A bicyclic peptide targeting BCMA, its preparation method and application

Abstract

This invention discloses a bicyclic peptide targeting BCMA, its preparation method, and its applications. The bicyclic peptide of this invention exhibits high affinity for BCMA protein and can specifically target and recognize BCMA-positive tumors. The BCMA-targeting bicyclic peptide of this invention can be further designed as a tumor-targeting imaging agent, immunotherapy drug, or peptide-conjugated drug, etc., in combination with anticancer agents or contrast agents, providing new ideas for early tumor diagnosis, dynamic monitoring of immune checkpoints, and targeted tumor therapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a bicyclic peptide targeting BCMA, its preparation method, and its application. Background Technology

[0002] Multiple myeloma (MM) is a malignant plasma cell proliferative disorder, usually accompanied by excessive production of monoclonal immunoglobulins or light chains (M protein), and its clinical manifestation is often bone destruction. MM is more common in the elderly, and is often diagnosed at an advanced stage. It lacks specific symptoms and mainly relies on bone marrow biopsy for diagnosis, but this examination is invasive and cannot reflect the overall condition of the tumor. With the aging of my country's population, the incidence of MM is rising year by year and has surpassed that of acute leukemia.

[0003] B-cell maturation antigen (BCMA) is highly selectively expressed on the surface of malignant plasma cells in multiple myeloma (MM), making it an important target for MM diagnosis and treatment. Studies have shown that BCMA expression levels are relatively consistent across different stages (including relapsed and refractory MM), and it is not expressed in normal tissues or is expressed at low levels only on a few mature B cells and plasma cells. Therefore, BCMA has become an ideal target for MM diagnosis and treatment. In recent years, various BCMA-targeting drugs have been developed, including antibody-drug conjugates (ADCs), bispecific antibodies (BsAbs), and CAR-T cell therapy. However, the application of these drugs can be accompanied by adverse reactions such as cytokine release syndrome and off-target effects, and there is a lack of effective means to monitor efficacy. Therefore, there is an urgent need for a non-invasive method to assess BCMA expression at the in vivo level.

[0004] Traditional 18 F-FDG PET / CT imaging has significant limitations in MM, with low GLUT1 and HK-2 expression in MM cells leading to low imaging sensitivity. Furthermore, other imaging agents such as 11C-acetate and choline lack specificity, and while dynamic contrast-enhanced MRI can show bone marrow tumor infiltration, the false-positive problem remains unresolved. Therefore, there is an urgent need for imaging targets that can specifically label BCMA to facilitate molecular imaging research in MM.

[0005] ImmunoPET imaging combines the high sensitivity of PET / CT with the specificity of antibodies, enabling non-invasive evaluation of the in vivo biodistribution of targets. However, probes based on intact antibodies often suffer from limitations such as high blood background, long imaging time, high preparation costs, and strong immunogenicity, and the large molecular weight of antibodies leads to poor tumor penetration. Peptides, due to their small structure, ease of modification, and good penetration, have gradually become a key research focus in the field of radiographic probes. However, traditional linear peptides are easily degraded by proteases in vivo, resulting in a short half-life in the blood and affecting imaging quality. In contrast, the unique bicyclic constraint structure of cyclic peptides links the N-terminal and C-terminal amino acid residues to the main chain through cyclization, forming a more stable backbone structure. This structural rigidity reduces spatial conformational changes in the peptide chain, enhances resistance to enzymatic degradation, and prolongs the in vivo half-life, making it superior to linear peptides in terms of targeting and stability. Furthermore, the three-dimensional structure of cyclic peptides allows for tighter binding of target molecules, improving specificity and affinity, and effectively penetrating tumor tissue. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a bicyclic peptide targeting BCMA. This cyclic peptide, through rational structural design, possesses high selectivity and high affinity, enabling it to specifically bind to various MM tumor cells that highly express BCMA and accumulate at the tumor site in the bloodstream. Compared to traditional antibodies and linear peptide probes, this cyclic peptide exhibits stronger stability, superior tumor penetration, and lower immunogenicity. Furthermore, this probe can be labeled with radionuclides to achieve non-invasive, highly sensitive imaging of MM tumors, providing a new solution for early diagnosis and treatment monitoring of MM. Simultaneously, this targeting cyclic peptide can be conjugated with anticancer drugs to form drug-peptide conjugates (PDCs), improving tumor site penetration and providing new insights for targeted therapy of MM.

[0007] This invention analyzes the crystal structure of the BCMA complex, extracts the binding hotspot amino acid sites, performs single-point mutagenesis, and designs a peptide library using computer simulation. Candidate peptides are initially screened using docking scores and binding energies. Subsequently, a series of high-affinity BCMA peptides are screened using surface plasmon resonance (SPRi) technology.

[0008] After extensive experimental verification and screening, the BCMA-targeting bicyclic peptide of this invention was finally obtained, with the molecular formula C0. 67 H 96 N 16 O 42 S, this polypeptide has a specific affinity for BCMA protein and selectively binds to tumor cells that highly express BCMA. This invention also provides products derived from this polypeptide that specifically bind to BCMA, and the uses of said polypeptide and its derivatives in tumor treatment, diagnosis, and imaging.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a bicyclic peptide that targets BCMA.

[0011] The bicyclic peptide targeting BCMA includes a polypeptide and a molecular scaffold.

[0012] The polypeptide has the following amino acid sequence (from N-terminus to C-terminus):

[0013] Gly-Gly-Ser-Gly-Cys-Glu-Glu-Tyr-Cys-Phe-Tyr-Asp-Pro-Tyr-Phe-Cys (SEQID No. 1);

[0014] The cysteine ​​residues at positions 5, 9, and 16 of the polypeptide are covalently bonded to the molecular scaffold, thereby forming two polypeptide rings on the molecular scaffold.

[0015] Furthermore, the molecular scaffold may be 1,3,5-tris(bromomethyl)benzene (TBMB) or 1,3,5-triacryloylhexahydro-1,3,5-triazine (TATA); preferably TBMB.

[0016]

[0017] When the molecular scaffold is TBMB, the bromine atoms in the TBMB molecular scaffold can react with the thiol groups of the three Cys in the polypeptide to form two polypeptide rings.

[0018] The amino acid residues of a polypeptide can be L-type, D-type, mirror-image structure, or a mixture of L-type and D-type, as well as sequence variations of mirror-image structures, cyclic peptide structures, and variations modified by PEG or fatty acid chains.

[0019] The bicyclic peptide targeting BCMA has high affinity and specificity for BCMA.

[0020] According to an embodiment of the present invention, the structural formula of the BCMA-targeting bicyclic peptide is shown in Formula I:

[0021]

[0022] In a second aspect, the present invention provides isomers, derivatives, mixtures, pharmaceutically acceptable salts, hydrates or solvates of the bicyclic peptide;

[0023] The derivative is a divalent or multivalent form of the bicyclic peptide.

[0024] The aforementioned divalent or multivalent can target BCMA.

[0025] Preferably, the divalent or multivalent is formed by linking molecules via covalent or non-covalent linkage, or by mixing with polymers via non-covalent linkage.

[0026] Preferably, the linker is polyethylene glycol (PEG), GSGS, or 8-aminooctanoic acid.

[0027] More preferably, the covalently linked linker is at least one of fluorescein isothiocyanate, 6-tert-butoxycarbonylhydrazinenicotinic acid, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide or N-hydroxysuccinimide, and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid.

[0028] More preferably, the non-covalently linked linking molecules include, but are not limited to, lipophilic near-infrared dyes such as ICG and IRDye800.

[0029] More preferably, the polymer is at least one of polyethylene glycol (PEG), polyvinyl alcohol (PVA), cyclodextrin, polyamide-amine dendritic polymer (PAMAM), polylactic acid (PLA), polylactic acid-ethanolamine (PLGA), and liposomes.

[0030] Thirdly, the present invention provides a method for preparing the BCMA-targeting bicyclic peptide described in the first aspect.

[0031] The method for preparing the BCMA-targeting bicyclic peptide provided by this invention includes the following steps:

[0032] 1) The polypeptide shown in SEQ ID No. 1 was synthesized using the Fmoc solid-phase polypeptide synthesis method;

[0033] 2) React the cysteine ​​residues in the polypeptide with the molecular scaffold to obtain the bicyclic peptide targeting BCMA.

[0034] Fourthly, the present invention provides a polypeptide that targets BCMA.

[0035] The polypeptide has the following amino acid sequence (from N-terminus to C-terminus): Gly-Gly-Ser-Gly-Cys-Glu-Glu-Tyr-Cys-Phe-Tyr-Asp-Pro-Tyr-Phe-Cys (SEQ ID No. 1).

[0036] Fifthly, the present invention provides a nucleic acid encoding the polypeptide described in the fourth aspect.

[0037] Sixthly, the present invention provides a biomaterial.

[0038] The biomaterial includes the bicyclic peptide targeting BCMA, or isomers, derivatives, mixtures, pharmaceutically acceptable salts, hydrates or solvates of the bicyclic peptide, or the nucleic acid; preferably, the biomaterial is a carrier, expression cassette, transposon, host cell or transgenic cell line.

[0039] The vectors include, but are not limited to, cloning vectors, expression vectors, and plasmid vectors. All vectors containing at least one copy of the nucleic acid encoding the BCMA-targeting polypeptide of the present invention are within the scope of protection of the present invention.

[0040] The host cell or transgenic cell line may be a cell or cell line derived from microorganisms, plants or animals; the plant cell or plant cell line has lost the ability to develop into a complete plant individual.

[0041] In a seventh aspect, the present invention provides a drug.

[0042] The medicament provided by this invention comprises a pharmaceutically acceptable excipient and at least one selected from the following components: the bicyclic peptide targeting BCMA, or an isomer, derivative, mixture of the bicyclic peptide, a pharmaceutically acceptable salt, hydrate or solvate, or the nucleic acid, or the biological material.

[0043] Preferably, the active ingredient of the drug further includes an agent capable of killing tumor cells.

[0044] More preferably, the agent capable of killing tumor cells is at least one of the following: chemical drugs, biological drugs, nanomedicines, radiopharmaceuticals, photothermal therapy or photodynamic therapy drugs capable of killing tumor cells; or, at least one of the following: alkylating agents, antimetabolites, antitumor natural drugs, antitumor antibiotics, hormones, metal complexes or tumor radiotargeting markers.

[0045] More preferably, the drug further includes a carrier conjugated to or mixed with the bicyclic peptide or a derivative thereof.

[0046] The carrier includes, but is not limited to, carriers used for preparing targeted drugs.

[0047] The carrier includes at least one of nanomaterials, liposomes, and oily compounds.

[0048] Eighthly, the present invention provides a coupling.

[0049] The conjugate provided by the present invention comprises a carrier and at least one of the following components: the bicyclic peptide targeting BCMA, or an isomer, derivative, mixture, pharmaceutically acceptable salt, hydrate or solvate of the bicyclic peptide targeting BCMA, or the nucleic acid, or the biomaterial.

[0050] The conjugate is obtained by covalently or non-covalently linking or interacting the bicyclic peptide targeting BCMA or a derivative thereof with a carrier.

[0051] Preferably, the carrier is at least one selected from chelating agents, fluorescein, antibodies, polymers, high molecular weight materials, nanomaterials, liposomes, oily compounds, and inorganic materials.

[0052] More preferably, the polymeric material is at least one of polyester, polyanhydride, polyamide phospholipid polymer micelles, polylactic acid-glycolic acid copolymer, polyethylene glycol, and chitosan.

[0053] More preferably, the inorganic material is at least one of nano-gold, carbon materials, calcium materials, magnetic materials, mesoporous silicon materials, and quantum dots.

[0054] Furthermore, the conjugate is a bicyclic peptide nucleoside ligand that targets BCMA.

[0055] The BCMA-targeting bicyclic peptide nuclide ligand is a ligand with a radionuclide modified on the first glycine residue in the polypeptide of the BCMA-targeting bicyclic peptide.

[0056] The ligands for the radionuclides may be selected from DOTA, NOA, DTPA, DFO, and their variants.

[0057] According to an embodiment of the present invention, the structural formula of the BCMA-targeting bicyclic peptide nuclide ligand is shown in Formula II:

[0058]

[0059] In a ninth aspect, the present invention provides an imaging formulation comprising an imaging agent and at least one selected from the following components: the said bicyclic peptide targeting BCMA, or an isomer, derivative, mixture, pharmaceutically acceptable salt, hydrate, or solvate of the said bicyclic peptide targeting BCMA, or the said nucleic acid, or the said biomaterial, or the said drug, or the said conjugate; wherein the imaging agent is at least one selected from the following: a radionuclide, a radionuclide label, a fluorescent molecule, a magnetic resonance contrast agent, or a molecular imaging formulation.

[0060] Preferably, the fluorescent molecule is at least one selected from IRDye800CW, Cy7, Cy5.5, rhodamine, and indocyanine green (ICG), and the radionuclide is... 131 I, 177 Lu、 64 Cu、 99m Tc, 18 F and 68At least one of Ga.

[0061] Preferably, the BCMA-targeting bicyclic peptide, or isomers, derivatives, mixtures, pharmaceutically acceptable salts, hydrates or solvates of the BCMA-targeting bicyclic peptide, or the nucleic acid, or the biomaterial, or the drug, or the conjugate is coupled, conjugated or mixed with the imaging agent.

[0062] Since the bicyclic peptide targeting BCMA has the function of targeting BCMA protein, it can be used as a homing peptide conjugated with small molecule drugs or a drug-carrying carrier; or conjugated with various imaging molecules such as radionuclides to form tumor contrast agents, thus providing more possibilities for tumor treatment and imaging diagnosis.

[0063] According to an embodiment of the present invention, the imaging agent is a bicyclic peptide nuclide probe targeting BCMA.

[0064] The BCMA-targeting bicyclic peptide nuclide probe is formed by the BCMA-targeting bicyclic peptide nuclide ligand shown in Formula II, through which the ligand of the radionuclide interacts with the radionuclide. 68 Ga is obtained through a coordination reaction.

[0065] In a tenth aspect, the present invention provides a reagent or kit comprising the said bicyclic peptide targeting BCMA, or isomers, derivatives, mixtures, pharmaceutically acceptable salts, hydrates or solvates of the said bicyclic peptide targeting BCMA, or the said nucleic acid, or the said biomaterial, or the said drug, or the said conjugate, or the said imaging preparation.

[0066] Preferably, the reagent or kit is used for the diagnosis of BCMA-related diseases.

[0067] In a ninth aspect, the present invention provides the use of the said BCMA-targeting bicyclic peptide, or isomers, derivatives, mixtures, pharmaceutically acceptable salts, hydrates or solvates of the said BCMA-targeting bicyclic peptide, or the said nucleic acid, or the said biomaterial, or the said drug, or the said conjugate, or the said imaging formulation or the said reagent or kit in at least one of the following aspects:

[0068] (1) Detect the expression level of BCMA in cells;

[0069] (2) Preparation of reagents for detecting BCMA expression levels in cells;

[0070] (3) Preparation of pharmaceutical products; said pharmaceutical products are used for the diagnosis, prevention or treatment of diseases marked by BCMA;

[0071] (4) Preparation of diagnostic reagents, diagnostic kits, or imaging agents;

[0072] (5) Prepare products for detecting disease staging or auxiliary staging using BCMA as a marker.

[0073] The specific peptides of this invention have extremely high affinity for BCMA, enabling them to be used to detect BCMA expression levels, diagnose BCMA-related tumors, predict treatment efficacy, or be directly used to treat related tumors. In particular, the BCMA-targeting specific probes prepared in this invention exhibit significant affinity for tumor uptake while showing low non-specific uptake in normal tissues, enabling non-invasive, accurate, and efficient detection of human BCMA expression.

[0074] Compared with the prior art, the present invention has the following beneficial effects:

[0075] (1) The peptide of this invention is a novel bicyclic peptide that specifically targets BCMA and is reported for the first time. It exhibits high selectivity, small molecular weight, high biocompatibility, low immunogenicity, and high tumor permeability. This peptide can be synthesized using chemical synthesis methods, which are simple to operate and have low production costs. Small molecule peptides are easier to design and modify for drugs and can be further optimized into multifunctional targeting materials, showing strong practicality and application prospects.

[0076] (2) The peptides of this invention can be combined with imaging agents and clinically translated for use as molecular probes to detect BCMA expression in tumor cells, thereby monitoring the efficacy of immunotherapy in real time. They can also serve as predictive and companion diagnostic reagents for BCMA immunotherapy. Furthermore, they can be used as homing peptides, combined with anticancer agents to form peptide-conjugated drugs for targeted and combined therapy of various tumors. Attached Figure Description

[0077] Figure 1 This is the structural formula of the DOTA-BCMA bicyclic peptide in this invention.

[0078] Figure 2 This is the LC-MS spectrum of the bicyclic peptide targeting BCMA in this invention.

[0079] Figure 3 for 68 Radiochemical purification and in vitro stability analysis of Ga-DOTA-BCMA bicyclic peptide.

[0080] Figure 4 In this invention 68 Results of Ga-DOTA-BCMA bicyclic peptide cellular uptake.

[0081] Figure 5 PET imaging of small animals 68 In vivo detection results of Ga-DOTA-BCMA bicyclic peptide in BCMA-positive and BCMA-negative tumors.

[0082] Figure 6 For injection 68 Biodistribution of tumors and organs in BCMA-positive and negative tumor models after 30 and 60 minutes of Ga-DOTA-BCMA bicyclic peptide treatment.

[0083] Figure 7 BCMA immunofluorescence staining for H929 and K562 tumor tissues.

[0084] Figure 8 for 68 Pharmacokinetics of Ga-DOTA-BCMA bicyclic peptide in normal rhesus monkeys.

[0085] Figure 9 for 68 Whole-body static PET / CT imaging results of Ga-DOTA-BCMA bicyclic peptide in normal rhesus monkeys at 30 and 60 minutes. Detailed Implementation

[0086] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0087] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0088] Example 1: Preparation of DOTA-BCMA bicyclic peptide and its application 68 Ga mark

[0089] The structure of the DOTA-BCMA bicyclic peptide is as follows: Figure 1 As shown, and subjected to 68 Ga marker.

[0090] 1. Preparation of a bicyclic peptide targeting BCMA (as shown in Formula I)

[0091] a) Synthesis of the bicyclic peptide: The linear peptide shown in SEQ ID No. 1 was synthesized using the standard Fmoc solid-phase synthesis method. When cleaving the linear peptide from the resin, a TFA cleavage mixture containing a suitable protecting group scavenger was used. The linear peptide was precipitated by diethyl ether and dissolved in a 50:50 acetonitrile / water solution. 1 mM of the linear peptide was mixed with 1.3 equivalents of the scaffold TATA or TBMB, and a cyclization reaction was catalyzed by 100 mM amino carbonate as a base. After the reaction was complete, the cyclization reaction was terminated using N-acetylcysteine ​​(10 equivalents). The reaction solution was lyophilized and purified by HPLC and LC-MS (e.g., LC-MS). Figure 2 As shown in the figure, the sample was verified and frozen.

[0092] 2. Preparation of DOTA-BCMA bicyclic peptide

[0093] DOTA was reacted with a bicyclic peptide targeting BCMA. The Fmoc protecting group was removed using 20% ​​Piperidine / DMF, exposing the terminal amino group of the peptide. Two molar excesses of DOTA-COOH were weighed and dissolved in anhydrous DMF. HBTU (1-[Bis(dimethylamino)methylene]-1H-benzotriazolium hexafluorophosphate(1-),3-oxide) and DIEA activation solution were added, and the mixture was allowed to stand at room temperature for 10 minutes. The activated DOTA-COOH solution was added to the deprotected peptide resin and incubated on a shaker at room temperature for 1 hour. The resin was washed 3-5 times alternately with DMF and DCM to remove unreacted reagents and byproducts. 95% TFA was added, and the mixture was treated at room temperature for 2 hours to complete peptide cleavage and removal of side-chain protecting groups. The crude peptide was precipitated with cold anhydrous diethyl ether and purified by HPLC to obtain the DOTA-BCMA bicyclic peptide.

[0094] 3. The bicyclic peptide of DOTA-BCMA was subjected to... 68 Ga mark

[0095] Rinse with 0.05M high-purity hydrochloric acid 68 Ge- 68GaCl3 solution was obtained using a Ga germanium-gallium generator. 1 mL of Ga-68 solution was added to 100 μL of sodium acetate (1M), the container was capped, and the solution was mixed. The pH was measured to be 4-4.5 using 0-6 precision pH paper. 70 μg of the molecule to be labeled (the bicyclic peptide of DOTA-BCMA) was added to the prepared solution. The mixture was heated at 90°C for 10 min and then eluted. After cooling the reaction solution, it was added to an activated Sep-Pak LightC18 column (5 mL deionized water, 5 mL ethanol, 5 mL deionized water, activated using a water-ethanol-water ratio). Impurities were rinsed with 3.0 mL of pure water and discarded. A 0.22 μm sterile microporous membrane was added, and the product was collected into a sterile vacuum bottle with 0.5 mL of ethanol solution. 5.0 mL of physiological saline was added to the system for later use.

[0096] 68 The Ga-DOTA-BCMA bicyclic peptide-labeled product was further determined by high-performance liquid chromatography (HPLC). Chromatographic conditions: C18 column (4.6 × 150 mm, 5 μm, XBridge, Waters); mobile phase A: deionized water (0.1% trifluoroacetic acid); mobile phase B: acetonitrile (0.1% trifluoroacetic acid); flow rate: 1.0 mL / min. Specific analytical methods were: 0–2 min, 10% B; 2–10 min, 10%–60% B; 10–12 min, 60% B; 12–15 min, 60%–10% B. Radiometric spectra were collected. The purified product was then... 68 Ga-DOTA-BCMA bicyclic peptides (2.96–3.7 MBq) were incubated at 37 °C in 200 μL of 0.01 M phosphate-buffered saline (PBS, pH 7.4) and fetal bovine serum (FBS) for 60 and 120 minutes, respectively. Samples at each time point were analyzed by high-performance liquid chromatography.

[0097] HPLC analysis results showed that the BCMA bicyclic peptide probe was effective in labeling... 68 Ga has a radiochemical purity of over 95%. Furthermore, 68 The Ga-DOTA-BCMA bicyclic peptide exhibited good in vitro stability in physiological saline or FBS solution within 2 hours. See the attached table for details. Figure 3 .

[0098] Example 2 68 Ga-labeled BCMA bicyclic peptide cellular uptake results

[0099] Human myeloma cell line H929 was selected as the BCMA-positive cell line, and human chronic myeloid leukemia cell line K562 was used as the negative control. Both cell lines were cultured in RPMI 1640 medium containing 10% FBS. In the cell uptake assay, approximately 740 kBq of... 68Ga-DOTA-BCMA bicyclic peptide and 1×10 6 Cells were incubated at 37°C for 30 minutes. After incubation, cells were washed three times with PBS to remove unbound probes. For the blocking group, 740 kBq of [a specific reagent / method] was used. 68 Ga-DOTA-BCMA bicyclic peptide and 1×10 -4 M BCMA bicyclic peptide and 1×10 6 Cells were incubated at 37°C for 30 minutes. After incubation, the cells were washed three times with PBS. The washed cell suspension was collected, and radioactivity was measured using a gamma counter (Hidex). Each data point represents the average of three replicates.

[0100] Figure 4 The results show that 68 The mean binding rate of Ga-DOTA-BCMA bicyclic peptide to H929 cells was 0.497±0.06%, while the mean binding rates in the H929 blocking group and the K562 non-blocking group were 0.225±0.03% (P<0.01) and 0.255±0.04% (P<0.01), respectively, significantly lower than that in the H929 non-blocking group. Furthermore, there was no significant difference between the non-blocking and blocking groups in K562 cells. These data indicate that the radiolabeled BCMA bicyclic peptide can specifically bind to BCMA under in vitro conditions.

[0101] Example 3: Small Animal PET / CT Imaging Methods and Result Analysis

[0102] All animal experiments were conducted in accordance with laboratory animal guidelines and approved by the Animal Committee of Peking University First Hospital (Approval No.: 2022018). Multiple myeloma subcutaneous tumor models were established using NCG-severe immunodeficient mice (female, 4–6 weeks old, purchased from GemPharmatech Ltd.) lacking T cells, B cells, and NK cells. Mice were randomly divided into two groups: the experimental group (H929) and the control group (K562), with 5 mice in each group. A subcutaneous injection of 1×10⁻⁶ NCG-containing compound was administered into the right axilla. 7 A subcutaneous tumor model was established using a 100 μL L Atrigel suspension per cell. The health status and tumor size of the mice were monitored every other day. When the tumor volume reached 1 cm, the tumor was considered closed. 3 In vivo imaging and biodistribution studies were then initiated. One-hour dynamic PET / CT scans were performed on mice with H929-positive and K562-negative tumor models using a micro-PET / CT scanner (Novel Medical, China). Mice were injected via tail vein. 68Ga-DOTA-BCMA bicyclic peptide (11.1 MBq, dissolved in 200 μL PBS) was administered to patients under 2% isoflurane anesthesia. Scanning was performed at 10, 20, 30, 40, 50, and 60 minutes post-injection (pi, n=3). Regions of interest (ROIs) were delineated using NMSoft-AIWSV.1.7 (Beijing Novel Medical) software.

[0103] The results are as follows Figure 5 As shown, the maximum intensity projection (MIP) results indicate that in the BCMA-positive H929 tumor model, 68 Ten minutes after injection of the Ga-DOTA-BCMA bicyclic peptide, significant radioactive accumulation was observed in the tumor, with high uptake remaining from 10 to 60 minutes. In contrast, no significant radioactive accumulation was observed in the K562 tumor model, which was negative for BCMA expression.

[0104] Example 4 68 In vivo biodistribution analysis of Ga-DOTA-BCMA bicyclic peptide

[0105] The experimental method is the same as in Example 3, during injection. 68 Mice were sacrificed 30 and 60 minutes after administration of the Ga-DOTA-BCMA bicyclic peptide for biodistribution analysis. Blood and major organs (including heart, lung, liver, kidney, spleen, bladder, stomach, bone, muscle, small intestine, brain, and tumor) were collected, weighed, and their radioactivity was determined using a gamma counter (Hidex). Results are expressed as %ID / g (mean ± standard deviation). The content of the radioactive probe is expressed as the percentage of radioactive count per gram of tissue relative to the total injected radioactive count (%ID / g), representing the radioactive uptake.

[0106] Biological distribution results as follows Figure 6 As shown in a and 6b, 68 The uptake of the Ga-DOTA-BCMA bicyclic peptide probe in H929 tumors (1.38±0.08% ID g-1 at 30 minutes and 1.05±0.04% ID g-1 at 60 minutes, respectively) was significantly higher than that in K562 tumors (0.75±0.07% ID g-1 at 30 minutes and 0.45±0.07% ID g-1 at 60 minutes, respectively). Furthermore, the uptake of the Ga-DOTA-BCMA bicyclic peptide probe in H929 tumors was significantly higher than that in K562 tumors (0.75±0.07% ID g-1 at 30 minutes and 0.45±0.07% ID g-1 at 60 minutes, respectively). 68 There was a statistically significant difference in Ga-DOTA-BCMA uptake (P<0.001).

[0107] Example 5: BCMA immunofluorescence staining of H929 and K562 tumor tissues.

[0108] The expression level of BCMA in tumor tissue was assessed by immunofluorescence staining. Figure 7 In the immunofluorescence staining results, H929 tumor tissue showed significant BCMA expression with strong green fluorescence intensity. This indicates that the expression level of BCMA in H929 tumor cells is high. Compared with H929 tumor, K562 tumor tissue showed lower BCMA expression, weaker immunofluorescence intensity, and less obvious green fluorescence signal. This indicates that the expression level of BCMA in K562 tumor cells is relatively low. Immunohistochemical results further validated this. 68 Ga-DOTA-BCMA bicyclic peptide targets BCMA specifically.

[0109] The above results demonstrate that the bicyclic peptide probe of the present invention has the characteristic of rapidly targeting BCMA and has good tumor penetration ability, enabling high-sensitivity in vivo imaging of various BCMA-positive tumors.

[0110] Example 6 68 Pharmacokinetics of Ga-DOTA-BCMA bicyclic peptide in normal rhesus monkeys

[0111] Healthy adult male rhesus monkeys, weighing 7.5 kg, were used in the experiment. All animals underwent health screening prior to the experiment to ensure they were free of underlying diseases that could affect pharmacokinetics. All experimental animals were fasted for 6 hours before the PET / CT scan to minimize the impact of basal metabolism. Each rhesus monkey was administered an intravenous injection. 68Ga-DOTA-BCMA bicyclic peptide, at a dose of 5 MBq / kg. PET / CT (uMI780; United Imaging Health Care) scanning was initiated immediately upon injection, covering the area from head to abdomen. Continuous scanning lasted 60 minutes. During imaging, low-dose CT (tube voltage 120 kV, tube current 100 mA, matrix size 512 × 512) was used for attenuation correction and to provide anatomical reference. PET scans were performed at four bed positions, each with a matrix size of 192 × 192, at a scan rate of 1.5 minutes per bed. All PET data were reconstructed using the OSEM (Ordered Subset Expectation Maximization) algorithm with two iterations and 20 subsets. Reconstructed images were registered with anatomical reference images to ensure analytical accuracy. Image post-processing was performed on a professional workstation (uXceed, version R001; United Imaging Health Care) to ensure standardized and consistent data processing. Image acquisition time points included 2, 5, 10, 20, 30, 40, 50, and 60 minutes. Changes in the standard uptake value (SUV) in multiple tissues (heart, liver, kidney, muscle, bone, and aorta) were recorded. The SUVmean curve was calculated over time to compare the uptake and clearance characteristics of different tissues.

[0112] like Figure 8 As shown in a, within 2 minutes, 68 The Ga-DOTA-BCMA bicyclic peptide rapidly distributes to the heart, kidneys, and large blood vessels. Over time, the radioactive signal in the heart and blood vessels gradually decreases, while the kidneys maintain a higher level, exhibiting rapid uptake and slow clearance characteristics, suggesting that the kidneys are the primary clearance pathway. Figure 8 As shown in b, the peak SUV value for the kidneys was highest at 5 minutes (19.76 ± 8.91), reaching its maximum at 3 minutes, and then gradually decreased to approximately 3.25 ± 1.61 (60 minutes). The SUV values ​​for the liver and heart remained stable after 30 minutes. The SUV values ​​for bones and muscles remained relatively stable, with low overall uptake (SUVmean < 2). The SUV value for the aorta decreased rapidly in the early stages, suggesting rapid distribution of the peptide from the blood to the tissues.

[0113] Static scanning further confirmed this trend. For example... Figure 9As shown, the whole-body static PET / CT imaging results at 30 and 60 minutes indicate that the kidneys remain the primary site of radioactivity accumulation, with the SUV value decreasing from 4.41±0.58 at 30 minutes to approximately 2.34±0.04 at 60 minutes—a change consistent with the trend observed in dynamic scans. The radioactive signal in the heart and large blood vessels significantly decreased in static scans, with SUV values ​​significantly lower than the dynamic data from 30 minutes prior, suggesting that the peptides had been largely cleared from the bloodstream at this time point. The SUV value of the liver remained at approximately 1.0 without significant change, while the SUV values ​​of muscle and bone remained at low levels (<1.0), indicating low overall uptake in systemic tissues. These results demonstrate the rapid clearance of radioactive tracers from normal organs, consistent with the metabolic characteristics of small molecule peptides.

[0114] In summary, the bicyclic peptide probe of this invention can specifically target various tumor cells expressing BCMA, exhibiting significant specificity and affinity. Therefore, in practical applications, this peptide probe can serve as a homing peptide, binding with anticancer drugs or imaging agents, making it suitable for targeted therapy and high-resolution imaging of various tumors, and possessing the potential to become an integrated molecular probe for tumor diagnosis and treatment.

[0115] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A bicyclic peptide targeting BCMA, comprising a polypeptide and a molecular scaffold; The polypeptide has the amino acid sequence shown in SEQ ID No. 1; in, The cysteine ​​residues at positions 5, 9, and 16 of the polypeptide are covalently bonded to the molecular scaffold, thereby forming two polypeptide rings on the molecular scaffold. The molecular scaffold is 1,3,5-tris(bromomethyl)benzene (TBMB); the bromine atom in the TBMB reacts with the thiol groups on the three cysteine ​​residues in the polypeptide to form two polypeptide rings; 。 2. The derivative of the BCMA-targeting bicyclic peptide according to claim 1, a pharmaceutically acceptable salt or solvate; The derivative is a divalent or multivalent form of the bicyclic peptide.

3. The method for preparing the BCMA-targeting bicyclic peptide according to claim 1, comprising the following steps: 1) The polypeptide shown in SEQ ID No. 1 was synthesized using the Fmoc solid-phase polypeptide synthesis method; 2) React the cysteine ​​residues in the polypeptide with the molecular scaffold to obtain the bicyclic peptide targeting BCMA.

4. A drug, characterized in that: The drug comprises a pharmaceutically acceptable excipient and at least one selected from the following components: the bicyclic peptide of claim 1, or a derivative of the bicyclic peptide of claim 2, a pharmaceutically acceptable salt, or a solvate.

5. A coupling agent, characterized in that: The conjugate comprises a carrier and at least one of the following components: the bicyclic peptide of claim 1, or a derivative of the bicyclic peptide of claim 2, a pharmaceutically acceptable salt, or a solvate. The carrier is any one or more of the following: fluorescein, antibody, polymer, high molecular weight material, nanomaterial, liposome, oily compound, and inorganic material.

6. A developing agent, characterized in that: The imaging formulation comprises an imaging agent and at least one selected from the following components: the bicyclic peptide of claim 1, a derivative of the bicyclic peptide of claim 2, a pharmaceutically acceptable salt or solvate, the drug of claim 4, and the conjugate of claim 5. The imaging agent is at least one of a radionuclide, a radionuclide label, a fluorescent molecule, a magnetic resonance contrast agent, or a molecular imaging preparation.

7. The use of the bicyclic peptide of claim 1, or a derivative of the bicyclic peptide of claim 2, a pharmaceutically acceptable salt or solvate, or the drug of claim 4, or the conjugate of claim 5, or the imaging formulation of claim 6, in at least one of the following aspects: (1) Prepare reagents for detecting BCMA expression levels in cells; (2) Preparation of diagnostic reagents, diagnostic kits, or imaging agents; (3) Prepare products for detecting disease staging or auxiliary staging using BCMA as a marker.

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