Targeting Trop2 polypeptide and molecular probe and application thereof

Abstract

The invention relates to the technical field of tumor molecular imaging, nuclear medicine diagnosis and targeted therapy, and discloses a Trop2-targeted polypeptide, a Trop2-targeted molecular probe and application of the Trop2-targeted polypeptide and the Trop2-targeted molecular probe. The polypeptide can be coupled with different imaging elements to construct molecular probes or radiopharmaceuticals for tumor targeted imaging, so that noninvasive visual detection on the Trop2 expression level in tumor tissues is realized. The probe can specifically recognize Trop2 positive solid tumors, and a new technical means is provided for noninvasive diagnosis and dynamic monitoring of various Trop2 high-expression tumors such as pancreatic cancer, lung cancer, breast cancer and thyroid cancer. The probe disclosed by the invention has the advantages of simple preparation process, low cost, high specificity and stability, short imaging period, low radiation dosage, easiness in clinical transformation and the like, and has a wide application prospect in precise diagnosis and individualized treatment of tumors.

Description

Technical Field

[0001] This invention relates to the fields of tumor molecular imaging, nuclear medicine diagnostics and targeted therapy, and more specifically, to a peptide and molecular probe targeting Trop2 and their applications. Background Technology

[0002] Cancer poses a serious threat to human life and health. How to achieve early diagnosis, precise classification, and personalized treatment of tumors to improve patient prognosis has always been a crucial issue that urgently needs to be addressed in the field of oncology. Molecular imaging technology, through non-invasive methods, tracks the expression and distribution of specific molecular targets in the body, providing an important tool for the early detection, staging assessment, and efficacy monitoring of tumors.

[0003] Tropocyte surface antigen 2 (Trop2) is a transmembrane glycoprotein that is abnormally highly expressed in various solid malignant tumors. Its expression level is closely related to tumor invasiveness, metastatic risk, and poor prognosis. Studies have shown that Trop2 is significantly higher in various tumor tissues, including breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, thyroid cancer, prostate cancer, cervical cancer, head and neck squamous cell carcinoma, nasopharyngeal carcinoma, and ovarian cancer, than in normal tissues, making it a highly promising tumor diagnostic biomarker and therapeutic target.

[0004] Given the high expression of Trop2 in tumors, targeted therapy strategies against Trop2 have attracted widespread attention. Currently, anti-Trop2 antibody-drug conjugates (ADCs) have been used in research to treat various metastatic tumors, including triple-negative breast cancer, non-small cell lung cancer, and small cell lung cancer. However, this type of treatment strategy still faces challenges such as systemic toxicity, drug tolerance, and heterogeneity of efficacy, and its therapeutic effect often depends on the expression level of Trop2 in tumor tissue. Therefore, precise stratification of patients based on Trop2 expression status is considered a key step in improving the efficacy of Trop2-targeted therapy and promoting the development of personalized medicine. However, current Trop2 detection methods mainly rely on tissue biopsy and immunohistochemical analysis, which have limitations such as invasive sampling, difficulty in dynamic monitoring, and the influence of human factors on results. Furthermore, there is still a lack of a unified and standardized threshold for "Trop2 positivity," which to some extent restricts the further development of Trop2-targeted therapy strategies.

[0005] Molecular imaging technology is considered a promising solution for achieving non-invasive, dynamic, and quantitative assessment of Trop2 expression levels. Current Trop2 targeting strategies mainly focus on monoclonal antibodies, antibody fragments, single-domain antibodies, and antibody-drug conjugates (ADCs). Although these large molecular systems have made some progress in therapy and imaging, they generally suffer from problems such as large molecular weight, limited tumor tissue penetration, slow in vivo clearance, long imaging cycles, and high preparation costs, limiting their further application in rapid imaging and integrated diagnosis and treatment.

[0006] In contrast, peptide-based targeted molecules, due to their advantages such as small molecular weight, good biocompatibility, low immunogenicity, strong tissue penetration, and ease of chemical modification, are gradually becoming an important research direction for constructing next-generation molecular imaging probes and targeted therapy carriers. Peptides not only retain some of the targeted recognition functions of protein molecules, but can also be prepared efficiently and at low cost using mature solid-phase synthesis techniques. Through rational structural design, targeted peptides can be coupled with different imaging groups or therapeutic payloads for precise PET or SPECT imaging of tumors, and hold promise for integrated diagnostic and therapeutic applications.

[0007] With the rapid development of precision oncology and immunotherapy, there is an urgent clinical need for a non-invasive molecular imaging tool that can reflect the molecular subtyping and target expression status of tumors to assist in patient screening, efficacy evaluation, and treatment strategy adjustment. However, the number of publicly available Trop2-targeting peptides is limited, and most of them are derived from traditional screening methods. There is a lack of peptide systems designed de novo based on artificial intelligence that can be used for both imaging and therapy. Summary of the Invention

[0008] The purpose of this invention is to provide a peptide and molecular probe that target Trop2 and their applications.

[0009] To achieve the objectives of this invention, in a first aspect, this invention provides a polypeptide targeting Trop2, said polypeptide being: (a) The amino acid sequence is shown in SEQ ID NO:1 (LFRLQAETWMDP) or SEQ ID NO:2 (KYDETWRSQNC); (b) A polypeptide derived from (a) with one or more amino acids substituted, deleted or added to the sequence shown in SEQ ID NO:1 or 2 and having the same function; (c) A polypeptide derived from (a) that has more than 70% homology with the amino acid sequence of (a) or (b), preferably more than 80% homology, more preferably more than 90% homology, and has the same function.

[0010] Secondly, the present invention provides variants of the polypeptide, wherein the variants are: 1) Stereochemical variant: One or more amino acid residues in the polypeptide are L-type, D-type, or a mixture of L-type and D-type; 2) Structural modification variants: Cyclic derivatives, bicyclic derivatives, covalently modified derivatives of the polypeptide, or derivatives modified at its N-terminus, C-terminus or side chain by polyethylene glycol, fatty acid chains, fluorescent groups or biotin. 3) The polypeptide forms a divalent or multivalent.

[0011] Thirdly, the present invention provides a modified polypeptide or a pharmaceutically acceptable salt thereof, said modified polypeptide being obtained by coupling a bifunctional chelating agent to the N-terminus of said polypeptide or said variant via a linker; The bifunctional chelating agent is selected from DOTA, Nota, HYNIC, DTPA, TETA, DOTAGA, or NODAGA, etc. Preferably, the modified polypeptide is TRP1 or TRP69, with the following structure: TRP1:DOTA-acp-LFRLQAETWMDP; TRP69:DOTA-acp-KYDETWRSQNC; Wherein, acp represents 6-aminocaproic acid. The structural formula of TRP1 is shown in equation (1): Equation (1) The structural formula of TRP69 is shown in equation (2): Equation (2) More preferably, the modified polypeptide is a covalent probe precursor formed by introducing a tyrosine residue modified with a fluorosulfonyl (-SO2F) group into TRP1, with the structure: DOTA-acp-Tyr(SO2F)-LFRLQAETWMDP.

[0012] Fourthly, the present invention provides a conjugate or molecular probe comprising the polypeptide or the variant or the modified polypeptide or a pharmaceutically acceptable salt thereof, and an active agent conjugated to the polypeptide, variant or modified polypeptide.

[0013] Furthermore, the active agent is an imaging agent, which is a radionuclide complexed with a bifunctional chelating agent; Preferably, the radioactive nuclide is 68 Ga、 99m Tc, 89 Zr、 64 Cu or18 F, etc.

[0014] Furthermore, the bifunctional chelating agent also contains a linker between itself and the polypeptide, variant, or modified polypeptide; The linker is PEG. n Click on the chemical linker (Mal-PEG) n -DOTA, Tz-TCO, Tetrazine-PEG n -DOTA, DBCO-PEG n -NHS) or aminocaproic acid (ε-Ahx); where n is an integer between 1 and 50.

[0015] Furthermore, the active agent is a therapeutic agent, which is selected from radioactive nuclides, cytotoxic agents, photosensitizers, chemotherapeutic drugs, immunomodulators, or antitumor small molecule drugs, etc. Preferably, the radioactive therapeutic nuclide is 177 Lu、 90 Y、 131 I, 225 Ac or 212 Pb.

[0016] Preferably, the antitumor small molecule drug is selected from microtubule inhibitors, DNA damaging agents, or topoisomerase inhibitors, etc.

[0017] Fifthly, the present invention provides a pharmaceutical composition comprising the polypeptide or the variant or the modified polypeptide or a pharmaceutically acceptable salt thereof or the conjugate or molecular probe, and one or more pharmaceutically acceptable carriers or excipients.

[0018] In a sixth aspect, the present invention provides any of the following applications of the said polypeptide or the said variant or the said modified polypeptide or a pharmaceutically acceptable salt thereof or the said conjugate or molecular probe: (1) Products used for the diagnosis or treatment of Trop2-positive tumors; (2) Reagents used to prepare for measuring or evaluating Trop2 expression levels in cells; (3) Used to prepare drugs for the diagnosis, prevention and / or treatment of diseases using Trop2 as a biomarker; (4) Diagnostic reagents, diagnostic kits or imaging agents used to prepare for the detection of Trop2-related diseases; (5) For the preparation of detection products for disease staging or auxiliary staging based on Trop2 biomarkers; (6) For the preparation of tracers for surgical navigation based on Trop2 markers (to achieve precise tumor resection).

[0019] Furthermore, the Trop2-positive tumors are selected from breast cancer, lung cancer, thyroid cancer, head and neck squamous cell carcinoma, kidney cancer, urothelial carcinoma, prostate cancer, ovarian cancer, colorectal cancer, pancreatic cancer, liver cancer, stomach cancer, or esophageal cancer, etc.

[0020] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention provides a novel peptide targeting Trop2. This peptide, obtained through an AI-assisted structural design method, exhibits high selectivity and affinity, enabling it to specifically recognize tumor cells highly expressing Trop2. Furthermore, the peptide can be labeled with a radionuclide to construct a molecular probe for nuclear medicine imaging, achieving non-invasive, highly sensitive, and visual detection of Trop2-positive tumors. This provides a new technical means for the early diagnosis, efficacy monitoring, and personalized treatment of Trop2-related tumors.

[0021] The peptides targeting Trop2 in this invention have small molecular weights, well-defined structures, and low immunogenicity, making them suitable for rapid distribution and clearance in vivo. Compared to antibodies or antibody fragments, they offer shorter imaging cycles and lower background signals. They are also easy to radiolabel and conjugate with drugs, making them compatible for both diagnostic and therapeutic applications. Based on AI-assisted design, they overcome the limitations of traditional screening methods in terms of sequence space and structural optimization, significantly shortening the development cycle, reducing costs, and improving the targeting and stability of the probes. Attached Figure Description

[0022] Figure 1 The preferred embodiment of the present invention shows the structural formula and synthetic identification of peptide TRP1. (A) Molecular structure of TRP1; (B) HPLC detection of TRP1 after synthesis; (C) MS analysis of TRP1 after synthesis.

[0023] Figure 2 The preferred embodiment of the present invention shows the structural formula and synthetic identification of peptide TRP69. (A) Molecular structure of TRP69; (B) HPLC detection of TRP69 after synthesis; (C) MS analysis of TRP69 after synthesis.

[0024] Figure 3 In a preferred embodiment of the present invention, the affinity between the peptide and human Trop2 protein was detected using the surface plasmon resonance (SPRi) method. (A) Binding curve of TRP1 to Trop2 protein; (B) Binding curve of TRP69 to Trop2 protein.

[0025] Figure 4This invention provides a preferred embodiment for detecting the specific binding of peptides to Trop2-positive and Trop2-negative cell lines. (A) Confocal imaging of FITC-labeled TRP1 with Trop2-positive and Trop2-negative cells in this invention; (B) Confocal imaging of FITC-labeled TRP69 with Trop2-positive and Trop2-negative cells in this invention.

[0026] Figure 5 In a preferred embodiment of the present invention 68 Radiolabeling and stability analysis of Ga-DOTA-TRP1. (A) Analysis by thin-layer chromatography (TLC) 68 Ga-DOTA-TRP1 radiochemical yield; (B) 68 Stability analysis of Ga-DOTA-TRP1 in physiological saline; (C) 68 Stability analysis of Ga-DOTA-TRP1 in HSA.

[0027] Figure 6 The polypeptide probe is a preferred embodiment of the present invention. 68 PET / CT imaging of Ga-DOTA-TRP1 in BXPC3 and A549 tumor models. (A) 68 PET images of Ga-DOTA-TRP1 in bilateral tumor models BXPC3 and A549, respectively; (B) 68 Quantitative analysis of SUVmax in tumor tissues after 30 min in Ga-DOTA-TRP1 and Block treatment groups; express P <0.001; (C) 68 Quantitative analysis of SUVmax in tumors treated with Ga-DOTA-TRP1 and Block at different time points; (D) IHC staining analysis of TROP2 in BXPC3 and A549 tumor tissues.

[0028] Figure 7 These are PET images of the TRP1 variant-covalent structure at different time points in the BXPC3 model, representing a preferred embodiment of the present invention.

[0029] Figure 8 In a preferred embodiment of the present invention 68 PET imaging of Ga-DOTA-TRP1 in the BCPAP thyroid cancer model. (A) 68 PET images of Ga-DOTA-TRP1 and block treatment in the BCPAP thyroid cancer model; (B) 68(C) Quantitative analysis of SUVmax of Ga-DOTA-TRP1 probe in tumor tissue; (D) IHC staining analysis of TROP2 in BCPAP tumor tissue; 68 Biodistribution of Ga-DOTA-TRP1 in BCPAP thyroid cancer model mice; express P <0.01.

[0030] Figure 9 In a preferred embodiment of the present invention 68 PET images of Ga-DOTA-TRP1 in the H292 lung cancer model and Trop2 immunohistochemical staining of tumor tissue sections.

[0031] Figure 10 The polypeptide probe is a preferred embodiment of the present invention. 68 PET / CT imaging of Ga-DOTA-TRP69 in the BXPC3 tumor model. (A) 68 PET images of Ga-DOTA-TRP69 in a BXPC model; (B) 68 Biodistribution of Ga-DOTA-TRP69 in the BXPC3 tumor model. Detailed Implementation

[0032] This invention provides a polypeptide targeting trophoblast cell surface antigen 2 (Trop2) and its application in tumor diagnosis and treatment. The polypeptide can be conjugated with different imaging elements to construct molecular probes or radiopharmaceuticals for tumor-targeted imaging, enabling non-invasive and visual detection of Trop2 expression levels in tumor tissues. This probe can specifically identify Trop2-positive solid tumors, providing a new technical means for the non-invasive diagnosis and dynamic monitoring of various Trop2-high-expressing tumors such as pancreatic cancer, breast cancer, and thyroid cancer. The probe of this invention has advantages such as simple preparation process, low cost, high specificity and stability, short imaging cycle, low radiation dose, and easy clinical translation, showing broad application prospects in precision tumor diagnosis and personalized treatment.

[0033] The present invention adopts the following technical solution: This invention provides a systematic technical solution, the core of which lies in obtaining a novel Trop2-binding polypeptide using an innovative computational design method, and also provides the application of the above-mentioned polypeptide and its derivatives in tumor diagnosis and imaging.

[0034] This invention provides a short peptide capable of specifically binding to the Trop2 protein. Its amino acid sequence and structural formula are as follows:

[0035] The peptide was obtained through artificial intelligence-assisted molecular design methods and screened using high-throughput SPRi technology, combining Trop2 protein structure information with peptide-protein interaction prediction models. It has the ability to specifically recognize and bind to Trop2.

[0036] The present invention also provides a DNA fragment comprising a nucleotide sequence encoding the polypeptide.

[0037] The present invention also provides variants of the peptide. These variants involve conserved substitutions of 1-4 amino acid sites without affecting Trop2 binding ability; or the introduction of linker groups or functional groups through N-terminal or C-terminal modification. The peptide may contain variants having at least 70%, 80%, or 90% sequence homology with TRP1 or TRP69, which retain specific binding ability to Trop2.

[0038] The variants include any of the following forms: (a) Stereochemical variant: one or more amino acid residues in the polypeptide are L-type, D-type, or a mixture of L-type and D-type; (b) Sequence-derived variants: the mirror image structure of the polypeptide, or the sequence obtained by substitution, deletion, addition or mutation of one or more conserved amino acids based on the sequences shown in SEQ ID NO:1 and SEQ ID NO:2; (c) Structural modification variants: Cyclic forms, bicyclic forms, or derivatives of the polypeptide modified with polyethylene glycol, fatty acid chains, biotin, or reporter groups at its N-terminus, C-terminus, or side chains. For example, TRP1 modified with the fluorosulfate-L-tyrosine (FSY) covalent modifier DOTA-acp-Tyr(SO2F)-LFRLQAETWMDP.

[0039] The present invention also provides conjugates of the said polypeptide or the said derivative, the conjugates comprising: (a) Imaging agent conjugate: obtained by conjugating the polypeptide or the derivative with an imaging agent via a linker; the imaging agent is a radionuclide, a fluorescent group, a magnetic resonance contrast agent, or an ultrasound contrast agent; (b) Therapeutic agent conjugate: obtained by conjugating the polypeptide or the derivative with a therapeutic agent via a linker; the therapeutic agent is a radionuclide therapeutic agent, photosensitizer, chemotherapeutic drug or immunomodulator.

[0040] The present invention also provides a polypeptide-drug conjugate comprising: the above-mentioned Trop2-targeting polypeptide; a linker; and an antitumor small molecule drug. The antitumor small molecule drug is selected from microtubule inhibitors, DNA damaging agents, or topoisomerase inhibitors.

[0041] The linker can be cleavable or non-cleavable; the polypeptide conjugate can selectively kill tumor cells through Trop2-mediated targeted enrichment.

[0042] (c) Therapeutic conjugate: Obtained by coupling the polypeptide or its derivative with a radionuclide used for imaging and a radionuclide used for treatment via the same or different linkers; wherein the radionuclide used for imaging is preferably... 68 Ga、 99m Tc, 89 Zr、 64 Cu or 18 F, the preferred radionuclide for treatment 177 Lu、 90 Y、 131 I, 225 Ac or 212 Pb.

[0043] More preferably, the polypeptide, the derivative, or the conjugate is linked to the radionuclide via a bifunctional chelating agent.

[0044] The bifunctional chelating agent is selected from one or more of DOTA, NOTA, HYNIC, DTPA, TETA, DOTAGA, and NODAGA.

[0045] The bifunctional chelating agent also contains a linker between itself and the polypeptide; the linker is PEG. n Click on the chemical linker (Mal-PEG5-DOTA, Tz-TCO, Tetrazine-PEG5-DOTA, DBCO-PEG) n -NHS) or 6-aminohexanoic acid (Acp). n is an integer between 1 and 50.

[0046] The present invention also provides derivatives of the polypeptide, which are divalents or multivalents formed from the polypeptide, wherein the divalents or multivalents have the property of targeting Trop2 protein; Preferably, the divalent or multivalent is formed by linking molecules through covalent or non-covalent linkage, or by mixing with polymers through covalent or non-covalent linkage; The present invention also provides a polypeptide composite material comprising the polypeptide, isomers of the polypeptide, derivatives, mixtures, pharmaceutically acceptable salts, hydrates or solvates.

[0047] The present invention also provides an imaging formulation comprising the polypeptide or the derivative or the conjugate; The imaging preparation further includes an imaging agent, which is any one of a radionuclide, a radionuclide marker, a magnetic resonance contrast agent, or a molecular imaging preparation.

[0048] Preferably, the polypeptide, the derivative, or the conjugate is coupled to or mixed with the radionuclide.

[0049] More preferably, the polypeptide, the derivative, or the conjugate is linked to the radionuclide via a chelating agent.

[0050] This invention also provides a molecular probe targeting Trop2, comprising: the aforementioned Trop2-targeting peptide; a bifunctional chelating agent; and a radionuclide. The bifunctional chelating agent can be attached to the N-terminus or C-terminus of the peptide for stable binding to the radionuclide. The radionuclide can be selected from diagnostic radionuclides (…). 68 Ga、 99m Tc, 89 Zr、 64 Cu、 18 F), therapeutic radionuclides ( 177 Lu、 90 Y、 131 I, 225 Ac、 212 Pb) or combinations thereof; the molecular probe is suitable for PET imaging, SPECT imaging or nuclear medicine therapy.

[0051] The chelating agent is preferably HYNIC, DTPA, DFO, DOTA, NOA, or TETA.

[0052] In addition, it is compatible with fluorescent dyes and magnetic resonance contrast agents (such as Gd). 3+ Coordination with complexes is used for optical or MRI imaging.

[0053] The present invention also provides conjugates of the polypeptide or the derivative thereof, wherein the conjugates are obtained by linking or interacting the polypeptide or the derivative with a carrier in a covalent or non-covalent manner; Preferably, the carrier includes any one or more of the following: fluorescein, polymer, high molecular weight material, liposome, oily compound, and inorganic material.

[0054] The present invention also provides a polypeptide drug comprising the polypeptide, the derivative, or the conjugate. It includes: the aforementioned Trop2-targeting polypeptide; a linker; and an antitumor small molecule drug.

[0055] Preferably, the active ingredient of the drug further includes an agent capable of killing tumor cells.

[0056] More preferably, the agent capable of killing tumor cells is any one or more of the following: chemical drugs, biological drugs, nanomedicines, radiopharmaceuticals, photothermal therapy drugs, or photodynamic therapy drugs capable of killing tumor cells. Preferably, the drug is selected from: cytotoxic agents, radiolabelers, fluorophores, imaging agents, microtubule inhibitors, DNA damaging agents, topoisomerase inhibitors, immunomodulators, angiogenesis inhibitors, ferroptosis inducers, or protein degradation inducers, and any combination thereof.

[0057] The present invention also provides that the polypeptide, nucleic acid, biomaterial, derivative, or conjugate can be used to develop drugs / reagents / medications for the treatment and diagnosis of diseases related to the Trop2 signaling pathway.

[0058] Includes any of the following applications: (1) Reagents used to prepare for determining or evaluating Trop2 expression levels in cells; (2) For the preparation of drugs for the diagnosis, prevention and / or treatment of diseases using Trop2 as a biomarker; (3) Used to prepare diagnostic reagents, diagnostic kits or imaging agents for the detection of Trop2-related diseases; (4) Used to prepare detection products for disease staging or auxiliary staging based on Trop2 biomarkers; (5) Used to prepare tracers for precise tumor resection in surgical navigation based on Trop2 markers.

[0059] Preferably, the disease is a Trop2-overexpressing tumor, which includes one or more of the following: breast cancer, lung cancer, pancreatic cancer, thyroid cancer, head and neck squamous cell carcinoma, kidney cancer, urothelial carcinoma, prostate cancer, ovarian cancer, colorectal cancer, liver cancer, gastric cancer, and esophageal cancer.

[0060] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0061] Example 1: Design and Synthesis of Trop2-Targeted Peptides This embodiment provides a peptide targeting Trop2. Based on the AI ​​model PocketX-Mol, peptides with high affinity for Trop2 were generated de novo, and TRP1 and TRP69 were obtained through screening. The amino acid sequences of this peptide are shown in SEQ ID NO:1 and SEQ ID NO:2. To improve in vivo stability and targeting performance, various optimization designs were employed, such as replacing all amino acids with D-type non-natural amino acids, mutating individual sites, and introducing Acp, PEG linkers, or the covalently binding unit FSY. The structural formula is shown below. Figure 1 and Figure 2 As shown, the peptides synthesized by solid-phase synthesis methods were identified by MS and purified by HPLC to obtain the correct products for subsequent experiments.

[0062] Example 2: Detection of the affinity between the peptide and human Trop2 protein using the surface plasmon resonance (SPRi) method. Surface plasmon resonance imaging (SPRi) was used to detect the binding affinity of a peptide to human Trop2 protein. The synthesized peptide was modified by introducing a cysteine ​​residue at its C-terminus, immobilized on a bare gold chip surface via a gold-sulfur bond, and then non-specifically blocked. Using PBST as the running buffer, different concentrations of Trop2 protein solution were sequentially passed through the chip, and the binding and dissociation signals were recorded in real time. The binding-dissociation constant (KD) was calculated by curve fitting. Figure 3 The results showed that the SPRi signal increased with increasing Trop2 protein concentration, and the KD values ​​of TRP1 and TRP69 were 26.8 nM and 24 nM, respectively, indicating that the peptide can bind specifically to human Trop2 protein with high affinity.

[0063] Example 3: Verification of Trop2 affinity of the targeting peptide at the cellular level After the final deprotection step of solid-phase synthesis of TRP1 and TRP69, a portion of the synthesized resin was taken and reacted with aminocaproic acid. After deprotection was complete, FITC was dissolved in a mixed solution of pyridine / DMF / DCM = 12:7:5 (v:v) and reacted overnight at room temperature in the dark. After lysis, the FITC-labeled peptide was purified by HPLC for later use.

[0064] The BxPC3 pancreatic cancer cell line, which highly expresses Trop2, was selected and cultured in 1640 medium containing 10% FBS. The 293T negative cell line, which expresses low Trop2, was cultured in DMEM medium containing 10% FBS. The peptide was co-incubated with both positive (BxPC3) and negative (293T) cells, and the binding was investigated using confocal microscopy. The specific experimental steps were as follows: Both cell lines were seeded onto confocal microplates and cultured overnight at 37°C in a 5% CO2 incubator. After cell adhesion, the culture medium was discarded, and nuclei were stained using the Hoechst 33342 kit. The cells were incubated at room temperature for 10 min and washed twice with PBS. Then, 50 μg / mL of the peptide FITC conjugate was added, and the cells were incubated at 4°C in the dark for 20 min, followed by washing three times with PBS. The fluorescence distribution in the cells was detected using a laser scanning confocal microscope.

[0065] like Figure 4 As shown, it can be clearly seen that FITC-labeled TRP1 and TRP69 can bind well to the BxPC3 cell membrane and emit a strong fluorescent signal, while no signal is emitted on negative cells, proving that peptides TRP1 and TRP69 can target and recognize TROP2 positive cells with good specificity.

[0066] Example 4: Radiolabeling and stability analysis of peptides Firstly, from 68 Ge- 68 Ga generator (Eckert & Ziegler) elution 68 GaCl3 (0.1 M HCl solution). Mix 1300 µL of eluent (370-555 MBq) with 50 µg of TRP1 or TRP69 precursor, adjust the pH to 3.5-4.0 with sodium acetate solution (1.25 M), and react at 95 °C for 10 min. Radiochemical yield was determined by thin-layer chromatography (TLC). Before tail vein injection in mice, [the following text is incomplete and requires further context: "..."] 68 The Ga-labeled product was diluted with physiological saline and filtered through a sterile 0.22 μm filter membrane. To assess in vitro stability, [the following was observed]... 68 Ga-DOTA-TRP1 was added to physiological saline and buffer containing HSA, incubated at 37°C, and its stability within 120 minutes was analyzed by radio-high performance liquid chromatography (Radio-HPLC).

[0067] The results are as follows Figure 5 As shown, thin-layer chromatography analysis confirmed that its radiochemical yield was excellent (>95%). After incubation in physiological saline at room temperature for 120 minutes, 68 Ga-DOTA-TRP1 and 68The radiochemical purity of Ga-DOTA-TRP69 remained unchanged and remained at a stable level, indicating that the probe is stable in vitro.

[0068] Example 5 68 Ga-DOTA-TRP1 and its variants for in vivo PET imaging in pancreatic cancer models Trop2-positive cells (BxPC3) and Trop2-negative cells (A549) were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS). After confluence, the cells were digested with trypsin, collected, and counted. Simultaneously, Balb / c mice were inoculated to establish a tumor model. When the tumor volume reached 150 mm², the tumor was cultured. 3 The probe solution was injected via the tail vein into each mouse (n=5, injection dose was 10 MBq, 100 μL). The tumor-bearing mouse model was then imaged using a small animal PET / CT system (Mediso Medical Solutions, Inc.) under isoflurane-oxygen anesthesia (30, 45, and 60 minutes). Regions of interest (ROIs) of major organs (liver, lung, kidney, muscle) were delineated on the reconstructed PET images using the OsiriX Lite image processing workstation, and the radioactive uptake of important organs was calculated in %ID / g (percent of injected dose per gram). In the blocking imaging group, the mouse model was co-injected with a sufficient amount of peptide (200 µg). Furthermore, tumor tissue was sectioned and immunohistoscopy was performed to observe Trop2 expression.

[0069] The results are as follows Figure 6 As shown, 68 The Ga-DOTA-TRP1 radiotracer showed significantly higher uptake of the probe in BxPC3 tumors than in A549 tumors in the same mouse. Clear and persistent signals were observed at 30, 45, and 60 minutes post-injection. In contrast, tumor uptake was negligible in negative A549 xenografts. A sharp decrease in tumor uptake was observed after blocking with an unlabeled peptide, confirming the specific binding of the radiotracer to its target. Analysis of tumor SUVmax values ​​revealed significantly higher uptake in Trop2-positive tumors than in negative tumors. Immunohistochemical staining using a Trop2-specific antibody further confirmed high Trop2 expression within BxPC3 tumors and low expression in A549 tumors. Figure 6 D).

[0070] 68The Ga-DOTA-TRP1 imaging signal decays rapidly within approximately 1 hour after injection, limiting its application in therapeutic radionuclide labeling. For example... Figure 7 The result shown is formed after covalent modification and introduction of FSY. 68 The Ga-DOTA-TRP1 variant significantly prolonged the retention time of the probe in BxPC3 tumors without significantly affecting targeting specificity, with tumor uptake lasting up to 2 hours. These results demonstrate that the structural modification effectively improves the in vivo stability and tumor retention properties of the peptide probe, paving the way for subsequent introduction... 177 Therapeutic radionuclides such as Lu provide the necessary pharmacokinetic basis for targeted radiotherapy. The structural optimization strategy of the peptide probe described in this invention has clear functional gains and significant translational value for integrated diagnosis and treatment.

[0071] Example 6 68 Ga-DOTA-TRP1 for in vivo PET imaging of thyroid cancer models Trop2-positive thyroid cancer cells were inoculated into Balb / c mice using BCPAP to establish a tumor model. Each mouse (n=3, injection dose: 7.5 MBq, 100 μL) was anesthetized with isoflurane-oxygen and imaged using a small animal PET / CT system (Mediso Medical Solutions, Inc.) at 30, 45, and 60 minutes. Regions of interest (ROIs) of major organs were delineated on the reconstructed PET images using an OsiriX Lite image processing workstation, and radioactive uptake values ​​of important organs were calculated in %ID / g. In the blocking imaging group, mice were co-injected with a sufficient amount of peptide (200 µg). Furthermore, tumor tissue was sectioned and immunohistoscopy was performed to observe Trop2 expression. Mice were sacrificed 30 minutes after injection (0.74 MBq, 0.1 mL), and blood samples and organs of interest were collected. Radioactivity counts were measured using a gamma counter to characterize the tumor. 68 Biological distribution of Ga-DOTA-TRP1 in a mouse model. Radioactive counts in the heart, liver, lungs, kidneys, spleen, stomach, bones, muscles, small intestine, and tumors were analyzed using a gamma counter.

[0072] The results are as follows Figure 8 As shown, 68The Ga-DOTA-TRP1 radiotracer showed significant uptake in both BCPAP tumors of the same mouse. Clear and persistent signals were observed at 30, 45, and 60 minutes post-injection. Tumor uptake decreased sharply after blockade with an unlabeled peptide, confirming the specific binding of the radiotracer to its target. Analysis of the tumor SUVmax value revealed that uptake in the right tumor was significantly higher than in the left. Trop2 immunohistochemical staining confirmed intratumoral heterogeneity. In vivo biodistribution results indicated… 68 Ga-DOTA-TRP1 was primarily enriched in the kidneys and bladder, suggesting that it is mainly excreted via the renal-urinary route. In addition to these excretion-related organs, its radioactive uptake levels in other non-target tissues were low and remained below the blood background signal throughout the observation period, demonstrating good in vivo clearance characteristics. This radiotracer offers advantages in pharmacokinetics and dosimetry, including rapid excretion and low accumulation in non-target tissues, which is beneficial for achieving high signal-to-noise ratio and high-contrast imaging within a clinically feasible timeframe.

[0073] Example 7 68 Ga-DOTA-TRP1 for in vivo PET imaging of lung cancer models Trop2-positive lung cancer cells (H292) were used to inoculate Balb / c mice to construct a tumor model. Imaging methods were the same as above.

[0074] The results are as follows Figure 9 As shown, 68 The Ga-DOTA-TRP1 radiotracer showed significant uptake in H292 tumors and was able to characterize tumor boundaries, revealing spatial heterogeneity in TROP2 expression within the tumor. Clear and persistent signals were observed at 30, 45, and 60 minutes post-injection. Immunohistochemical staining confirmed the intratumoral heterogeneity of H292 tumors, with Trop2 primarily expressed at the tumor periphery.

[0075] Example 8 68 Ga-DOTA-TRP69 for in vivo PET imaging of pancreatic cancer models 68 The Ga-DOTA-TRP69 radiotracer was used in the same way as above for tumor imaging in BxPC3.

[0076] The results are as follows Figure 10 As shown, 68 The Ga-DOTA-TRP69 radiotracer showed significant uptake in BxPC3 tumors, with clear and persistent signals visible 15-60 minutes post-injection. In vivo biodistribution analysis indicated... 68 Ga-DOTA-TRP69 primarily accumulates in the kidneys and bladder, indicating that it is mainly cleared via the renal-urinary pathway.

[0077] In summary, the polypeptide of the present invention has the characteristic of targeting Trop2-positive tumor cells. In practical applications, the polypeptide of the present invention can be used as a targeting molecule and combined with anticancer drugs or imaging agents for targeted tumor therapy and imaging.

[0078] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A peptide targeting Trop2, characterized in that, The polypeptide is: (a) The amino acid sequence is as shown in SEQ ID NO:1 or 2; (b) A polypeptide derived from (a) with one or more amino acids substituted, deleted or added to the sequence shown in SEQ ID NO:1 or 2 and having the same function; (c) A polypeptide derived from (a) that has more than 70% homology with the amino acid sequence of (a) or (b), preferably more than 80% homology, more preferably more than 90% homology, and has the same function.

2. A variant of the polypeptide of claim 1, characterized in that, The variant is: 1) Stereochemical variant: One or more amino acid residues in the polypeptide are L-type, D-type, or a mixture of L-type and D-type; 2) Structural modification variants: Cyclic derivatives, bicyclic derivatives, covalently modified derivatives of the polypeptide, or derivatives modified at its N-terminus, C-terminus or side chain by polyethylene glycol, fatty acid chains, fluorescent groups or biotin. 3) The polypeptide forms a divalent or multivalent.

3. A modified polypeptide or a pharmaceutically acceptable salt thereof, characterized in that, The modified polypeptide is obtained by coupling a bifunctional chelating agent to the N-terminus of the polypeptide of claim 1 or the variant of claim 2 via a linker; The bifunctional chelating agent is selected from DOTA, Nota, HYNIC, DTPA, TETA, DOTAGA, or NODAGA; Preferably, the modified polypeptide is TRP1 or TRP69, with the following structure: TRP1:DOTA-acp-LFRLQAETWMDP; TRP69:DOTA-acp-KYDETWRSQNC; Wherein, acp represents 6-aminocaproic acid; More preferably, the modified polypeptide is a covalent probe precursor formed by introducing a fluorosulfonyl-modified tyrosine residue into TRP1, with the structure: DOTA-acp-Tyr(SO2F)-LFRLQAETWMDP.

4. A conjugate or molecular probe, characterized in that, It comprises the polypeptide of claim 1 or the variant of claim 2 or the modified polypeptide of claim 3 or a pharmaceutically acceptable salt thereof, and an active agent conjugated to the polypeptide, variant or modified polypeptide.

5. The conjugate or molecular probe according to claim 4, characterized in that, The active agent is an imaging agent, which is a radionuclide complexed with a bifunctional chelating agent; Preferably, the radioactive nuclide is 68 Ga、 99m Tc, 89 Zr、 64 Cu or 18 F.

6. The conjugate or molecular probe according to claim 5, characterized in that, The bifunctional chelating agent also contains a linker between itself and the polypeptide, variant, or modified polypeptide; The linker is PEG. n ,Mal-PEG-DOTA,Tz-TCO,Tetrazine-PEG n -DOTA, DBCO-PEG n -NHS or aminocaproic acid; where n is an integer between 1 and 50.

7. The conjugate or molecular probe according to claim 4, characterized in that, The active agent is a therapeutic agent, which is selected from radioactive nuclides, cytotoxic agents, photosensitizers, chemotherapeutic drugs, immunomodulators, or antitumor small molecule drugs. Preferably, the radioactive therapeutic nuclide is 177 Lu、 90 Y、 131 I, 225 Ac or 212 Pb. Preferably, the antitumor small molecule drug is selected from microtubule inhibitors, DNA damaging agents, or topoisomerase inhibitors.

8. A pharmaceutical composition, characterized in that, It comprises the polypeptide of claim 1 or the variant of claim 2 or the modified polypeptide of claim 3 or a pharmaceutically acceptable salt thereof or the conjugate or molecular probe of any one of claims 4-7, and one or more pharmaceutically acceptable carriers or excipients.

9. Any of the following applications of the polypeptide of claim 1, the variant of claim 2, the modified polypeptide of claim 3, or a pharmaceutically acceptable salt thereof, or the conjugate or molecular probe of any one of claims 4-7: (1) Products used for the diagnosis or treatment of Trop2-positive tumors; (2) Reagents used to prepare for measuring or evaluating Trop2 expression levels in cells; (3) Used to prepare drugs for the diagnosis, prevention and / or treatment of diseases using Trop2 as a biomarker; (4) Diagnostic reagents, diagnostic kits or imaging agents used to prepare for the detection of Trop2-related diseases; (5) For the preparation of detection products for disease staging or auxiliary staging based on Trop2 biomarkers; (6) For the preparation of tracers for surgical navigation based on Trop2 markers.

10. The application according to claim 9, characterized in that, The Trop2-positive tumors are selected from breast cancer, lung cancer, thyroid cancer, head and neck squamous cell carcinoma, kidney cancer, urothelial carcinoma, prostate cancer, ovarian cancer, colorectal cancer, pancreatic cancer, liver cancer, stomach cancer, or esophageal cancer.

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