A click chemistry-based signal enrichment polypeptide compound and application thereof

Abstract

The present application relates to the field of biomedicine and nuclear medicine, and particularly relates to a polypeptide compound for signal enrichment based on click chemistry and application thereof. The polypeptide compound of the present application comprises a targeting unit, an assembly polypeptide unit and a click chemistry unit. The targeting unit is used for specifically recognizing and combining with antigens or receptors expressed on the surface of tumor cells, thereby providing a spatial positioning basis for subsequent self-assembly and signal amplification. After self-assembly of the assembly polypeptide unit, the polypeptide compound is transformed from a single molecule state into a macroscopic fiber network composed of multiple molecules, thereby realizing signal amplification from "a single molecule carrying a few sites" to "a network structure carrying a large number of sites". The click chemistry unit can perform biological orthogonal click chemistry reaction, and finally realize efficient diagnosis and treatment through enrichment of target points.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and nuclear medicine, specifically to a peptide compound for signal enrichment based on click chemistry and its applications. Background Technology

[0002] Targeted radionuclide therapy (TRT) is an advanced treatment modality that achieves precision therapy by selectively delivering therapeutic radionuclides to tumor tissue. TRT can cause irreversible damage to target cells through the local deposition of high-energy radiation, thus attracting increasing attention for treating refractory tumors that are unresponsive to existing therapies, including but not limited to metastatic cancers. Several TRT drugs, such as Pluvicto, have been approved by the FDA for clinical treatment. However, traditional radionuclide therapies suffer from insufficient targeting, short residence time in tumors, and easy accumulation in non-target sites. Existing methods struggle to simultaneously achieve both targeted efficacy and therapeutic benefit.

[0003] Click chemistry, due to its high efficiency, specificity, and bioorthogonality, has become a promising tool in the field of nuclear medicine. However, this technology currently faces significant limitations, including insufficient signal amplification at single-site clicks. The current mainstream "pre-targeting" relies on a 1:1 stoichiometric reaction, meaning that one targeting molecule can bind to only one radionuclide. This limits the signal intensity and effective radiation dose at the tumor site, making it difficult to meet the demands for highly efficient treatment. Summary of the Invention

[0004] The purpose of this invention is to provide a polypeptide compound for signal enrichment based on click chemistry.

[0005] Another object of the present invention is to provide a composition based on the above-mentioned polypeptide compound.

[0006] Another object of the present invention is to provide the application of the above-mentioned polypeptide compounds and compositions in nuclear medicine.

[0007] According to a specific embodiment of the present invention, a peptide compound for signal enrichment based on click chemistry comprises a targeting unit, an assembly peptide unit, and a click chemistry unit linked by chemical bonds, wherein... Targeting units are used to specifically recognize and bind to tumor surface antigens; Assemble polypeptide units for self-assembly to enrich click chemical sites; Click on the chemical unit to perform IEDDA reaction, CuAAC reaction, SPAAC reaction, or sulfur-fluoride exchange chemical reaction.

[0008] The polypeptide compound of the present invention covalently links three functional units to the same molecular backbone through chemical bonds (e.g., amide bonds, ester bonds, or indirect linkages via polyethylene glycol linkers).

[0009] The function of the targeting unit is to specifically recognize and bind to antigens or receptors expressed on the surface of tumor cells. The targeting unit can be a small molecule ligand, peptide, or antibody fragment, the choice of which depends on the molecular phenotype of the target tumor. Once the targeting unit binds to the tumor surface antigen, the peptide compound is anchored to the tumor cell membrane, providing a spatial localization basis for subsequent self-assembly and signal amplification.

[0010] The function of the assembled polypeptide unit is to spontaneously assemble in an ordered manner between molecules under specific conditions to form an ordered structure at the nanoscale or microscale. The "specific conditions" refer to physiological temperature (approximately 37°C), physiological pH (approximately 7.4), or specific stimuli in the tumor microenvironment (such as catalysis by certain enzymes). The design principles of the assembled polypeptide unit are: (1) it has a tendency to self-assemble under physiological conditions; (2) the nanostructures formed after self-assembly (such as nanofibers, nanoribbons, or hydrogel networks) have a high specific surface area and a large number of exposed surface sites; (3) the self-assembly process does not depend on external physical stimuli (such as ultraviolet light, ultrasound, etc.) to ensure the feasibility of in vivo application. After the assembled polypeptide unit self-assembles, the polypeptide compound transforms from a single molecule state to a macroscopic fiber network composed of multiple molecules, realizing the signal amplification effect of the click chemical site from "a single molecule carrying a few sites" to "a network structure carrying a large number of sites".

[0011] The click chemistry unit functions to perform bioorthogonal click chemistry reactions, such as the IEDDA reaction, CuAAC reaction, SPAAC reaction, or sulfur-fluoride exchange chemistry. The IEDDA reaction (reverse electron-demanding Diels-Alder reaction) is a cycloaddition reaction between trans-cyclooctene and tetrazine, possessing the fastest known bioorthogonal reaction rate. The CuAAC reaction (copper-catalyzed azido-yne cycloaddition) is a cycloaddition reaction between an azido group and a terminal yne group under copper ion catalysis, representing a classic click chemistry model. The SPAAC reaction (strain-promoted azido-yne cycloaddition) is a cycloaddition reaction between an azido group and cyclooctyne compounds (such as dibenzocyclooctene and cyclopropanecyclooctene) under copper-free conditions, exhibiting good biocompatibility. The sulfur-fluoride exchange chemistry reaction is based on the formation and exchange of sulfur (VI)-fluorine bonds, exhibiting high selectivity and good stability. The selection of the click chemistry unit must form a complementary pairing with the reactive groups of the second component to ensure specific linkage between the two in vivo.

[0012] According to specific embodiments of the present invention, the peptide compounds for signal enrichment based on click chemistry include targeting units such as PPTQF, FAPI-04, FAPI-46, FAPI-74, and FAP-2286 targeting fibroblast activation protein; PSMA-617, PSMA-11, PSMA-1007, PSMA-I&T, and PSMA-ALB-56 targeting prostate-specific membrane antigen; DOTATATE, DOTATOC, DOTANOC, JR11, and LM3 targeting somatostatin receptor; poly-RGD cyclic peptide targeting integrin receptor; Girentuximab and Nota-cG250 targeting carbonic anhydrase IX; and Trastuzumab, Pertuzumab, HER2-targeting nanobody, EGFR antibody, and EGFR-targeting peptide targeting human epidermal growth factor receptor.

[0013] The selection of the targeting unit depends on the molecular phenotypic characteristics of the target tumor. This invention covers a variety of clinically important tumor target systems, specifically including: The FAPI series of ligands targeting fibroblast activating protein (FAP) includes FAPI-04 and FAPI-46. FAP is a type II transmembrane serine protease that is highly expressed on the surface of tumor-associated fibroblasts in over 90% of epithelial-derived tumors (such as breast cancer, pancreatic cancer, colorectal cancer, and lung cancer), but is almost not expressed in normal tissues. Both FAPI-04 and FAPI-46 are small molecule inhibitors based on a quinoline backbone, exhibiting nanomolar affinity for FAP and possessing suitable molecular weights (approximately 500-600 Da), facilitating rapid penetration into tumor tissue and rapid renal clearance. Compared to FAPI-04, FAPI-46 exhibits higher tumor uptake and longer tumor retention time, making it one of the preferred targeting units.

[0014] PSMA ligands targeting prostate-specific membrane antigen (PSMA) include PSMA-617, PSMA-11, and PSMA-1007. PSMA is a type II transmembrane glycoprotein that is specifically and highly expressed on the cell membrane of prostate cancer cells (expression levels can be 100-1000 times higher than in normal tissues), and its expression is further upregulated in metastatic prostate cancer, making it a gold standard target for prostate cancer diagnosis and treatment. PSMA-617 and PSMA-11 are small molecule inhibitors based on a urea backbone, exhibiting picomolar affinity for the enzymatic active site of PSMA; PSMA-1007, on the other hand, possesses higher lipophilicity and improved pharmacokinetic properties.

[0015] Ligands targeting the somatostatin receptor (SSTR) include DOTATATE, JR11, and LM3. SSTRs (primarily the SSTR2 subtype) are overexpressed on the surface of neuroendocrine tumor (NET) cells, but their expression is limited in normal tissues. DOTATATE, a derivative of the somatostatin analog octreotide, has a high affinity for SSTR2. JR11 and LM3 are novel SSTR2 antagonist ligands.

[0016] Polycyclic RGD peptides targeting integrin receptors. Integrin αvβ3 is highly expressed on the surface of tumor vascular endothelial cells and various tumor cells, playing a crucial role in tumor angiogenesis, invasion, and metastasis. The RGD (arginine-glycine-aspartic acid) tripeptide sequence is the specific recognition motif of integrin αvβ3. Polycyclic RGD peptides (such as dimer or tetramer forms) have stronger binding affinity and higher tumor uptake than monomeric RGD, making them effective targeting units for tumor angiogenesis and certain solid tumors.

[0017] It should be noted that the above-described targeting units are merely illustrative examples, and the present invention is not limited thereto. Any peptide, small molecule, or antibody fragment targeting ligand capable of specifically recognizing and binding to tumor surface antigens can be used as a targeting unit of the present invention and fall within the scope of protection of the present invention.

[0018] According to a specific embodiment of the present invention, a peptide compound for signal enrichment based on click chemistry is provided, wherein the assembled peptide unit includes GNNQQNY, KLVFF, FF, or RADA16.

[0019] The assembly of polypeptide units is driven by non-covalent intermolecular interactions (e.g., β-sheet hydrogen bonds, π-π stacking, and hydrophobic interactions). Specifically, this includes: GNNQQNY sequence: a heptapeptide fragment derived from the prion domain of yeast prion protein Sup35, which can form a highly ordered nanofiber structure through intermolecular β-sheet stacking under physiological conditions.

[0020] KLVFF sequence: derived from the core hydrophobic region of β-amyloid (Aβ), which can form stable nanofibers through β-sheet and hydrophobic interactions.

[0021] The FF sequence (diphenylalanine) consists of two phenylalanine residues. FF forms a stable nanostructure through π-π stacking interactions between benzene rings.

[0022] RADA 16Sequence: It is an ion-complementary self-assembling polypeptide with the sequence (RADA)4 (i.e., a repeating alternating arginine-alanine-aspartic acid-alanine motif), consisting of 16 amino acid residues. Under physiological ionic strength (such as in body fluid environments), it can form a stable hydrogel nanofiber network through intermolecular electrostatic interactions and β-sheets.

[0023] Those skilled in the art should understand that the above-described assembled polypeptides are merely preferred examples. Any short peptide sequence that has the ability to self-assemble under physiological conditions and can form ordered structures such as nanofibers can be used in this invention and also fall within the protection scope of this invention.

[0024] Preferably, the click chemical unit includes azide, alkynyl, dibenzocyclooctynyl, cyclopropanecyclooctynyl, transcyclooctene, or tetrazine.

[0025] Azide (-N3): A linear azide functional group containing three nitrogen atoms. In click chemistry, it can undergo CuAAC reaction with terminal alkynyl groups under copper catalysis, and it can also undergo SPAAC reaction with cyclooctyne compounds under copper-free conditions.

[0026] Alkyne (-C≡CH): A terminal functional group containing a carbon-carbon triple bond.

[0027] Dibenzocyclooctyne (DBCO): A strained cyclic alkyne that can pair with an azide group for pre-targeted applications in vivo.

[0028] Bicyclononyne (BCN): A click chemistry unit of cyclooctyne that can undergo the SPAAC reaction with azide groups without the need for a copper catalyst. DBCO and BCN can be selected according to specific requirements.

[0029] Trans-Cyclooctene (TCO): a highly strained cyclic olefin.

[0030] Tetraazine (Tz): A six-membered heterocycle containing four nitrogen atoms, used in combination with TCO.

[0031] In this invention, the specific selection of the click chemical unit depends on the reaction rate requirements and the in vivo application scenario. Preferably, the click chemical unit is trans-cyclooctene (TCO), which pairs with the tetrazine group on the second component to achieve the fastest in vivo labeling rate. The click chemical unit can be introduced into the N-terminus, C-terminus, or side-chain lysine residues of the peptide compound through chemical synthesis methods.

[0032] According to specific embodiments of the present invention, a click chemistry-based composition includes... The first component includes the aforementioned peptide compounds for signal enrichment based on click chemistry; The second component includes a reactive group loaded with a radionuclide; the first component and the second component are capable of undergoing a bioorthogonal click chemistry reaction.

[0033] The composition of the present invention employs a stepwise delivery and in-situ locking mechanism, comprising two components, a first component and a second component, which are applied in time and achieve functional coupling through complementary click chemistry pairing.

[0034] The first component is the aforementioned click chemistry-based signal enrichment peptide compound. In practical applications, the first component is administered to the subject first (usually via intravenous injection). After entering the body, the first component actively recognizes and binds to specific antigens on the surface of tumor cells through its targeting units, thereby localizing to the tumor tissue. Subsequently, under the physiological microenvironment conditions of the tumor site, the assembly peptide units on the first component initiate a self-assembly process, linking individual molecules distributed on the cell membrane surface into a three-dimensional nanofiber network. This fiber network covers the surface of tumor cells and is enriched with multiple click chemistry units (such as TCO), providing numerous binding sites for the second component.

[0035] The second component is a radionuclide delivery unit containing reactive groups loaded with radionuclides. After the first component has sufficiently accumulated at the tumor site and been cleared from the blood (typically 2-6 hours after administration of the first component), the second component is administered to the subject. At this point, the second component, during its circulation in vivo, encounters the fibrous network of the first component at the tumor site, i.e., it undergoes covalent bonding through a bioorthogonal reaction between the reactive groups and the click chemistry unit, and is thus captured and accumulated in the tumor region.

[0036] The term "bioorthogonal" refers to a reaction that occurs specifically within the complex environment of an organism, without interfering with normal biochemical processes or being affected by bioactive substances (such as enzymes and glutathione). Click chemistry reactions that meet these conditions include the IEDDA reaction and the SPAAC reaction.

[0037] In a preferred embodiment of the present invention, the click chemistry unit of the first component is TCO, and the reactive group of the second component is tetrazine. The two are linked together very quickly and specifically through an IEDDA reaction.

[0038] According to a specific embodiment of the composition of the present invention, the second component further includes a chelating group, wherein the radionuclide is loaded onto the reactive group via the chelating group.

[0039] The chelating group is a polydentate ligand containing multiple coordinating atoms (such as nitrogen and oxygen atoms), which can form a stable coordination complex with radioactive metal ions. The complete structure of the second component consists of three functional parts: a reactive group (for connection with the first component), a chelating group (for carrying the nuclide), and a radionuclide (functional effector molecule). The connection order of the three is: reactive group—connecting arm—chelating group—radionuclide (coordination complex).

[0040] In embodiments of the present invention, a linker may be further included between the reactive group and the chelating group. The linker is preferably a polyethylene glycol (PEG) chain or a short peptide chain (such as GGG). The length of the linker can be adjusted as needed to optimize hydrophilicity, reduce steric hindrance, and regulate pharmacokinetics in vivo.

[0041] Preferably, the chelating group includes DOTA, NOA, or DOTAGA. All of the above chelating groups form coordination bonds with the metal ion through a nitrogen atom and a carboxylate oxygen atom.

[0042] DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid): Capable of forming stable octacoordinate complexes with various radioactive metal ions. Radionuclides that DOTA can chelate include: 68 Ga (used in PET imaging) 177 Lu (used for beta-radiotherapy) 225 Ac (used for alpha radiation therapy) 64 Cu (used in PET imaging and treatment) 90 Y (used for beta-radiotherapy), etc.

[0043] NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid): A chelating agent containing a 9-membered ring skeleton and three carboxylic acid side arms. NOTA can... 18 F or 68 Ga and other substances were used for chelation.

[0044] DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-glutaric acid): is a structural derivative of DOTA, which adds an extra glutaric acid side arm to the DOTA skeleton.

[0045] In this invention, DOTA, NOTA, and DOTAGA are merely examples. Any nitrogen-containing heterocyclic polycarboxylic acid chelating agents (such as TRAP, HBED-CC, AAZTA, etc.) that have the function of chelating radioactive metal ions can be used in this invention and also fall within the protection scope of this invention.

[0046] According to a specific embodiment of the present invention, the radionuclide is selected from beta-ray, alpha-ray, or positron-emitting nuclides; the radionuclide is... 64 Cu、 68 Ga、 177 Lu、 223 Ra or 225 Ac.

[0047] In a specific embodiment of the present invention, when the second component is used for radiotherapy, a β or α emitting nuclide is preferably selected. 177 Lu or 225 Ac) is chelated with DOTA or DOTAGA. The second component can also carry both diagnostic and therapeutic radionuclides, or use diagnostic radionuclide imaging to screen suitable patients and then perform matched therapy with therapeutic radionuclides.

[0048] This invention provides the use of the above-described click chemistry-based signal enrichment polypeptide compound or the composition thereof in the preparation of a radiotherapy drug for treating tumors.

[0049] The "preparation of radioactive diagnostic and therapeutic tumor drugs" encompasses both diagnostic and therapeutic aspects. The compositions of this invention can be used in PET or SPECT imaging, utilizing positron emission tomography (PET) nuclides (such as...). 68 (Ga) or single-photon emission radionuclides can be used to visualize and locate lesions positive for tumor markers such as FAP, PSMA, and SSTR, and can be applied to early tumor diagnosis, staging assessment, detection of micrometastases, recurrence monitoring, and efficacy evaluation. In terms of therapeutic applications, the compositions of this invention can be used for radionuclide targeted therapy, utilizing beta or alpha rays to kill confirmed tumor lesions. They can be used alone or in combination with other treatments such as surgery, chemotherapy, and immunotherapy.

[0050] In the application of this invention, the administration scheme of the first component and the second component is as follows: the first component is administered at a first time point, and the second component is administered at a second time point after the first component is administered, with the second time point being later than the first time point. Preferably, the second time point is 2-7 hours after the first time point, more preferably 3-5 hours later.

[0051] The tumors include breast cancer, pancreatic cancer, glioma, esophageal tumor, colorectal tumor, lung cancer, stomach cancer, or metastatic cancer.

[0052] It should be emphasized that the tumor types described above are merely illustrative examples, and the application of this invention is not limited thereto. Any solid tumor positive for targets such as FAP, PSMA, SSTR, or integrins, regardless of its histological origin, can be adapted to this invention by selecting the corresponding targeting unit. In particular, this invention has significant application value for refractory, metastatic, or recurrent tumors that are unresponsive to existing therapies.

[0053] Compared with the prior art, the present invention has the following beneficial effects: The polypeptide compound of this invention covalently links three functional units—a targeting unit, an assembly polypeptide unit, and a click chemistry unit—within the same molecular backbone via chemical bonds. The targeting unit specifically recognizes and binds to antigens or receptors expressed on the surface of tumor cells, providing a spatial localization basis for subsequent self-assembly and signal amplification. After self-assembly, the assembly polypeptide unit transforms the polypeptide compound from a single molecule into a macroscopic fibrous network composed of multiple molecules, achieving signal amplification from "a single molecule carrying a few sites" to "a network structure carrying a large number of sites" in click chemistry. The click chemistry unit can perform bioorthogonal click chemistry reactions, ultimately achieving efficient diagnosis and treatment through target enrichment.

[0054] Based on the above principles, the composition of the present invention includes a first component and a second component. After administration of the first component, it targets the tumor or CAF cell membrane and forms in situ assembly enrichment through assembly sequence, thereby achieving signal amplification and retention enhancement. Subsequently, after administration of the second component, the click chemical group coupled with the metal ion chelating agent DOTA is enriched at the target site through click reaction, thereby achieving efficient diagnosis and treatment.

[0055] Specifically, this invention provides the polypeptide composition K(TCO)GNNQQNYPPTQF (TGP) and the corresponding Tz-DOTA. This invention conducted fibrinogenic kinetics experiments using thioflavin T (ThT) as a fluorescent probe, obtaining results showing that the fluorescence intensity of the TGP solution increased rapidly after the addition of fibroblast activating protein (FAP), and that it assembled into fibrils faster than the group without FAP. This demonstrates that TGP has excellent in-situ assembly (fibrinogenic) ability under target-triggered conditions.

[0056] This invention conducted a confocal microscopy observation experiment on the cell membrane of fluorescently labeled TGP, and obtained results showing that the cells still maintained a high TGP signal after 72 h, indicating that TGP has a stronger signal intensity and a longer retention time compared with target peptides without assembly sequences.

[0057] This invention conducted a fluorescence imaging experiment on Cy5-labeled TGP in tumor-bearing mice, and obtained results showing that the tumor tissue still maintained a strong fluorescence signal 72 h after injection, indicating that the split-type strategy significantly enhanced the tumor enrichment ability and retention time compared with the single target sequence.

[0058] This invention was performed on tumor-bearing mice. 68 In the Ga-labeled PET / CT imaging experiment, the tumor tissue signal was increased by about 3.3 times compared with the original targeted sequence radionuclide imaging, indicating that the peptide assembly amplification strategy can significantly improve the tumor enrichment efficiency of radiopharmaceuticals.

[0059] This invention was performed on tumor-bearing mice. 177The Lu-labeled SPECT / CT imaging and treatment experiments showed that the radionuclide tumor enrichment efficiency and retention time were significantly enhanced, and the ex vivo tumor images 14 days after treatment showed a significant tumor inhibition effect, indicating that the present invention has significant efficacy in tumor treatment. Attached Figure Description

[0060] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0061] Figure 1 These are the mass spectrometry results of the TGP polypeptide of this invention; Figure 2 The fibrinolytic kinetics curve of the K(TCO)GNNQQNYPPTQF (TGP) polypeptide of the present invention; Figure 3 These are the mass spectrometry results of the Tz-DOTA molecule of this invention; Figure 4 This is the invention 68 Paper chromatography results for Ga-labeled Tz-DOTA; Figure 5 This is a liquid phase diagram of the TGP and Tz-DOTA of the present invention after the click reaction occurs; Figure 6 This is a confocal microscopy image of the fluorescently labeled TGP of the present invention on the cell membrane (scale bar: 20 μm). Figure 7 This is a liquid phase diagram of the present invention after the sequential addition of TGP and Tz-DOTA to cells; Figure 8 These are fluorescence images of the fluorescently labeled TGP of the present invention remaining in mice; Figure 9 These are PET / CT imaging results of the TGP peptide assembly amplification strategy in tumor-bearing mice; Figure 10 These are SPECT / CT imaging results of the TGP peptide assembly amplification strategy in tumor-bearing mice; Figure 11 These are tumor images after treatment using the TGP peptide assembly amplification strategy; Figure 12 These are SPECT / CT imaging results of TG617 in tumor-bearing mice; Figure 13 These are tumor images after TG617 treatment; Figure 14 It is a marker177 HE staining results of Lu after Tz-DOTA treatment indicate that the present invention has good safety for non-target organs. Detailed Implementation

[0062] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0063] Example 1 Part 1 Synthesis of Polypeptides The PPTQF sequence targeting fibroblast activation protein was synthesized using solid-phase synthesis to link an assembled peptide containing the GNNQQNY sequence, and a TCO was coupled to the terminal lysine K. The synthesized peptide was purified by HPLC.

[0064] The specific synthesis steps are as follows: Peptide K(TCO)GNNQQNYPPTQF was synthesized using the Fmoc solid-phase peptide synthesis (Fmoc-SPPS) method. A resin pre-loaded with Fmoc-Phe-OH (optionally 2-CTC resin, Wang resin, or Rink Amide resin) was used as the solid-phase support, and amino acids were coupled sequentially from the C-terminus to the N-terminus. Before each round of coupling, the Fmoc protecting group was removed using a 20% piperidine / DMF solution (5 min + 15 min), followed by thorough washing of the resin with DMF. The coupling reaction used a HATU / DIPEA activation system, with each Fmoc-protected amino acid used in 4 equivalents, and the reaction was carried out at room temperature for 30–45 min. The amino acids were coupled sequentially in the following order: FQTPPYNQQNNGK(Dde).

[0065] The N-terminal lysine is protected with Fmoc-Lys(Dde)-OH, the α-amino group is protected with Fmoc, and the ε-amino group is protected with Dde, so as to facilitate subsequent selective modification of the side chain.

[0066] After the main chain synthesis was completed, the Fmoc protecting group on the α-amino group of lysine was first removed using 20% ​​piperidine / DMF. Subsequently, the Dde protecting group on the ε-amino group of the lysine side chain was selectively removed using 2% hydrazine hydrate / DMF solution. Each treatment lasted 5 min and was repeated 6 times. During each treatment, the resin was thoroughly washed with DMF to ensure that the Dde protecting group was completely removed without affecting other side chain protecting groups.

[0067] After Dde removal, the resin was washed several times with DMF. TCO-NHS ester (3–5 equiv) and DIPEA (6–10 equiv) were added to the DMF, and the mixture was shaken at room temperature for 2–4 h to allow TCO to undergo amidation coupling with the ε-amino group of the lysine side chain via the NHS ester. After the reaction, the resin was thoroughly washed with DMF, dichloromethane (DCM), and methanol sequentially to remove unreacted TCO-NHS and byproducts.

[0068] Finally, the peptide was lysed at room temperature for 2 h using a lysis buffer of TFA / TIS / H2 (95:2.5:2.5, v / v / v) to remove all acid-sensitive side-chain protecting groups and cleave the peptide from the resin. The filtrate was dried under nitrogen and concentrated, then pre-cooled anhydrous diethyl ether was added to precipitate the crude product. The precipitate was collected by centrifugation and washed again with cold diethyl ether. The obtained crude peptide was then subjected to C... 18 The target peptide K(TCO)GNNQQNYPPTQF was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and lyophilized to obtain the peptide. The purity was determined by analytical RP-HPLC, and the molecular weight was confirmed by ESI-MS or MALDI-TOF MS.

[0069] The molecular formula of TGP polypeptide is as follows: ; Its mass spectrometry verification results are as follows Figure 1 As shown.

[0070] The in vitro assembly ability of TGP peptides was verified using thiamine T, and the specific steps are as follows: The fibrosis kinetics of TGP peptides and TGP / FAP incorporating 9 nM fibroblast activation protein (FAP) were monitored using thioflavin T (ThT) as a fluorescent probe. TGP and TGP / FAP were dissolved in PBS buffer (10 mM, pH 7.4) to a final concentration of 100 μM. Freshly prepared ThT stock solution (1 mM, dissolved in deionized water) was added to each sample to achieve a final ThT concentration of 10 μM. The mixture was then immediately transferred to black 96-well plates (200 μL per well) and placed in a Thermo Fisher microplate reader pre-equilibrated to 37 °C for detection. Fluorescence signals were acquired at an excitation wavelength of 440 nm and an emission wavelength of 485 nm, with fluorescence intensity recorded every 5 min for 300 min. Between assays, the 96-well plates were continuously oscillated to maintain uniform sample dispersion.

[0071] The fibrillation kinetics curve was obtained by plotting the normalized fluorescence intensity (I / I0, where I0 is the initial fluorescence intensity at 0 min) against time.

[0072] like Figure 2 As shown, the fluorescence intensity was significantly enhanced after the addition of fibroblast-activating protein, indicating that the TGP peptide has excellent assembly ability.

[0073] Example 2 Preparation of Tz-DOTA nuclide labeling Tz-DOTA was synthesized in liquid phase using commercially available Tz-NHS and NH2-PEG4-DOTA. Subsequently, 100 μg of Tz-DOTA was dissolved in 100 μL of H2O and reacted with 10 mCurie's solution. 68 The GaCl3 mixture was adjusted to pH 4 using 0.01 mol / L HCl, followed by heating in a metal bath to 85°C for 15 minutes. C was then used to further adjust the pH. 18 Column separation labeling paper 68 Ga's Tz-DOTA.

[0074] The mass spectrometry verification results of Tz-DOTA are as follows: Figure 3 As shown. The results of the chromatography are as follows. Figure 4 As shown, this indicates that Tz-DOTA was successfully tagged. 68 Ga.

[0075] TGP and Tz-DOTA were mixed at a 1:1 ratio and reacted in water for 20 minutes. The click chemistry between the synthesized molecules was then verified by high performance liquid chromatography (C18 reversed-phase column, mobile phase of methanol and water, flow rate of 1.0 mL / min, detection wavelength of 220 nm).

[0076] The results are as follows Figure 5 As shown in the results, the peaks of the two reactants indicate that efficient click reactions can occur between molecules.

[0077] Following the methods described in Examples 1 and 2, the targeting sequence, assembly sequence, and click chemical groups were changed to verify the amplification effect of the obtained peptides in the click reaction: Target sequences: FAPI-04, PSMA-617, DOTATATE.

[0078] Assembly sequence: KLVFF, FF, RADA 16 .

[0079] Click on Chemistry: CuAAC reaction, SPAAC reaction. See Table 1 for details: Table 1

[0080] Experimental results show that the peptides in the table above all have excellent assembly ability and can undergo efficient click reactions.

[0081] Example 3 In vitro cell experiments FITC-labeled TGP peptides were obtained by co-incubation and dialysis. The fluorescently labeled peptides were added to the HT-1080-FAP cell culture system and incubated for one hour. The peptides were then washed off with PBS solution, and the assembly and retention time were observed under a confocal microscope.

[0082] In addition to the TGP group, we also set up a PBS control group and a FITC-conjugated PPTQF group. Compared to the TGP group, PPTQF is the target sequence of the TGP group.

[0083] like Figure 6 As shown, the results indicate that at 72 hours, the cells still had a higher TGP signal compared to the control group, suggesting that TGP has a stronger signal and retention time.

[0084] After incubating cells with TGP solution, Tz-DOTA was added, and the click enrichment effect was detected by high performance liquid chromatography analysis of cell lysates.

[0085] The specific experimental procedure was as follows: FAP-positive NIH 3T3 cells were seeded in 6-well plates, approximately 5 × 10⁶ cells per well. 5 Cells were cultured overnight at 37 °C in a 5% CO2 incubator until they adhered to the culture medium. After discarding the medium, the cells were gently washed twice with PBS buffer.

[0086] Add 2 mL of serum-free medium containing 100 μM TGP to each well and incubate at 37 °C for 30 min to allow TGP to bind to the cell surface and assemble in situ. After incubation, wash the cells three times with cold PBS to remove unbound TGP.

[0087] Fresh serum-free medium containing 100 μM Tz-DOTA was then added to each well, and the cells were incubated at 37 °C for 30 min to allow Tz-DOTA to undergo a reverse electron-demanding Diels-Alder (IEDDA) click reaction with TCO on the cell surface. After the reaction, the cells were washed five times with cold PBS to thoroughly remove unreacted free Tz-DOTA.

[0088] Cells were lysed by adding 200 μL of RIPA lysis buffer (containing protease inhibitors) and incubating on ice for 30 min, gently mixing by pipetting every 10 min. After collecting the lysis buffer, the cells were centrifuged at 12,000 rpm and 4 °C for 10 min. The supernatant was then filtered through a 0.22 μm filter membrane and used for high-performance liquid chromatography (HPLC) analysis.

[0089] HPLC uses C 18 A reversed-phase analytical column (4.6 × 250 mm, 5 μm) was used. Mobile phase A was 0.1% TFA aqueous solution, and mobile phase B was acetonitrile. Gradient elution (e.g., 5%–60% B, 30 min) was employed at a flow rate of 1.0 mL / min. The detection wavelength was determined based on the maximum absorption wavelength of Tz-DOTA (typically 254 nm or 280 nm). Qualitative analysis of the click reaction products in the cell lysate was performed by comparing the retention times of TGP, Tz-DOTA, and the click products.

[0090] like Figure 7 As shown, the chromatographic peaks detected in the experiment were consistent with those of the standard click product, indicating that TGP and Tz-DOTA can undergo a highly efficient bioorthogonal click reaction at the cellular level.

[0091] Example 4 Animal Experiment Cy5-labeled TGP peptides were injected into HT-1080-FAP mouse tumor models, and tumor uptake and TGP retention time in the tumor were observed using small animal fluorescence imaging equipment.

[0092] The specific experimental steps are as follows: Construct an HT-1080-FAP mouse tumor model, and when the tumor volume grows to approximately 100-150 mm... 3 Cy5-labeled TGP (Cy5-TGP) was injected into tumor-bearing mice via the tail vein (100 μL PBS, 5 nmol per mouse). A control group containing only the Cy5-labeled target sequence (Cy5-Target) was used, with the same injection dose.

[0093] Following injection, whole-body fluorescence imaging was performed at 0.5, 1, 2, 6, 12, 24, 48, and 72 h under continuous inhalation anesthesia with 2% isoflurane using a small animal in vivo fluorescence imaging system. The excitation wavelength of Cy5 was set to 640 nm, and the emission wavelength was set to 680 nm. The exposure time, binning, and F / stop parameters were kept consistent at all time points to ensure the comparability of fluorescence signals at different time points.

[0094] The Living Image software was used to perform quantitative analysis of the region of interest (ROI), calculate the average radiant efficiency or total fluorescence intensity of the tumor site at each time point, and plot the tumor fluorescence signal over time to evaluate the enrichment and retention dynamics of Cy5-TGP in tumor tissue.

[0095] After 72 hours of in vivo imaging, mice were euthanized, and tumors and major organs (heart, liver, spleen, lung, and kidney) were isolated for in vitro fluorescence imaging. The fluorescence signal intensity of each tissue was quantitatively analyzed using Living Image software.

[0096] like Figure 8 As shown, compared with the control group containing only the target sequence, Cy5-TGP exhibited higher uptake levels and more persistent retention in tumor tissue, and the tumor site still maintained a strong fluorescence signal 72 h after injection.

[0097] The strategy of this invention was used to treat a mouse HT-1080-FAP tumor model. 68 Ga-labeled PET / CT imaging, the specific experimental steps are as follows: A subcutaneous tumor-bearing nude mouse model was established using HT-1080-FAP. When the tumor volume grew to approximately 80–120 mm³, the tumor-bearing mice were randomly divided into PBS group and PPTQF group. 68 Group Ga and the imaging group of this invention, each group consisting of 5 to 8 individuals.

[0098] The PBS group received an equal volume of PBS via tail vein injection.

[0099] PPTQF- 68 Group Ga was injected directly via tail vein. 68 Ga-labeled PPTQF has a radioactive dose of 185 MBq / kg.

[0100] The imaging group of this invention first injects the first part of the invention, TGP, via the tail vein at a dose of 1–10 mg / kg. After injection, the TGP circulates in vivo for a predetermined time (preferably 2 hours) to allow it to accumulate sufficiently in the tumor tissue and induce the formation of an assembly network rich in TCO click sites under FAP-mediated regulation. Subsequently, the second part is injected via the tail vein. 68 Ga-labeled Tz-DOTA, with a radioactive dose of 185 MBq / kg.

[0101] Following the second part of drug injection, dynamic and static imaging was performed using a small animal PET / CT system at 60 min and 240 min (or other time points as needed for the experiment). Mice were anesthetized with continuous inhalation of 2% isoflurane before imaging, and their body temperature was maintained at 37 ℃. CT images were first acquired for anatomical localization, followed by PET scans. The PET images were then reconstructed in three dimensions and fused with the CT images for analysis.

[0102] Using the accompanying image analysis software (PMOD), the regions of interest (ROI) of the tumor and major organs were delineated, the radioactive uptake values ​​of the tumor tissue (SUV_mean, SUV_max or %ID / g) and the tumor / muscle (T / M) or tumor / blood (T / B) ratios were calculated, and the uptake dynamics and retention of radionuclides at different time points in the tumor site were analyzed. like Figure 9 As shown, imaging results at different time points demonstrate that the present invention significantly improves the enrichment efficiency of radiopharmaceuticals in tumors, increasing the signal of tumor tissue by approximately 3.3 times.

[0103] The strategy of this invention was used to treat mouse tumor models. 177 In the Lu-labeled treatment study, the first part involved an injection dose of 1-10 mg / kg, and the second part involved an injection dose of 1850 MBq / kg. The specific experimental procedures were as follows: A HT-1080-FAP subcutaneous tumor-bearing nude mouse model was established. The tumor volume was measured to be approximately 80–120 mm. 3 At that time, tumor-bearing mice were randomly divided into PBS group and PPTQF group. 177 Group Lu and the treatment group of this invention, each group consisting of 5 to 8 animals.

[0104] The PBS group received an equal volume of PBS via tail vein injection.

[0105] PPTQF- 177 Group Lu received direct injection via tail vein. 177 Lu-labeled PPTQF has a radioactive dose of 1850 MBq / kg.

[0106] In the treatment group of this invention, the first part of the invention, namely TGP, is first injected via tail vein at a dose of 1–10 mg / kg. After TGP has sufficiently accumulated in the tumor tissue and induced the formation of an assembly network rich in TCO click sites on the tumor surface (preferably 2 h), the second part is then injected via tail vein. 177 Lu-labeled Tz-DOTA, with a radioactive dose of 1850 MBq / kg.

[0107] Following the second part of the radiopharmaceutical injection, mice underwent sequential imaging using a small animal SPECT / CT system at 1, 4, 12, 24, 36, 48, and 72 hours. Mice were anesthetized with 2% isoflurane inhalation and kept at a constant body temperature before imaging. CT images were first acquired for anatomical localization, followed by SPECT scanning. Regions of interest (ROIs) were delineated in the tumor area using accompanying image analysis software. Radioactive uptake and tumor / background ratios at the tumor site were quantitatively analyzed to evaluate the enrichment efficiency and retention time of the radionuclide in the tumor.

[0108] SPECT / CT imaging results show that the present invention significantly improves the enrichment efficiency and retention time of radiopharmaceuticals in tumors, such as Figure 10 As shown.

[0109] During treatment, the long diameter (L) and short diameter (W) of the tumor were measured every 2 days using calipers, according to the formula V = L × W. 2 / 2 Calculate tumor volume and simultaneously record the weight and mental state of mice in each group.

[0110] All experimental animals were sacrificed 14 days after treatment. Tumor tissue was completely dissected, and photographs of the ex vivo tumors were taken and their weight measured. The tumor growth inhibition effects of each group were compared. The ex vivo tumor photograph on day 14 is shown below. Figure 11 As shown, this invention significantly improves the tumor treatment efficacy of radionuclides.

[0111] The therapeutic effect of TG617 was examined using the method described above, except that PPTQF was replaced with PSMA-617, TGP with TG617, and the HT-1080-FAP subcutaneous tumor-bearing nude mouse model was replaced with the 22Rv1 subcutaneous tumor-bearing nude mouse model, while keeping all other steps unchanged. SPECT / CT imaging results and tumor images during TG617 treatment are shown below. Figure 12 , 13 As shown, it significantly improved the tumor enrichment efficiency and retention time of radiopharmaceuticals, and enhanced the tumor efficacy of radionuclides. The compounds in Table 1 all exhibit comparable efficacy.

[0112] The specific experimental procedures for safety assessment were as follows: On day 30 post-treatment, mice were anesthetized with isoflurane and euthanized by cervical dislocation. Major organs, including the heart, liver, spleen, lungs, and kidneys, were rapidly isolated, and after gently rinsing the surface blood with cold PBS, they were fixed in 4% paraformaldehyde for 24 hours.

[0113] The fixed tissues were sequentially dehydrated with 70%, 80%, 90%, 95%, and 100% ethanol, each step lasting 30-60 minutes. They were then cleared twice with xylene, 15 minutes each time. Finally, they were immersed in molten paraffin and embedded. The tissues were then cut into continuous sections approximately 4 μm thick using a paraffin microtome and attached to glass slides.

[0114] The steps for HE staining are as follows: Slices were dried at 60 ℃ for 30 min; Dewaxing with xylene twice, 10 minutes each time; Rehydrated successively with 100%, 95%, 90%, 80%, and 70% ethanol; After rinsing with distilled water, stain with hematoxylin for 5 min; Rinse with tap water and then decompose in 1% hydrochloric acid alcohol for a few seconds; The water returns to blue in 5 minutes; Counterstain with eosin for 1–2 min; It was dehydrated again by 70%, 80%, 90%, 95% and 100% ethanol; After the xylene becomes clear, add neutral resin to seal the slide.

[0115] After staining, the structures of various organs and tissues were observed using an optical microscope at 20× magnification. The focus was on assessing for pathological changes such as inflammatory cell infiltration, cell necrosis, hepatocyte vacuolar degeneration, renal tubular damage, alveolar structural destruction, and abnormal spleen tissue.

[0116] Mark at 30 days 177 HE staining after Tz-DOTA treatment with Lu demonstrates the safety of this invention, such as Figure 14 As shown.

[0117] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A polypeptide compound for signal enrichment based on click chemistry, characterized in that, The polypeptide compound comprises a targeting unit, an assembly polypeptide unit, and a click chemistry unit linked by chemical bonds, wherein, Targeting units are used to specifically recognize and bind to tumor surface antigens; Assemble polypeptide units for self-assembly to enrich click chemical sites; Click on the chemical unit to perform IEDDA reaction, CuAAC reaction, SPAAC reaction, or sulfur-fluoride exchange chemical reaction.

2. The peptide compound for signal enrichment based on click chemistry according to claim 1, characterized in that, The targeting unit includes PPTQF, FAPI-04, FAPI-46, FAPI-74, and FAP-2286, which target fibroblast-activating proteins; PSMA-617, PSMA-11, PSMA-1007, PSMA-I&T, and PSMA-ALB-56 target prostate-specific membrane antigens; Dotatate, Dotatoc, DotaNOC, JR11, and LM3 target the somatostatin receptor; Poly-RGD cyclic peptide targeting integrin receptors; Girentuximab and Nota-cG250, which target carbonic anhydrase IX; Trastuzumab, Pertuzumab, HER2-targeting nanobodies, EGFR antibodies, and EGFR-targeting peptides targeting the human epidermal growth factor receptor.

3. The peptide compound for signal enrichment based on click chemistry according to claim 1, characterized in that, The assembled polypeptide unit includes GNNQQNY, KLVFF, FF, or RADA. 16 .

4. The peptide compound for signal enrichment based on click chemistry according to claim 1, characterized in that, Click chemical units include azide, alkynyl, dibenzocyclooctylene, cyclopropanecyclooctylene, transcyclooctene, or tetrazine.

5. A composition based on click chemistry, characterized in that, The composition includes The first component comprises the polypeptide compound for signal enrichment based on click chemistry as described in any one of claims 1-4; The second component includes a reactive group, which is loaded with a radionuclide. The first component and the second component can undergo a bioorthogonal click chemistry reaction.

6. The composition according to claim 5, characterized in that, The second component also includes a chelating group, through which the radionuclide is loaded onto the reactive group.

7. The composition according to claim 6, characterized in that, The chelating group includes DOTA, NOA, or DOTAGA.

8. The composition according to claim 6, characterized in that, The radionuclide is selected from beta-ray, alpha-ray, or positron-emitting nuclides; the radionuclide is... 64 Cu、 68 Ga、 177 Lu、 223 Ra or 225 Ac.

9. The use of the click chemistry-based peptide compounds of claims 1-4 or the compositions of claims 5-8 in the preparation of radiotherapy drugs for treating tumors.

10. The application according to claim 9, characterized in that, The tumors include breast cancer, pancreatic cancer, glioma, esophageal tumor, colorectal tumor, lung cancer, gastric cancer, or metastatic tumors.

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