High-affinity HER2 targeting polycyclic peptide molecule as well as preparation method and application thereof

Abstract

The invention discloses a high-affinity HER2 targeting polycyclic peptide molecule and a preparation method and application thereof.The preparation method comprises the steps that affinity optimization is conducted on initial HER2 targeting polycyclic peptides obtained through phage display library screening by combining a yeast display technology with an error-prone PCR directed evolution system, and a series of high-affinity disulfide bond-rich polycyclic peptides (DDMP) are obtained; the polycyclic peptide is coupled with a conjugate (DOTA or FITC) and a linker, so that a monovalent or divalent molecular probe can be constructed; the molecular probe has high specificity, high stability and good biocompatibility, can be used for accurate imaging and quantitative evaluation of HER2 positive cells / tumors and diagnosis and targeted therapy of HER2 signal path related diseases through radionuclide labeling or fluorescence labeling, and provides a novel tool and a transformation basis for early diagnosis and accurate treatment of HER2 positive solid tumors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a multi-loop peptide molecule targeting HER2 with high affinity as well as a preparation method and application thereof. BACKGROUND

[0002] Human epidermal growth factor receptor 2 (HER2 / ErbB2) belongs to the epidermal growth factor receptor family and is a transmembrane glycoprotein with tyrosine kinase activity. Studies have shown that HER2 is overexpressed in about 20-30% of breast cancers and some gastric cancers, ovarian cancers and other malignant tumors, and its overexpression is closely related to the increase of tumor malignancy, the increase of metastasis risk and the poor prognosis of patients.

[0003] In the prior art, although the targeted therapy drugs (such as trastuzumab, pertuzumab, etc.) for HER2-positive tumors have achieved certain effects, they still face technical bottlenecks in dynamic monitoring of HER2 expression levels in clinical applications. The HER2 detection techniques commonly used in the clinic mainly include immunohistochemical detection (IHC) and fluorescence in situ hybridization (FISH), and these traditional detection methods have obvious defects: tissue samples need to be obtained, which is invasive; only the local HER2 expression at the sampling time point can be reflected; and real-time dynamic monitoring in the whole body range cannot be achieved.

[0004] The molecular imaging technology based on radionuclide labeling (especially PET) provides a new technical idea for solving the above problems. At present, a variety of HER2-targeted molecular probes have been developed for PET imaging, mainly including radiolabeled monoclonal antibodies and their fragments, nanobody probes and polypeptide probes, etc., but these schemes have the following deficiencies: the antibody probes have large molecular weight, poor tissue penetration, slow blood clearance, and non-specific uptake problems; the existing polypeptide probes generally have insufficient affinity (K D d; and there is a lack of ideal probe molecules with high affinity and high stability.

[0005] Compared with traditional linear peptides and monocyclic peptides, cyclic peptides can achieve higher affinity target binding due to the limited conformation. Among them, multi-loop peptides (DDMP) rich in disulfide bonds are a special kind of constrained peptides, which have high affinity, high specificity and outstanding protease stability, and are expected to become a new molecular template for developing HER2 high-efficiency targeted peptides and promote the research and development of a new generation of HER2 diagnosis and treatment integrated radiopharmaceuticals. SUMMARY

[0006] To solve the above problems, the present application provides a multi-loop peptide molecule targeting HER2 with high affinity as well as a preparation method and application thereof.

[0007] To achieve the above purpose, the present application adopts the following technical solutions:

[0008] In the first aspect, a high-affinity polycyclic peptide molecule targeting HER2 is described. A random mutant yeast display library is constructed using yeast display technology combined with a fault-prone PCR-based directed evolution system. Positive cells are obtained by flow cytometry sorting. The amino acid sequence of the polycyclic peptide molecule contains the following general formula:

[0009] CPWX a CX b YX c CX d X e X f X g X h CSEVX i X j X k X l CPX m X n C,

[0010] Among them, X a For F, A; X b For I, T; X c For P, A; X d For K, R, E; X e For V, A; X f For E, Q; X g For P, A; X h For R, A; X i For Y, F; X j For A, E; X k For E, A, V, K, R; X l For H, A, Q, L; X m X is any amino acid; n It can be any amino acid; and all amino acids are L-type amino acids.

[0011] Preferably, X a For F; X b For T; X c For P; X d K; X e For V; X f For Q; X g For P; X h R; X i For Y; X j For A; X k For E; X l H; X m For A; X n R; or, the amino acid sequence of the high-affinity HER2-targeting polycyclic peptide molecule is selected from any of the following sequences:

[0012] CPWFCTYPCKVQPRCSEVYAEQCPQTC(SEQ ID No.1);

[0013] CPWFCTYPCKVEPRCSEVYAEQCPAVC(SEQ ID No.2);

[0014] CPWFCTYPCKVQPRCSEVYAVHCPQRC(SEQ ID No.3);

[0015] CPWFCTYPCKVQPRCSEVYAAQCPIGC(SEQ ID No.4);

[0016] CPWFCTYPCKVQPRCSEVYAEHCPARC(SEQ ID No.5);

[0017] CPWFCTYPCKVQPRCSEVYAKLCPQSC(SEQ ID No.6);

[0018] CPWFCTYPCKVQPRCSEVYAEHCPQRC(SEQ ID No.7);

[0019] CPWACTYPCKVQPRCSEVYAEHCPARC(SEQ ID No.8);

[0020] CPWFCTYACKVQPRCSEVYAEHCPARC(SEQ ID No.9);

[0021] CPWFCTYPCEVQPRCSEVYAEHCPARC(SEQ ID No.10);

[0022] CPWFCTYPCRVQPRCSEVYAEHCPARC(SEQ ID No.11);

[0023] CPWFCTYPCKAQPRCSEVYAEHCPARC(SEQ ID No.12);

[0024] CPWFCTYPCKVQARCSEVYAEHCPARC(SEQ ID No.13);

[0025] CPWFCTYPCKVQPACSEVYAEHCPARC(SEQ ID No.14);

[0026] CPWFCTYPCKVQPRCSEVYEEHCPARC(SEQ ID No.15);

[0027] CPWFCTYPCKVQPRCSEVYAAHCPARC(SEQ ID No.16);

[0028] CPWFCTYPCKVQPRCSEVYAEACPARC(SEQ ID No.17);

[0029] CPWFCTYPCKVQPRCSEVYAEHCPGRC(SEQ ID No.18);

[0030] CPWFCTYPCKVQPRCSEVYAEHCPAAC(SEQ ID No.19);

[0031] CPWFCTYPCEVQPRCSEVYAAHCPAAC(SEQ ID No.20); or

[0032] CPWFCTYPCEVQPRCSEVYAAHCPAKC (SEQ ID No. 21).

[0033] Secondly, a method for preparing a high-affinity HER2-targeting polycyclic peptide molecule includes the following steps:

[0034] S1A: Using the encoding genes of the initial polycyclic peptides M-1 and M-2 as templates, random mutations were introduced using error-prone PCR. After 10 rounds of error-prone PCR amplification, the PCR products from rounds 6 to 10 were collected and mixed to construct a random mutant yeast display library, Library 1. The amino acid sequence of polycyclic peptide M-1 is CPWFCIYPCKVEPRCSEVYAEQCPQTC; the amino acid sequence of polycyclic peptide M-2 is CPWFCIYPCKVEPRCSQVYEQECPAVC.

[0035] S2A. Flow cytometry was used to perform four rounds of HER2-targeted screening on the random mutant yeast display library Library 1. After each round of screening, high-throughput sequencing analysis was performed on the positive yeast cell population to obtain optimized peptide sequences, which include the sequences shown in SEQ ID No. 1 to 2.

[0036] S3A. Using the positive yeast plasmid obtained in the 4th round of screening of the random mutant yeast display library Library 1 as a template, 10 rounds of error-prone PCR were performed, and the product of the 10th round was used to construct the secondary random mutant yeast display library Library 2.

[0037] S4A. The secondary random mutant yeast display library Library 2 was subjected to four rounds of HER2-targeted flow cytometry sorting and high-throughput sequencing analysis to obtain the final optimized sequence, which includes the sequences shown in SEQ ID No. 3 to 7.

[0038] S5A. The structure-activity relationship of the peptide that obtained the highest affinity was studied to obtain the target peptide sequence, which includes the sequences shown in SEQ ID No. 8 to 21.

[0039] Thirdly, a polycyclic peptide molecular probe targeting HER2, characterized by the structure DLP; wherein, D is a conjugate selected from fluorescein isothiocyanate (FITC); L is 3-5 amino acid residues composed of Gly and Ser; and P is the high-affinity polycyclic peptide molecule targeting HER2 or the derived polypeptide.

[0040] Fourthly, a HER2-targeting divalent polycyclic peptide molecular probe is formed by coupling the high-affinity HER2-targeting polycyclic peptide molecule or the derived polypeptide via a linker DSS, wherein the DSS is bis(succinimide) octanoate; the C-terminus of the high-affinity HER2-targeting polycyclic peptide molecule is synonymous with the C-terminus, and the N-terminus is synonymous with the N-terminus; and the structure of the divalent polycyclic peptide molecular probe is any one of the following:

[0041]

[0042] Wherein, P is the high-affinity HER2-targeting polycyclic peptide molecule.

[0043] Fifthly, a labeled precursor of a HER2-targeting therapeutic drug, wherein the labeled precursor has an XRP structure, wherein X is a chelating agent, R is a linker arm, and P is a high-affinity HER2-targeting polycyclic peptide molecule or a divalent polycyclic peptide as described in claim 1 or 2; the amino side of the linker arm is connected to the chelating agent, and the carboxyl side of the linker arm is connected to the amino group of the first amino acid at the N-terminus of the polypeptide compound.

[0044] Sixthly, a labeling precursor for a therapeutic drug targeting HER2, wherein the connecting arm is selected from one of the compounds with the following structural formula:

[0045]

[0046] Where n is an integer in the range of 1-10; preferably, the connecting arm is... Where n = 2.

[0047] Seventhly, a labeled prodrug for a therapeutic drug targeting HER2, wherein the chelate is selected from one of the compounds with the following structural formula:

[0048]

[0049] Preferably, the chelate is

[0050] Eighthly, a radiopharmaceutical targeting HER2, wherein the radiopharmaceutical is obtained by radiolabeling the polycyclic peptide molecule or divalent polycyclic peptide molecule probe with a radionuclide, and the radionuclide for diagnostic and therapeutic purposes is selected from... 18 F, 32 P, 33 P, 45 Ti、 47 Sc、 52 Fe、 59 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 75 Se、 75 Sc、 77 As、 86 Y、 89 Sr、 89 Zr、 90 Y、 94 Tc, 99 mTc, 99 Mo、 105 Pd, 105 Rh、 109 Pd, 111 TCP1 111 In、 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm、 161 Tb, 166 Dy、 166 Ho、 169 Er、 175 Lu、 177 Lu、 186 Re、 188 Re、 189 Re、 194 Ir、 198 Au、 199 Au、 211 At、 211 Pb, 212 Bi、 212 Pb, 213Bi、 223 Ra or 225 Ac.

[0051] Ninthly, the method for preparing the labeled precursor of the HER2-targeting therapeutic drug includes the following steps:

[0052] S1B. Linear polypeptides were synthesized using a solid-phase synthesis method.

[0053] S2B: The polypeptide-containing solid resin synthesized in step S1B is added to DMF for swelling, DOTA-NHS or FITC is added, and the pH is adjusted to 8.0 with N,N-diisopropylethylamine (DIEA). The reaction is carried out overnight at 37°C.

[0054] S3B: Cut the solid resin after the reaction in step S2B to remove the side chain protecting groups of amino acids, and purify the linear peptide by preparative chromatography.

[0055] S4B. The linear polypeptide obtained in step S3B is mixed with oxidized glutathione (GSSG) at a molar ratio of 1:20, reacted at 37°C overnight, and then purified to obtain an oxidized polycyclic peptide coupled with DOTA or FITC.

[0056] S5B. If the conjugate is DOTA, the DOTA-conjugated polycyclic peptide obtained in step S4B is radiolabeled: the radionuclide elution buffer is mixed with the labeling precursor, the pH is adjusted to 4-10 with buffer solution, and the reaction is carried out at 20-110°C for 10 minutes to overnight; the buffer solution is sodium acetate, water, ethanol, phosphoric acid, hydrochloric acid buffer solution or dimethyl sulfoxide; if the conjugate is FITC, the FITC-conjugated polypeptide obtained in S4B is dissolved in dimethyl sulfoxide solution, and then diluted with PBS buffer to the required concentration.

[0057] Tenthly, the application of the high-affinity HER2-targeting polycyclic peptide molecule, the HER2-targeting polycyclic peptide molecular probe, the HER2-targeting divalent polycyclic peptide molecular probe, or the labeling precursor of the HER2-targeting therapeutic drug in the preparation of drugs, diagnostic reagents, or drug lead molecules for the diagnosis and / or treatment of HER2 signaling pathway-related diseases.

[0058] Preferably, the high-affinity HER2-targeting polycyclic peptide molecule is radiolabeled for in vivo imaging or targeted therapy of HER2-positive tumors. These diseases include, but are not limited to, breast cancer, pancreatic cancer, urothelial carcinoma, ovarian cancer, cervical cancer, renal cell carcinoma, bladder cancer, gastric cancer, colorectal cancer, esophageal cancer, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, melanoma, osteosarcoma, soft tissue sarcoma, glioma, neuroblastoma, medulloblastoma, oral cancer, nasopharyngeal carcinoma, laryngeal cancer, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia, choriocarcinoma, hepatoblastoma, choroidal melanoma, cavernous hemangioma, renal angiomyolipoma, teratoma, sarcoma, or other related tumors.

[0059] Preferably, the diagnostic method is nuclear medicine imaging, selected from positron emission tomography or single-photon emission computed tomography; the treatment method is targeted therapy based on radionuclides; the HER2 signaling pathway-related diseases include HER2-positive tumors or non-tumor diseases related to abnormal HER2 expression.

[0060] By adopting the above technical solution, the present invention has the following beneficial effects:

[0061] 1. This invention breaks through the limitations of traditional peptide probes in affinity and stability at the molecular design level, achieving high affinity and specific binding. The polycyclic peptide molecular framework of this invention is a three-membered cyclic peptide rich in disulfide bonds. Through yeast display technology combined with a mistake-prone PCR directed evolution system optimization, a more complex rigid structure and a larger target binding interface are formed, significantly improving the binding affinity to HER2. This is far superior to peptide probes with micromolar affinity in existing technologies, enabling precise identification of HER2-positive cells or tumors and reducing background interference caused by uptake from non-target tissues. Simultaneously, the multiple disulfide bonds in the polycyclic peptide are not only the core of conformational stability but also effectively resist protease degradation. Compared to linear peptides or monocyclic peptides, it has a longer half-life in complex biological environments such as blood and extracellular fluid, ensuring that the probe can effectively reach the target site and exert its function in vivo, solving the problems of easy degradation and short in vivo action time of traditional peptide probes.

[0062] 2. This invention offers high efficiency and controllability in screening and preparation, possessing the potential for large-scale production. Employing a yeast-display directed evolution screening strategy, combined with flow cytometry sorting and high-throughput sequencing, the construction of two rounds of mutant libraries (Initial mutation in Library 1, directed optimization in Library 2) and progressively increasing screening pressure (reducing the sorting frame, decreasing the HER2 protein concentration, and increasing the number of washes) rapidly enriches high-affinity sequences. This results in a short screening cycle and high specificity, avoiding the low efficiency and sequence redundancy issues of traditional random screening, thus achieving high efficiency in directed evolution screening. Furthermore, the preparation of polycyclic peptides and molecular probes is based on solid-phase synthesis technology, with clear and reproducible steps. Key steps such as linear peptide synthesis, conjugate (DOTA / FITC) reaction, oxidative folding, and radionuclide labeling are all performed using conventional laboratory procedures without the need for special equipment. The purification process is completed using preparative chromatography and high-performance liquid chromatography, resulting in high product purity (purity of oxidation products can reach over 95%), facilitating large-scale production and providing a technological foundation for subsequent clinical translation and commercialization.

[0063] 3. At the functional application level, this invention achieves integrated diagnosis and treatment, expanding the boundaries of clinical value. The molecular probes of this invention can achieve functional switching between diagnosis and treatment through "the same molecular framework, modified with different conjugates"—conjugation with FITC can serve as a fluorescent probe for quantitative detection of HER2 protein on the cell surface, supporting cell function research and mechanism analysis; conjugation with DOTA followed by labeling with radionuclides (such as...) 68 Ga is used in PET imaging. 177 Lu (used for targeted radiotherapy) can achieve early diagnosis, staging, and treatment response monitoring of HER2-positive tumors through PET / SPECT imaging, and can also directly kill tumor cells through targeted radiotherapy (TRT), constructing an integrated closed loop of "diagnosis-treatment-efficacy evaluation" to improve the efficiency of individualized treatment for patients. It can be used not only for the diagnosis and treatment of HER2-positive tumors (covering more than 20 types of solid tumors and hematologic malignancies such as breast cancer, pancreatic cancer, ovarian cancer, and gastric cancer), but also for the diagnosis and treatment of non-tumor diseases related to abnormal HER2 expression (such as cardiomyopathy, pulmonary fibrosis, and autoimmune diseases) through radionuclide labeling. This breaks through the limitations of existing HER2-targeted drugs, which are mostly confined to tumor treatment, and expands the boundaries of clinical value.

[0064] 4. At the clinical translational level, this invention meets the practical needs of precision diagnosis and treatment. Based on radionuclide-labeled molecular imaging probes, whole-body HER2 expression monitoring can be achieved via PET / CT or SPECT without obtaining tissue samples, avoiding the invasiveness of traditional IHC and FISH tests. Simultaneously, it supports dynamic observation at multiple time points and sites, reflecting in real time the heterogeneity of tumor HER2 expression and changes in expression during treatment, providing precise evidence for tumor subtyping, prognostic assessment, and treatment plan adjustment. The polycyclic peptides contain only natural L-type amino acids, conforming to the amino acid configuration in vivo, and have no exogenous toxic fragments; animal experiments have verified that... 68 After administration of Ga-labeled diagnostic probes, mice showed no significant damage to major organs (heart, liver, spleen, lungs, kidneys, etc.) (no pathological abnormalities were observed in H&E staining), while therapeutic probes (such as [ 177 Lu-peptide 2) effectively inhibits tumor growth without significantly affecting mouse body weight, demonstrating good safety and laying the foundation for clinical application. Attached Figure Description

[0065] Figure 1 The top 20 sequences were enriched by high-throughput sequencing in the fourth round of screening for Library 1 and Library 2;

[0066] Figure 2 These are the sequencing results of single clones selected in the third and fourth rounds of screening in Library 2;

[0067] Figure 3 Flow cytometry was used to screen yeast libraries for Library 1 and Library 2.

[0068] Figure 4 Chromatogram of peptide 1-21 oxidation;

[0069] Figure 5 SPR affinity curves for peptide 1-21 (oxidized form) and peptide probes;

[0070] Figure 6 Imaging image of HEK293T cells transfected with FITC-peptide 5 and HER2-mCherry.

[0071] Figure 7 Imaging images of FITC-peptide 5, FITC-divalent peptide, and SKOV3 / BxPC-3 cells;

[0072] Figure 8 Titration curves of FITC-peptide 5 and FITC-divalent peptide in SKOV3 / BxPC-3 cells;

[0073] Figure 9 for[ 68 Ga]-M-1、[ 68 Ga]-peptide 2、[ 68 Radiochromatogram of Ga]-peptide 5;

[0074] Figure 10 for[ 177 Lu]-M-1、[ 177 Radiochromatogram of Lu]-peptide 2;

[0075] Figure 11 for[ 68 Ga]- probe in SKOV3 tumor-bearing mice PET / CT imaging;

[0076] Figure 12 for[ 68 Ga]-probe in PET / CT imaging of BxPC-3 tumor-bearing mice;

[0077] Figure 13 for[ 68 Ga]-DOTA-divalent peptide in SKOV3 tumor-bearing mice PET / CT imaging;

[0078] Figure 14 for[ 68 Dynamic imaging analysis of Ga]-probe in SKOV3 tumor-bearing mice;

[0079] Figure 15 for[ 68 Biodistribution analysis of Ga]-probes in SKOV3 tumor-bearing mice;

[0080] Figure 16 for[ 68 H&E staining of major organs in mice after administration of Ga-probe;

[0081] Figure 17 for[ 177 Lu]-probe curve of tumor / body weight change in SKOV3 tumor-bearing mice;

[0082] Figure 18 for[ 177 Survival curve of Lu-probe in SKOV3 tumor-bearing mice. Detailed Implementation

[0083] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0084] like Figures 1 to 18As shown, a high-affinity polycyclic peptide molecule targeting HER2 was constructed using yeast display technology combined with a fault-prone PCR directed evolution system to create a random mutant yeast display library. Positive cells were obtained by flow cytometry sorting. The amino acid sequence of the polycyclic peptide molecule contains the following general formula:

[0085] CPWX a CX b YX c CX d X e X f X g X h CSEVX i X j X k X l CPX m X n C,

[0086] Among them, X a For F, A; X b For I, T; X c For P, A; X d For K, R, E; X e For V, A; X f For E, Q; X g For P, A; X h For R, A; X i For Y, F; X j For A, E; X k For E, A, V, K, R; X l For H, A, Q, L; X m X is any amino acid; n It can be any amino acid; and all amino acids are L-type amino acids.

[0087] D stands for aspartic acid; W for tryptophan; H for histidine; T for threonine; Y for tyrosine; V for valine; E for glutamic acid; S for serine; F for phenylalanine; G for glycine; M for methionine; N for asparagine; I for isoleucine; L for leucine; A for alanine; R for arginine; K for lysine; P for proline; C for cysteine; and Q for glutamine.

[0088] Preferably, X a For F; X b For T; X c For P; X d K; X e For V; X f For Q; X g For P; X h R; X i For Y; Xj For A; X k For E; X l H; X m For A; X n R; or, the amino acid sequence of the high-affinity HER2-targeting polycyclic peptide molecule is selected from any of the following sequences:

[0089] CPWFCTYPCKVQPRCSEVYAEQCPQTC(SEQ ID No.1);

[0090] CPWFCTYPCKVEPRCSEVYAEQCPAVC(SEQ ID No.2);

[0091] CPWFCTYPCKVQPRCSEVYAVHCPQRC(SEQ ID No.3);

[0092] CPWFCTYPCKVQPRCSEVYAAQCPIGC(SEQ ID No.4);

[0093] CPWFCTYPCKVQPRCSEVYAEHCPARC(SEQ ID No.5);

[0094] CPWFCTYPCKVQPRCSEVYAKLCPQSC(SEQ ID No.6);

[0095] CPWFCTYPCKVQPRCSEVYAEHCPQRC(SEQ ID No.7);

[0096] CPWACTYPCKVQPRCSEVYAEHCPARC(SEQ ID No.8);

[0097] CPWFCTYACKVQPRCSEVYAEHCPARC(SEQ ID No.9);

[0098] CPWFCTYPCEVQPRCSEVYAEHCPARC(SEQ ID No.10);

[0099] CPWFCTYPCRVQPRCSEVYAEHCPARC(SEQ ID No.11);

[0100] CPWFCTYPCKAQPRCSEVYAEHCPARC(SEQ ID No.12);

[0101] CPWFCTYPCKVQARCSEVYAEHCPARC(SEQ ID No.13);

[0102] CPWFCTYPCKVQPACSEVYAEHCPARC(SEQ ID No.14);

[0103] CPWFCTYPCKVQPRCSEVYEEHCPARC(SEQ ID No.15);

[0104] CPWFCTYPCKVQPRCSEVYAAHCPARC(SEQ ID No.16);

[0105] CPWFCTYPCKVQPRCSEVYAEACPARC(SEQ ID No.17);

[0106] CPWFCTYPCKVQPRCSEVYAEHCPGRC(SEQ ID No.18);

[0107] CPWFCTYPCKVQPRCSEVYAEHCPAAC(SEQ ID No.19);

[0108] CPWFCTYPCEVQPRCSEVYAAHCPAAC(SEQ ID No.20); or

[0109] CPWFCTYPCEVQPRCSEVYAAHCPAKC (SEQ ID No. 21).

[0110] A method for preparing a high-affinity HER2-targeting polycyclic peptide molecule includes the following steps:

[0111] S1A: Using the encoding genes of the initial polycyclic peptides M-1 and M-2 as templates, random mutations were introduced using error-prone PCR. After 10 rounds of error-prone PCR amplification, the PCR products from rounds 6 to 10 were collected and mixed to construct a random mutant yeast display library, Library 1. The amino acid sequence of polycyclic peptide M-1 is CPWFCIYPCKVEPRCSEVYAEQCPQTC; the amino acid sequence of polycyclic peptide M-2 is CPWFCIYPCKVEPRCSQVYEQECPAVC.

[0112] S2A. Flow cytometry was used to perform four rounds of HER2-targeted screening on the random mutant yeast display library Library 1. After each round of screening, high-throughput sequencing analysis was performed on the positive yeast cell population to obtain optimized peptide sequences, which include the sequences shown in SEQ ID No. 1 to 2.

[0113] S3A. Using the positive yeast plasmid obtained in the 4th round of screening of the random mutant yeast display library Library 1 as a template, 10 rounds of error-prone PCR were performed, and the product of the 10th round was used to construct the secondary random mutant yeast display library Library 2.

[0114] S4A. The secondary random mutant yeast display library Library 2 was subjected to four rounds of HER2-targeted flow cytometry sorting and high-throughput sequencing analysis to obtain the final optimized sequence, which includes the sequences shown in SEQ ID No. 3 to 7.

[0115] S5A. The structure-activity relationship of the peptide that obtained the highest affinity was studied to obtain the target peptide sequence, which includes the sequences shown in SEQ ID No. 8 to 21.

[0116] A polycyclic peptide molecular probe targeting HER2 is characterized by the structure DLP; wherein, D is a conjugate selected from or fluorescein isothiocyanate (FITC); L is 3-5 amino acid residues composed of Gly and Ser; and P is a high-affinity polycyclic peptide molecule targeting HER2.

[0117] A HER2-targeting divalent polycyclic peptide molecular probe is formed by coupling a high-affinity HER2-targeting polycyclic peptide molecule or its derivative via a linker DSS, wherein the DSS is bis(succinimide) octanoate; the C-terminus of the high-affinity HER2-targeting polycyclic peptide molecule is synonymous with the C-terminus, and the N-terminus is synonymous with the N-terminus; and the structure of the divalent polycyclic peptide molecular probe is any one of the following:

[0118]

[0119] P is a polycyclic peptide molecule with high affinity targeting HER2.

[0120] The preparation method of the divalent polycyclic peptide molecular probe includes the following steps:

[0121] S1C: GEGEGK is attached to the C-terminus of a high-affinity HER2-targeting polycyclic peptide molecule as a linker. DSS is coupled to the amino group on the C-terminal lysine side chain of the peptide via an amide bond to form a divalent peptide. During synthesis, the lysine on the peptide is selected to be an amino acid with a Dde-protected side chain, and the lysine on the linker is selected to be an amino acid with a Boc-protected side chain. One peptide is attached to the N-terminus with FITC, and the other peptide is attached to the N-terminus with Ac protection. A linear peptide is synthesized using a solid-phase synthesis method.

[0122] S2C: The polypeptide-containing solid resin synthesized in S1C is added to DMF for swelling, DOTA-NHS or FITC is added, the pH is adjusted to 8.0 with DIEA, and the reaction is carried out overnight at 37°C.

[0123] S3C: The solid-phase resin after the S2C reaction is cut to remove the side chain protecting groups of amino acids, and the linear peptide is purified by preparative chromatography.

[0124] S4C: The linear peptide obtained in S3C is added to GSSG at a molar ratio of 1:20, reacted at 37°C overnight, and then purified to obtain an oxidized polycyclic peptide coupled with DOTA or FITC.

[0125] The method for purifying peptides with S5C and N-terminus blocked by Ac is the same as that in steps S3C and S4C.

[0126] S6C. Dissolve 1 mM Ac-peptide and 2 mM DSS in 50 μL DMF, add triethylamine to adjust the pH to about 8.0, react at room temperature for 1 h, and then separate and purify by high performance liquid chromatography to obtain Ac-peptide-DSS.

[0127] S7C. For DOTA-divalent peptides, 1 mM MAc-peptide and 2 mM DOTA-peptide were dissolved in 50 μL DMF, and triethylamine was added to adjust the pH to approximately 8.0. After reacting overnight at room temperature, the peptides were separated and purified by high-performance liquid chromatography (HPLC). For FITC-divalent peptides, 1 mM MAc-peptide and 2 mM FITC-peptide were dissolved in 50 μL DMF, and triethylamine was added to adjust the pH to approximately 8.0. After reacting overnight at room temperature, the peptides were separated and purified by HPLC.

[0128] S8C. Dissolve DOTA-divalent peptide or FITC-divalent peptide in DMF, take 100 μL of 100 μM peptide, add 2% hydrazine hydrate, react at room temperature for 15 min to remove the Dde protecting group, and separate and purify by high performance liquid chromatography to obtain DOTA-divalent peptide or FITC-divalent peptide.

[0129] S9C. The DOTA-divalent polypeptide obtained in S8C is radiolabeled using a radionuclide method. The radionuclide elution buffer is eluted, and the eluting precursor is mixed with the eluting buffer. The pH is adjusted to 4–10 using a buffer solution, which may be sodium acetate, water, ethanol, phosphoric acid, hydrochloric acid buffer, or dimethyl sulfoxide. The reaction is carried out at 20–110°C for 10 minutes to overnight. The FITC-conjugated polypeptide obtained in S8C is dissolved in dimethyl sulfoxide solution and then diluted with PBS buffer to a series of concentrations.

[0130] Applications of high-affinity HER2-targeting polycyclic peptide molecules and molecular probes in the preparation of drugs, diagnostic reagents or drug lead molecules for the diagnosis and / or treatment of HER2 signaling pathway-related diseases.

[0131] The diagnostic method is nuclear medicine imaging, selected from positron emission tomography or single-photon emission computed tomography; the treatment method is targeted therapy based on radionuclides, selected from alpha nuclide therapy or beta nuclide therapy; the HER2 signaling pathway-related diseases include HER2-positive tumors or non-tumor diseases related to abnormal HER2 expression.

[0132] Example 1: Construction and Screening of Yeast Display Libraries

[0133] 1) Construction of Library 1: Using the M-1 and M-2 coding genes as templates, sequence mutations were performed using a commercial error-prone PCR kit. After 10 rounds of amplification, the products from rounds 6-10 were ligated into the yeast expression vector pYD1, transformed into E. coli DH5α, and the plasmid was extracted and transfected into Saccharomyces cerevisiae EBY100 to construct Library 1, with a library size of approximately 1×10⁻⁶. 8 CFU;

[0134] 2) Screening process: Take 1×10 8 Yeast cells were divided into two groups. The experimental group was given 4 μL of Myc antibody and 10-fold biotinylated HER2 protein, while the control group was given only Myc antibody. After incubation at room temperature for 1 h, 2.5 μL of Alexa Fluor 647-streptavidin and 4 μL of LeraLite 488 anti-mouse secondary antibody were added. The cells were incubated at 4 °C for 30 min and then sorted by flow cytometry (0.7% in the first round, 0.5% in the second round, 0.3% in the third round, and 0.1% in the fourth round were positive cells).

[0135] 3) Construction and screening of Library 2: Extract the positive yeast plasmid from the fourth round of Library 1, perform 10 rounds of error-prone PCR, and use the product from the 10th round to construct Library 2. During screening, decrease the HER2 protein concentration (10-fold → 2-fold) and increase the number of washes (1 time → 10 times) in each round, repeating the sorting process for 4 rounds (e.g., ...). Figure 3 );

[0136] 4) Sequencing Validation: High-throughput sequencing and monoclonal sequencing analysis were performed on the positive cells obtained after the third and fourth rounds of screening to confirm the sequence enrichment trend (e.g., Figure 1 ), and select single clones for sequencing verification (e.g. Figure 2 ).

[0137] Example 2: Peptide Synthesis and Oxidation

[0138] 1) Solid-phase synthesis: A CEM LibertyBlue microwave synthesizer was used with RinkAmide MBHA resin (loading = 0.345 mmol / g) at a scale of 0.025 mmol. The Fmoc protecting group was removed with 20% piperidine. Coupling reaction was carried out with 20 times excess DIC, Oxyma and 5 times excess FmocAA-OH, sequentially from C-terminus to N-terminus.

[0139] 2) Cutting and purification: After synthesis, the peptide was cut with cutting solution (TFA:phenol:TIS:H2O = 88:5:2:5) at 37℃ for 2 h. The crude peptide was precipitated with anhydrous diethyl ether and purified by semi-preparative HPLC (mobile phase: water / acetonitrile, containing 0.1% TFA). The molecular weight was confirmed by MALDI-TOF mass spectrometry.

[0140] 3) Oxidative folding: The linear peptide was dissolved in DMSO, and 20-fold excess GSSG was added. The reaction was carried out overnight at 37°C in 30% DMSO / 70% PB buffer (100 mM, pH 7.4). The oxidation product was purified by HPLC (e.g., ...). Figure 4 The molecular weight was confirmed by mass spectrometry.

[0141] Example 3: Molecular probe preparation and performance verification

[0142] 1) Preparation of FITC-labeled probe: Take the purified linear peptide solid-phase resin, add FITC (5 times excess) and DIEA (10 times excess), swell with DMF, react overnight at 37°C, cut and purify, and then oxidize to obtain FITC-polycyclic peptide;

[0143] 2) DOTA labeling and radionuclide labeling: Linear peptide resin reacts with DOTA-NHS (5-fold excess), followed by cleavage, purification, and oxidation. 20 μg of DOTA-polycyclic peptide is then added to […]. 68 Ga] eluent (0.5M HCl eluent generator), pH adjusted to 4.5 with sodium acetate buffer, reacted at 100℃ for 10 min, purified by C18 column, and purity determined by Radio-HPLC (e.g., Ga). Figure 9 );

[0144] 3) Affinity assay: Using the Biacore 1K+ system, biotinylated HER2 protein was immobilized on a CAPture chip (response value 400-1500 RU), and the peptide was serially diluted and injected into the channel. The K value was obtained by fitting the SPR curve. D Value (e.g.) Figure 5 );

[0145] 4) Cell imaging: A plasmid fused with the mCherry tag of human HER2 protein was transiently transfected into HEK293T cells, 10 nM FITC-probe was added, and the cells were incubated at 37°C for 1 h. Confocal imaging (e.g., ...) was then performed. Figure 8(As shown); SKOV3 / BxPC-3 cells were plated for 24 hours, 100 nM FITC-probe was added, and the cells were incubated at 37°C for 1 hour. Hoechst 33342 staining was performed, and the cells were imaged using a confocal microscope (as shown). Figure 6 In the flow cytometry titration experiment, cells were incubated with gradient concentration probes at 4°C for 1 hour, and the fluorescence intensity was detected (e.g., ...). Figure 7 ).

[0146] Example 4: Small Animal PET / CT Imaging and Therapy Experiment

[0147] 1) Tumor-bearing model construction: SKOV3 / BxPC-3 cells (1×10⁻⁶) 6 ( / mouse) Subcutaneously inoculate nude mice until the tumor volume reaches 100-300 mm. 3 Time experiment;

[0148] 2) PET / CT imaging: 150 μCi injected via tail vein [ 68 Ga]-probe was used to perform MicroPET / CT scans at 60, 120, and 240 min to quantitatively analyze tumor uptake (%ID / g) (e.g.) Figure 11 , 12 13); Dynamic imaging lasted for 60 minutes, recording changes in radioactivity distribution (e.g., Figure 14 );

[0149] 3) Biodistribution: Inject 50 μCi [ 68 Ga]-probe, mice were sacrificed at different time points, organs / tumors were harvested and weighed, radioactivity was measured using a gamma counter, and %ID / g was calculated (e.g., Ga-probe, mice were sacrificed at different time points, organs / tumors were harvested and weighed, radioactivity was measured using a gamma counter, and %ID / g was calculated). Figure 15 H&E staining assesses organ safety (e.g.) Figure 16 );

[0150] 4) Treatment Experiment: SKOV3 tumor-bearing mice were divided into a control group (physiological saline) and [ 177 Lu-probe set (200, 400, 600 μCi), once via tail vein injection, tumor volume and body weight measured every two days (e.g.) Figure 17 Record survival time (e.g.) Figure 18 ).

[0151] Therefore, the high-affinity HER2-targeting polycyclic peptide molecule and molecular probe of the present invention have a mature preparation process (solid-phase synthesis and radionuclide labeling are both conventional techniques) and are easy to scale up; they have both diagnostic and therapeutic functions, enabling an integrated diagnostic and therapeutic strategy of "same molecular framework, different radionuclide labeling" to reduce clinical translation costs; and they have shown high targeting and safety in animal experiments, providing a new tool for the precision diagnosis and treatment of HER2-positive diseases, with significant clinical value and commercial potential.

Claims

1. A high-affinity polycyclic peptide molecule targeting HER2, characterized in that, A random mutant yeast display library was constructed using yeast display technology combined with a fault-prone PCR-based directed evolution system. Positive cells were obtained by flow cytometry sorting. The amino acid sequence of the polycyclic peptide molecule contains the following general formula: CPWX a CX b YX c CX d X e X f X g X h CSEVX i X j X k X l CPX m X n C, Among them, X a For F, A; X b For I, T; X c For P, A; X d For K, R, E; X e For V, A; X f For E, Q; X g For P, A; X h For R, A; X i For Y, F; X j For A, E; X k For E, A, V, K, R; X l For H, A, Q, L; X m X is any amino acid; n It can be any amino acid; and all amino acids are L-type amino acids.

2. The high-affinity HER2-targeting polycyclic peptide molecule as described in claim 1, characterized in that, X a For F; X b For T; X c For P; X d K; X e For V; X f For Q; X g For P; X h R; X i For Y; X j For A; X k For E; X l H; X m For A; X n R; or, the amino acid sequence of the high-affinity HER2-targeting polycyclic peptide molecule is selected from any of the following sequences: CPWFCTYPCKVQPRCSEVYAEQCPQTC(SEQ ID No.1); CPWFCTYPCKVEPRCSEVYAEQCPAVC(SEQ ID No.2); CPWFCTYPCKVQPRCSEVYAVHCPQRC(SEQ ID No.3); CPWFCTYPCKVQPRCSEVYAAQCPIGC(SEQ ID No.4); CPWFCTYPCKVQPRCSEVYAEHCPARC(SEQ ID No.5); CPWFCTYPCKVQPRCSEVYAKLCPQSC(SEQ ID No.6); CPWFCTYPCKVQPRCSEVYAEHCPQRC(SEQ ID No.7); CPWACTYPCKVQPRCSEVYAEHCPARC(SEQ ID No.8); CPWFCTYACKVQPRCSEVYAEHCPARC(SEQ ID No.9); CPWFCTYPCEVQPRCSEVYAEHCPARC(SEQ ID No.10); CPWFCTYPCRVQPRCSEVYAEHCPARC(SEQ ID No.11); CPWFCTYPCKAQPRCSEVYAEHCPARC(SEQ ID No.12); CPWFCTYPCKVQARCSEVYAEHCPARC(SEQ ID No.13); CPWFCTYPCKVQPACSEVYAEHCPARC(SEQ ID No.14); CPWFCTYPCKVQPRCSEVYEEHCPARC(SEQ ID No.15); CPWFCTYPCKVQPRCSEVYAAHCPARC(SEQ ID No.16); CPWFCTYPCKVQPRCSEVYAEACPARC(SEQ ID No.17); CPWFCTYPCKVQPRCSEVYAEHCPGRC(SEQ ID No.18); CPWFCTYPCKVQPRCSEVYAEHCPAAC(SEQ ID No.19); CPWFCTYPCEVQPRCSEVYAAHCPAAC(SEQ ID No.20); or CPWFCTYPCEVQPRCSEVYAAHCPAKC (SEQ ID No. 21).

3. A method for preparing a high-affinity HER2-targeting polycyclic peptide molecule as described in claim 1 or 2, characterized in that, Includes the following steps: S1A: Using the encoding genes of the initial polycyclic peptides M-1 and M-2 as templates, random mutations were introduced using error-prone PCR. After 10 rounds of error-prone PCR amplification, the PCR products from rounds 6 to 10 were collected and mixed to construct a random mutant yeast display library, Library 1. The amino acid sequence of polycyclic peptide M-1 is CPWFCIYPCKVEPRCSEVYAEQCPQTC; the amino acid sequence of polycyclic peptide M-2 is CPWFCIYPCKVEPRCSQVYEQECPAVC. S2A. Flow cytometry was used to perform four rounds of HER2-targeted screening on the random mutant yeast display library Library 1. After each round of screening, high-throughput sequencing analysis was performed on the positive yeast cell population to obtain optimized peptide sequences, which include the sequences shown in SEQ ID No. 1 to 2. S3A. Using the positive yeast plasmid obtained in the 4th round of screening of the random mutant yeast display library Library 1 as a template, 10 rounds of error-prone PCR were performed, and the product of the 10th round was used to construct the secondary random mutant yeast display library Library 2. S4A. The secondary random mutant yeast display library Library 2 was subjected to four rounds of HER2-targeted flow cytometry sorting and high-throughput sequencing analysis to obtain the final optimized sequence, which includes the sequences shown in SEQ ID No. 3 to 7. S5A. The structure-activity relationship of the peptide that obtained the highest affinity was studied to obtain the target peptide sequence, which includes the sequences shown in SEQ ID No. 8 to 21.

4. A polycyclic peptide molecular probe targeting HER2, characterized in that: The structural feature is DLP; wherein, D is a conjugate selected from fluorescein isothiocyanate; L is 3-5 amino acid residues composed of Gly and Ser; and P is the high-affinity HER2-targeting polycyclic peptide molecule as described in claim 1 or 2.

5. A divalent polycyclic peptide molecular probe targeting HER2, characterized in that: The high-affinity HER2-targeting polycyclic peptide molecule described in claim 1 or 2 is formed by coupling via a linker DSS, where DSS is bis(succinimide) octanoate; the C-terminus of the high-affinity HER2-targeting polycyclic peptide molecule is synonymous with the C-terminus, and the N-terminus is synonymous with the N-terminus; and the structure of the divalent polycyclic peptide molecular probe is any one of the following: Wherein, P is the high-affinity HER2-targeting polycyclic peptide molecule as described in claim 1 or 2.

6. A labeling prodrug for a therapeutic drug targeting HER2, characterized in that: The labeled precursor is an XRP structure, wherein X is a chelating agent, R is a linker arm, and P is a high-affinity HER2-targeting polycyclic peptide molecule or a divalent polycyclic peptide as described in claim 1 or 2; the amino side of the linker arm is connected to the chelating agent, and the carboxyl side of the linker arm is connected to the amino group of the first amino acid at the N-terminus of the polypeptide compound.

7. The labeling prodrug for the HER2-targeting therapeutic drug as described in claim 6, characterized in that, The connecting arm is selected from one of the compounds with the following structural formula: Where n is an integer in the range of 1 to 10.

8. The labeling prodrug for a HER2-targeting therapeutic drug as described in claim 6, characterized in that, The chelate is selected from one of the compounds with the following structural formula:

9. A radiotherapeutic drug targeting HER2, characterized in that: The radiopharmaceutical is obtained by radiolabeling the polycyclic peptide molecule or divalent polycyclic peptide molecular probe described in claim 1, 2 or 5 with a radionuclide. For diagnostic and therapeutic purposes, the radionuclide is selected from... 18 F, 32 P, 33 P, 45 Ti、 47 Sc、 52 Fe、 59 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 75 Se、 75 Sc、 77 As、 86 Y、 89 Sr、 89 Zr、 90 Y、 94 Tc, 99 mTc, 99 Mo、 105 Pd, 105 Rh、 109 Pd, 111 TCP1 111 In、 123 I, 124 I, 125 I, 131 I, 142 Pr、 143 Pr、 149 Pm, 153 Sm、 161 Tb, 166 Dy、 166 Ho、 169 Er、 175 Lu、 177 Lu、 186 Re、 188 Re、 189 Re、 194 Ir、 198 Au、 199 Au、 211 At、 211 Pb, 212 Bi、 212 Pb, 213 Bi、 223 Ra or 225 Ac.

10. The method for preparing the labeled precursor of the HER2-targeting therapeutic drug as described in claim 6, characterized in that, Includes the following steps: S1B. Linear polypeptides were synthesized using a solid-phase synthesis method. S2B: The polypeptide-containing solid resin synthesized in step S1B is added to DMF for swelling, DOTA-NHS or FITC is added, the pH is adjusted to 8.0 with N,N-diisopropylethylamine, and the reaction is carried out overnight at 37°C. S3B: Cut the solid resin after the reaction in step S2B to remove the side chain protecting groups of amino acids, and purify the linear peptide by preparative chromatography. S4B. The linear polypeptide obtained in step S3B is mixed with oxidized glutathione at a molar ratio of 1:20, reacted at 37°C overnight, and then purified to obtain an oxidized polycyclic peptide coupled with DOTA or FITC. S5B. If the conjugate is DOTA, the DOTA-conjugated polycyclic peptide obtained in step S4B is radiolabeled: the radionuclide elution buffer is mixed with the labeling precursor, the pH is adjusted to 4-10 with buffer solution, and the reaction is carried out at 20-110°C for 10 minutes to overnight; the buffer solution is sodium acetate, water, ethanol, phosphoric acid, hydrochloric acid buffer solution or dimethyl sulfoxide; if the conjugate is FITC, the FITC-conjugated polypeptide obtained in S4B is dissolved in dimethyl sulfoxide solution, and then diluted with PBS buffer to the required concentration.

11. The use of the high-affinity HER2-targeting polycyclic peptide molecule of claim 1 or 2, the HER2-targeting polycyclic peptide molecular probe of claim 4, the HER2-targeting divalent polycyclic peptide molecular probe of claim 5, or the labeling precursor of the HER2-targeting therapeutic drug of claim 6 in the preparation of drugs, diagnostic reagents, or drug lead molecules for the diagnosis and / or treatment of HER2 signaling pathway-related diseases.

12. The application as described in claim 11, characterized in that: The high-affinity HER2-targeting polycyclic peptide molecules are radiolabeled for in vivo imaging or targeted therapy of HER2-positive tumors, including but not limited to breast cancer, pancreatic cancer, urothelial carcinoma, ovarian cancer, cervical cancer, renal cell carcinoma, bladder cancer, gastric cancer, colorectal cancer, esophageal cancer, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, melanoma, osteosarcoma, soft tissue sarcoma, glioma, neuroblastoma, medulloblastoma, oral cancer, nasopharyngeal carcinoma, laryngeal cancer, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia, choriocarcinoma, hepatoblastoma, choroidal melanoma, cavernous hemangioma, renal angiomyolipoma, teratoma, sarcoma, or other related tumors.

13. The application as described in claim 12, characterized in that: The diagnostic method is nuclear medicine imaging, selected from positron emission tomography or single-photon emission computed tomography; the treatment method is targeted therapy based on radionuclides; the HER2 signaling pathway-related diseases include HER2-positive tumors or non-tumor diseases related to abnormal HER2 expression.

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