at a flow rate of 10 μL / min.

[0123] (6) Repeat steps (3)-(5) for channel 1 used as a reference, except that in step (4), use an acetate buffer without protein.

[0124] 2.2 Solution Calibration

[0125] Select 1.PBS-P+ containing 5% DMSO as the running buffer for the analyte. Replace the original running buffer in the left tray of the system with 1.PBS-P+ containing 5% DMSO, and insert the corresponding inlet tubes. Prepare a 5% DMSO concentration calibration curve by mixing 4.5% and 5.8% mother liquors according to Table 2.

[0126] Table 2. Configuration Table of Solvent Calibration Solution

[0127]

[0128] 2.3 Analyte Determination

[0129] (1) Dilute each analyte to several concentrations in a 96-well plate, and couple it with 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 flowed through, regenerate the chip with a 10 mM glycine hydrochloride (pH 2.0) solution for 5 min, and repeat this process until all the corresponding concentrations of the analytes have been run.

[0131] (3) Data of the samples were collected using BIAcore T200 Control software (v.2.0, GE Healthcare) and the data of the reference channel was subtracted from it. The association and dissociation constants were obtained by globally fitting the data to a 1:1 Langmuir binding model using BIAcore T200 evaluation software.

[0132] The affinity between the polypeptide and PDL1 protein was determined according to the dissolved concentration, and the SPR detection of the affinity between the polypeptide and PDL1 protein was carried out. The results are shown in Table 3. The SPR results are as Figure 2 shown. The targeting polypeptide has a high affinity for PDL1. Among them, pep1 has the highest affinity for binding to PDL1. Pep1 is a cyclic polypeptide, and its structural formula is as Figure 3A and Figure 3B shown.

[0133] Table 3. Affinity between polypeptide and PDL1 determined by SPR

[0134]

[0135] 3. Molecular docking

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

[0137] Mouse PDL1:

[0138] FTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQVIQF VAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISYG GADYKRITLKVNAPY, SEQ ID NO: 11.

[0139] PDP#1: ACKHEWSGEC was docked with Mouse PDL1.

[0140] In this project, AlphaFold3 Server was used for protein-polypeptide docking. Mouse PDL1 was docked with PDP#1 polypeptide, and the best conformation (model_0) was selected from the docking results for analysis respectively.

[0141] The three-dimensional structure 6SRU of Mouse PDL1 obtained by experimental analysis is basically consistent with the predicted structure by AlphaFold3. The RMSD after superimposing the predicted structure and the experimental structure is 0.299, as Figure 4As shown. Analysis of the docking mode between PDP#1 polypeptide and Mouse PDL1 protein is as follows Figure 5 As shown.

[0142] Example 3 Preparation and Characterization of a Molecular Imaging Probe Targeting the PDL1 Ligand

[0143] (1) Preparation of polypeptide lipids: The PDL1 polypeptide was synthesized by solid-phase synthesis (during the polypeptide synthesis process, an AC (acetyl) group was added at the beginning and an NH2 (amino) group was added at the end to block the polypeptide ends). The polypeptide sequence used for preparing the molecular imaging probe was: PDP#1: AC-ACKHEWSGEC-NH2. The molar ratio of DSPE-PEG2000-NHS to the PDL1 polypeptide was 1:2.8. Weigh 6.37 mg of DSPE-PEG2000-NHS and 7.18 mg of PDP#1, dissolve them in DMSO, incubate at room temperature for 1 h, and freeze-dry for later use.

[0144] (2) Weigh 27.99 mg of hydrogenated soy lecithin, 10.33 mg of cholesterol, 5.5 mg of DSPE-mPEG2000, 6.18 mg of the lipid polypeptide prepared in step (1), and 1 mL (100 mM) of the magnetic resonance contrast agent. Dissolve all the 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 for hydration, rotate on a rotary evaporator for 60 min, and ultrasonicate in an ice-water bath with a probe-type ultrasonic device for 1 min to obtain a liposome solution encapsulating the magnetic resonance contrast agent. Ultrafilter 4 times with a 3.5 KD pore size ultrafiltration tube, 40 min each time, to remove the unencapsulated magnetic resonance contrast agent in the solution and obtain a targeted liposome solution encapsulating the magnetic resonance contrast agent. Freeze-dry for later use.

[0145] (3) Use TEM to characterize the morphology of the molecular imaging probe prepared in this example. The molecular imaging probe forms nanoparticles with regular morphology and individual dispersion, as Figure 6 shown.

[0146] (4) Use a nanoparticle size, concentration, and ZETA potential analyzer to characterize the particle size and potential of the molecular imaging probe prepared in this example. The average particle size of the molecular imaging probe prepared in this example is 138.71 ± 11.96 nm, and the average charge is -10.60 ± 1.51 mV, as Figure 7 shown.

[0147] (5) In vitro experiment of the molecular imaging probe targeting PDL1 protein: To verify the specific binding between the PDL1 targeting probe and PDL1 protein, co-culture the targeting and non-targeting molecular probes with HL-1 cells infected with LV-PDL1 (high expression of PDL1). PDL1 highHL-1 cells were seeded in 6-well plates at a density of 10 5 cells per well for 24 hours. A targeted molecular imaging probe labeled with DIO or a non-targeted molecular imaging probe labeled with DIL was added to the cell culture plates and incubated at 37 °C for 4 hours. Subsequently, the cells were washed three times with PBS to remove unbound liposomes. The cells were fixed with 4% PFA, and the cell 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, as Figure 8 shown.

[0148] (6) In vivo evaluation of the efficacy of the molecular imaging probe for immune myocardial injury: Female BALB / c mice, 6-8 weeks old, were used to establish an immune myocardial injury model by combined subcutaneous injection of MyHc polypeptide 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 2 groups, and 150 μL of different samples, targeted and non-targeted molecular imaging probes, were injected via the tail vein (150 μL per mouse). Cardiac magnetic resonance imaging was performed 10 minutes after administration. The results were as Figure 9 shown. The non-targeted probe had poor cardiac targeting ability, while the targeted probe had good imaging effect for myocardial injury.

[0149] The PDL1-targeted peptide provided in this application can bind to the PDL1 protein. By preparing a targeted molecular imaging probe, it can be used for the evaluation of diseases with high PDL1 expression.

[0150] In vitro cell-level experiments and experiments on immune myocardial injury mice in this application showed that the PDL1-targeted peptide had significant ex vivo and in vivo PDL1 protein targeting ability. It can be used to prepare molecular imaging probes targeting PDL1, and even anti-tumor drugs, with good prospects for clinical translation.

[0151] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, 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, it should be considered as the scope described in this specification.

[0152] The above-described embodiments only represent several implementation manners of this application. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of this application, several modifications and improvements can be made, and these all belong to the protection scope of this application. Therefore, the protection scope of this application patent should be subject to the appended claims, and the description can be used to explain the content of the claims.

Claims

1. A PDL1 targeting polypeptide, characterized in that The PDL1 targeting polypeptide is shown in SEQ ID NO:

1.

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.

4. The lipid carrier according to claim 3, characterized in that The PDL1 targeting polypeptide is connected to the DSPE-PEG2000-NHS via an amide bond.

5. The lipid carrier according to claim 4, characterized in that The molar ratio of the PDL1 targeting polypeptide to the DSPE-PEG2000-NHS is 1:2.

8.

6. The lipid carrier according to any one of claims 2 to 5, characterized in that The PDL1 targeting polypeptide is modified on the surface of the lipid carrier.

7. The lipid carrier according to any one of claims 3 to 5, characterized in that The lipid carrier also includes excipients.

8. The lipid carrier according to claim 7, characterized in that The auxiliary materials include one or more of hydrogenated soybean lecithin, cholesterol and DSPE-PEG2000.

9. The lipid carrier according to claim 8, characterized in that In the lipid carrier, the molar ratio of hydrogenated soybean lecithin, cholesterol, DSPE-PEG2000 and targeting lipid is 55:40:3:

2.

10. 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 9, and further comprises a signal molecule.

11. The molecular imaging probe according to claim 10, characterized in that: The signal molecule is encapsulated in the interior of the lipid carrier.

12. The molecular imaging probe according to claim 11, characterized in that: The signal molecule has an imaging function.

13. The molecular imaging probe according to claim 12, characterized in that: The imaging function includes at least one of fluorescence imaging, magnetic resonance imaging, nuclear imaging, ultrasound imaging, photoacoustic imaging and electronic computer tomography imaging.

14. The molecular imaging probe according to claim 13, characterized in that: The signal molecule with 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.

15. The method for preparing a molecular imaging probe according to any one of claims 10 to 14, characterized in that: The preparation method comprises preparing the molecular imaging probe by a thin film dispersion method.

16. The preparation method according to claim 15, characterized in that: The steps include: 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.

17. The preparation method according to claim 16, 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.

18. Use of the PDL1 targeting polypeptide according to claim 1, the lipid carrier according to any one of claims 2 to 9, or the molecular imaging probe according to any one of claims 10 to 14, the use comprising one or more of the following aspects: (1) Use in the preparation of reagents for diagnosis or tracing imaging of tumors or immune myocardial damage, wherein the tumor is melanoma, lung cancer or renal cell carcinoma; (2) Use in the preparation of a drug for preventing and / or treating a tumor, wherein the tumor is melanoma, lung cancer or renal cell carcinoma; and (3) Application in the preparation of an evaluation product for a disease associated with abnormally high expression of PDL1, wherein the disease associated with abnormally high expression of PDL1 is selected from immune myocardial injury and tumor, and the tumor is melanoma, lung cancer or renal cell carcinoma.

19. 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 9, or the molecular imaging probe according to any one of claims 10 to 14.

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