BCMA-targeted bicyclic peptide as well as preparation method and application thereof

Abstract

The invention discloses a BCMA-targeted bicyclic peptide as well as a preparation method and application thereof. The bicyclic peptide provided by the invention has high affinity to BCMA protein, and can specifically recognize tumors with positive BCMA expression in a targeting manner. The targeted BCMA bicyclic peptide can be combined with an anti-cancer preparation or a contrast agent to be further designed into a tumor targeted imaging agent, an immunotherapy drug, a polypeptide coupling drug and the like, and a new thought is provided for early diagnosis of tumors, dynamic monitoring of immune checkpoints and tumor targeted therapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a bicyclic peptide targeting BCMA and a preparation method and application thereof. Background Art

[0002] Multiple myeloma (MM) is a malignant plasma cell proliferative disease, usually accompanied by excessive production of monoclonal immunoglobulins or light chains (M proteins), and clinical manifestations are mostly bone destruction. MM is more common in the elderly, and is usually diagnosed in the middle and late stages. It lacks specific symptoms and mainly relies on bone marrow biopsy for diagnosis. However, this examination is traumatic and difficult to reflect the overall condition of the tumor. With the aging of the population in my country, the incidence of MM has increased year by year and has exceeded that of acute leukemia.

[0003] B cell maturation antigen (BCMA) is highly selectively expressed on the surface of MM malignant plasma cells and is an important target for MM diagnosis and treatment. Studies have shown that the expression level of BCMA is relatively consistent in different stages (including relapsed and refractory MM), and it is not expressed in normal tissues or is only expressed at low levels on a few mature B cells and plasma cells. Therefore, BCMA has become an ideal target for MM diagnosis and treatment. In recent years, a variety of BCMA-targeted drugs have been developed, including antibody-drug conjugates (ADCs), bispecific antibodies (BsAbs), and CAR-T cell therapy, but the use 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 for monitoring efficacy. There is an urgent need for a non-invasive method to evaluate the expression of BCMA at the in vivo level.

[0004] Traditional 18 F-FDG PET / CT imaging has significant limitations in MM. The low expression of GLUT1 and HK-2 in MM cells leads to low sensitivity of imaging. In addition, other imaging agents such as 11C-acetate and choline also lack specificity. Although dynamic contrast-enhanced (DCE) MRI can show bone marrow tumor infiltration, the problem of false positives has not been solved. Therefore, there is an urgent need for imaging targets that can specifically label BCMA to carry out molecular imaging studies in MM.

[0005] ImmunoPET imaging combines the high sensitivity of PET / CT with the specificity of antibodies, and can non-invasively evaluate the biodistribution of targets in vivo. However, probes based on intact antibodies often have limitations such as high blood background, long imaging time, high preparation cost, and strong immunogenicity, and the large molecular weight of antibodies leads to poor tumor penetration. Peptides have gradually become the focus of research in the field of radioactive imaging probes due to their small structure, easy modification, and good penetration. However, traditional linear peptides are easily degraded by proteases in the body, resulting in a short half-life in the blood, which affects the imaging quality. In contrast, the unique bicyclic constraint structure of cyclic peptides connects the N-terminal and C-terminal amino acid residues to the main chain through cyclization to form a more stable skeleton structure. This structural rigidity reduces the spatial conformational changes of the peptide chain, enhances resistance to enzymatic hydrolysis, and prolongs the half-life in vivo, making it superior to linear peptides in terms of targeting and stability. In addition, the three-dimensional structure of cyclic peptides can bind to target molecules more tightly, improve specificity and affinity, and can effectively penetrate tumor tissues. Summary of the invention

[0006] In view of the defects of the prior art, the purpose of the present invention is to provide a bicyclic peptide targeting BCMA. The cyclic peptide has high selectivity and high affinity through reasonable structural design, can specifically bind to a variety of MM tumor cells with high expression of BCMA, and is enriched in the tumor site in the blood circulation. Compared with traditional antibodies and linear peptide probes, the cyclic peptide has stronger stability, excellent tumor penetrability and lower immunogenicity. In addition, the probe can be labeled with radionuclides to achieve non-invasive and highly sensitive imaging of MM tumors, providing a new solution for early diagnosis and efficacy monitoring of MM. At the same time, the targeted cyclic peptide can also be coupled with anticancer drugs to form a polypeptide-coupled drug (PDC), which improves the permeability of the tumor site and provides a new idea for the targeted treatment of MM.

[0007] The present invention analyzes the crystal structure of the BCMA complex, extracts the binding hotspot amino acid sites, performs single-point mutations, and designs a peptide library with computer simulation assistance. The candidate peptides are initially screened out by combining the Docking score and the binding energy. Subsequently, a series of BCMA high-affinity peptides are screened out using surface plasmon resonance technology (SPRi).

[0008] After a large number of experimental verifications and screening, the BCMA-targeted bicyclic peptide of the present invention was finally obtained, and its molecular formula is C 67 H 96 N 16 O 42 S, the polypeptide can specifically bind to BCMA protein and selectively bind to tumor cells with high BCMA expression. The present invention also provides products derived from the polypeptide that can specifically bind to BCMA and the use of the polypeptide and its derivatives in tumor treatment, diagnosis, and imaging.

[0009] In order to achieve the above object, the present invention adopts the following technical solution:

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

[0011] The bicyclic peptide targeting BCMA comprises a polypeptide and a molecular scaffold;

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

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

[0014] The cysteine ​​residues at positions 5, 9 and 16 in the polypeptide are covalently bonded to the molecular scaffold, so that two polypeptide rings are formed 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 three Cys in the polypeptide to form two polypeptide rings.

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

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

[0020] According to an embodiment of the present invention, the structural formula of the bicyclic peptide targeting BCMA 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 body formed by the bicyclic peptide.

[0024] The bivalent or multivalent antibody can target BCMA.

[0025] Preferably, the divalent or multivalent body is formed by covalent or non-covalent bonding through linker molecules, or is formed by non-covalent bonding through mixing with a polymer.

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

[0027] More preferably, the covalently linked linker molecule is at least one of fluorescein isothiocyanate, 6-tert-butyloxycarbonylhydrazinonicotinic 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 linker molecules include but are not limited to lipophilic near-infrared dyes, such as ICG, IRDye800.

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

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

[0031] The method for preparing the bicyclic peptide targeting BCMA provided by the present invention comprises the following steps:

[0032] 1) synthesizing the polypeptide shown in SEQ ID No. 1 by Fmoc solid phase peptide synthesis method;

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

[0034] In a fourth aspect, the present invention provides a polypeptide targeting BCMA.

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

[0036] In a fifth aspect, the present invention provides a nucleic acid encoding the polypeptide of the fourth aspect.

[0037] In a sixth aspect, the present invention provides a biomaterial.

[0038] The biological material comprises the bicyclic peptide targeting BCMA, or an isomer, derivative, mixture, pharmaceutically acceptable salt, hydrate or solvate of the bicyclic peptide, or the nucleic acid; preferably, the biological material is a vector, an expression cassette, a transposon, a host cell or a transgenic cell line.

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

[0040] The host cell or transgenic cell line may be a cell or cell line derived from a microorganism, a plant or an animal; 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 medicine.

[0042] The drug provided by the present 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, pharmaceutically acceptable salt, hydrate or solvate of the bicyclic peptide, or the nucleic acid, or the biological material.

[0043] Preferably, the active ingredient of the drug also includes a preparation capable of killing tumor cells.

[0044] More preferably, the preparation capable of killing tumor cells is at least one of chemical drugs, biological drugs, nanomedicines, radioactive drugs, photothermal therapy or photodynamic therapy drugs capable of killing tumor cells; or, it is at least one of alkylating agents, antimetabolites, anti-tumor natural drugs, anti-tumor antibiotics, hormones, metal complexes or tumor radiation targeting markers.

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

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

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

[0048] In an eighth aspect, the present invention provides a conjugate.

[0049] The conjugate provided by the present invention comprises a carrier and at least one selected from the following components: the bicyclic peptide targeting BCMA, or the 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 connecting or acting the bicyclic peptide targeting BCMA or the derivative of the bicyclic peptide targeting BCMA with a carrier in a covalent or non-covalent manner.

[0051] Preferably, the carrier is at least one of a chelating agent, fluorescein, antibody, polymer, high molecular material, nanomaterial, liposome, oily compound, and inorganic material.

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

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

[0054] Furthermore, the conjugate is a bicyclic peptide nuclide ligand targeting BCMA.

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

[0056] The ligand of the radionuclide may be selected from DOTA, NOTA, DTPA, DFO and variants thereof.

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

[0058]

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

[0060] Preferably, the fluorescent molecule is at least one of 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 bicyclic peptide targeting BCMA, or the isomer, derivative, mixture, pharmaceutically acceptable salt, hydrate or solvate of the bicyclic peptide targeting BCMA, 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 BCMA-targeting bicyclic peptide has the function of targeting BCMA protein, it can be used as a homing peptide to couple small molecule drugs or a carrier carrying drugs; or it can be coupled with various imaging molecules such as radionuclides to form tumor contrast agents, etc., which provides more possibilities for tumor treatment and imaging diagnosis.

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

[0064] The bicyclic peptide nuclide probe targeting BCMA is a bicyclic peptide nuclide ligand targeting BCMA shown in Formula II, wherein the radioactive nuclide ligand is coupled to the radioactive nuclide 68 Ga is obtained by coordination reaction.

[0065] In the tenth aspect, the present invention provides a reagent or kit, which includes the bicyclic peptide targeting BCMA, or the isomer, derivative, mixture, pharmaceutically acceptable salt, hydrate or solvate of the bicyclic peptide targeting BCMA, or the nucleic acid, or the biomaterial, or the drug, or the conjugate, or the imaging agent.

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

[0067] In the ninth aspect, the present invention provides the use of the bicyclic peptide targeting BCMA, or the isomer, derivative, mixture, pharmaceutically acceptable salt, hydrate or solvate of the bicyclic peptide targeting BCMA, or the nucleic acid, or the biomaterial, or the drug, or the conjugate, or the imaging preparation, or the reagent or kit in at least one of the following aspects:

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

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

[0070] (3) Preparation of drugs for diagnosing, preventing or treating diseases for which BCMA is a marker;

[0071] (4) preparing diagnostic reagents, diagnostic kits, or imaging preparations;

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

[0073] The specific polypeptide of the present invention has a very high affinity for BCMA, and can be used to detect the expression level of BCMA, diagnose BCMA-related tumors, predict treatment effects, or directly treat related tumors. In particular, the specific probe targeting BCMA prepared by the present invention shows significant affinity for tumor uptake, and has low non-specific uptake in normal tissues, which can achieve 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 polypeptide of the present invention is a novel bicyclic peptide specifically targeting BCMA reported for the first time, with high selectivity, small molecular weight, high biosafety, low immunogenicity, and high tumor permeability. The polypeptide can be synthesized by chemical synthesis, which is simple to operate and has low production cost. Small molecule polypeptides are easier to design and modify drugs, and can be further optimized into multifunctional targeting materials, which have strong practicality and application prospects.

[0076] (2) The polypeptide of the present invention can be combined with an imaging agent for clinical transformation and application, and used as a molecular probe to detect the expression of BCMA in tumor cells to monitor the efficacy of immunotherapy in real time, and can be used as a predictive and companion diagnostic reagent for BCMA immunotherapy. It can also be used as a homing peptide and combined with an anticancer agent to form a polypeptide-coupled drug for targeted therapy and combined therapy of various tumors. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0080] Figure 4 For the present invention 68 Ga-DOTA-BCMA bicyclic peptide cellular uptake results.

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

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

[0083] Figure 7 This is BCMA immunofluorescence staining of H929 and K562 tumor tissues.

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

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

[0086] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0087] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0088] Example 1. Preparation of DOTA-BCMA bicyclic peptide and its 68 Ga labeling

[0089] The DOTA-BCMA bicyclic peptide structure is as follows Figure 1 shown, and 68 Ga marking.

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

[0091] a) Synthesis of bicyclic peptide: The linear peptide shown in SEQ ID No. 1 was synthesized using a standard Fmoc solid phase synthesis method. When the linear peptide was cleaved from the resin, a TFA cleavage mixture containing an appropriate protecting group scavenger was used, and the linear peptide was precipitated by ether and dissolved in a 50:50 acetonitrile / water solution. 1 mM linear peptide was mixed with 1.3 equivalents of the scaffold TATA or TBMB, and the cyclization reaction was catalyzed in 100 mM amino carbonate as a base. After the reaction was completed, N-acetylcysteine ​​(10 equivalents) was used to terminate the cyclization reaction. The reaction solution was freeze-dried and purified by HPLC and LC-MS (such as Figure 2 Validated and stored frozen.

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

[0093] DOTA was reacted with a bicyclic peptide targeting BCMA. 20% photopic acid (Piperidine) / DMF was used to remove the Fmoc protecting group and expose the terminal amino group of the peptide. A 2-fold molar excess of DOTA-COOH was weighed and dissolved in anhydrous DMF. HBTU (1-[Bis(dimethylamino)methylene]-1H-benzotriazolium hexafluorophosphate(1-),3-oxide) and DIEA activation solution were added and 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 alternately with DMF and DCM for 3-5 times to remove unreacted reagents and by-products. 95% TFA was added and treated at room temperature for 2 hours to complete the peptide cleavage and removal of the side chain protecting groups. The crude peptide product was precipitated with cold anhydrous ether and purified using HPLC to obtain the bicyclic peptide of DOTA-BCMA.

[0094] 3. Bicyclic peptide of DOTA-BCMA 68 Ga labeling

[0095] Use 0.05M high purity hydrochloric acid for elution 68 Ge- 68Ga-germanium-gallium generator obtains GaCl3 solution, takes 1mL Ga-68 solution, adds 100 microliters of sodium acetate (1M), covers the lid, mixes well, uses 0-6 precision pH test paper to measure pH to 4-4.5, adds 70μg of the molecule to be labeled (bicyclic peptide of DOTA-BCMA) to the prepared solution. Heat at 90℃ for 10min and elute. After cooling the reaction solution, add it to the activated Sep-Pak LightC18 column (5mL deionized water, 5mL ethanol, 5mL deionized water, activated according to water-ethanol-water). Rinse the impurities with 3.0mL pure water and discard. Add a 0.22μm sterile microporous filter membrane, collect the product with 0.5mL ethanol solution into a sterile vacuum bottle, add 5.0mL physiological saline to the system and set aside.

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

[0097] HPLC analysis results showed that the BCMA bicyclic peptide probe was labeled 68 Ga has a radiochemical purity of over 95%. 68 The Ga-DOTA-BCMA bicyclic peptide has good in vitro stability in saline solution or FBS solution within 2 hours. The specific results are shown in Figure 3 .

[0098] Embodiment 2, 68 Results of cellular uptake of Ga-labeled BCMA bicyclic peptide

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

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

[0101] Example 3: Small animal PET / CT imaging method and result analysis

[0102] All animal experiments followed the guidelines for experimental animals and were approved by the Animal Committee of Peking University First Hospital (approval number: 2022018). NCG severely immunodeficient mice (female, 4–6 weeks old, purchased from GemPharmatech Ltd.) lacking T cells, B cells, and NK cells were selected to construct a subcutaneous tumor model of multiple myeloma. The mice were randomly divided into two groups, the experimental group (H929) and the control group (K562), with 5 mice in each group. The right armpit was subcutaneously injected with 1×10 7 The subcutaneous tumor model was established by using 100 μL Matrigel suspension of 100 cells. The health status and tumor size of the mice were monitored every other day. When the tumor volume reached 1 cm 3 In vivo imaging and biodistribution studies were performed at 1 hr. H929-positive tumor and K562-negative tumor model mice were subjected to 1-hour dynamic PET / CT scanning using a micro-PET / CT scanner (NovelMedical, China). 68Ga-DOTA-BCMA bicyclic peptide (11.1MBq, dissolved in 200μL PBS) and anesthetized with 2% isoflurane. Scanning time points were 10, 20, 30, 40, 50 and 60 minutes after injection (pi, n=3). Tumor regions of interest (ROIs) were depicted using NMSoft-AIWSV.1.7 (Beijing NovelMedical) software.

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

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

[0105] The experimental method is the same as in Example 3. 68 Mice were killed 30 minutes and 60 minutes after the administration of Ga-DOTA-BCMA bicyclic peptide for biodistribution analysis. Blood and major organs (including heart, lungs, liver, kidneys, spleen, bladder, stomach, bones, muscles, small intestine, brain and tumors) were collected, and their radioactivity was measured using a gamma counter (Hidex) after weighing. The results are expressed as %ID / g (mean ± standard deviation). The content of the radioactive probe is expressed as the percentage of radioactive counts per gram of tissue to the total injected radioactive counts (%ID / g), representing the amount of radioactive uptake.

[0106] Biodistribution results are as follows Figure 6 a and 6b, 68 The uptake of Ga-DOTA-BCMA bicyclic peptide probe in H929 tumors (1.38±0.08%ID g-1 at 30 min and 1.05±0.04%ID g-1 at 60 min) was significantly higher than that in K562 tumors (0.75±0.07%ID g-1 at 30 min and 0.45±0.07%ID g-1 at 60 min). 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 tumors was evaluated by immunofluorescence staining of tumor tissues. Figure 7 In the results of immunofluorescence staining, H929 tumor tissue showed significant BCMA expression, and its green fluorescence intensity was strong. This indicates that the expression level of BCMA in H929 tumor cells is high. Compared with H929 tumors, K562 tumor tissue has lower BCMA expression, its immunofluorescence intensity is weak, and the green fluorescence signal is not obvious. This indicates that the expression level of BCMA in K562 tumor cells is relatively low. The immunohistochemical results further verified 68 Ga-DOTA-BCMA bicyclic peptide targets BCMA specifically.

[0109] The above results prove that the bicyclic peptide probe of the present invention has the characteristics of rapid targeting of BCMA and good tumor penetration ability, and can achieve high-sensitivity in vivo imaging of various BCMA-positive tumors.

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

[0111] Healthy male adult rhesus monkeys weighing 7.5 kg were used. All animals were screened for health before the experiment to ensure that they had no underlying diseases that would affect pharmacokinetics. Before the experiment, all experimental animals fasted for 6 hours before PET / CT scanning to reduce the impact of basal metabolism. Each rhesus monkey was injected intravenously 68Ga-DOTA-BCMA bicyclic peptide was administered at a dose of 5 MBq / kg. PET / CT (uMI780; United Imaging Health Care) scanning was performed at the beginning of the injection, covering the head to the abdomen. The scanning was continuous for 60 minutes. During the imaging process, low-dose CT (tube voltage 120 kV, tube current 100 mA, matrix size 512 × 512) was used for attenuation correction and anatomical reference. PET scanning was completed using 4 bed positions, with a matrix size of 192 × 192 for each bed and a scanning speed of 1.5 minutes / bed. All PET data were reconstructed using the OSEM (ordered subset expectation maximization) algorithm with reconstruction parameters of 2 iterations and 20 subsets. The reconstructed images were registered with the anatomical reference images to ensure the accuracy of the analysis. Image post-processing was completed on a professional workstation (uXceed, version R001; United Imaging Health Care) to ensure standardization and consistency of data processing. Image acquisition time points included: 2, 5, 10, 20, 30, 40, 50, 60 minutes. Changes in the standard uptake value (SUV) in multiple tissues (heart, liver, kidney, muscle, bone, and aorta) were recorded. The standard uptake value (SUVmean) curve over time was calculated to compare the uptake and clearance characteristics of different tissues.

[0112] like Figure 8 As shown in a, within 2 minutes, 68 Ga-DOTA-BCMA bicyclic peptide was rapidly distributed to the heart, kidneys and large blood vessels. As time went on, the radioactive signals in the heart and blood vessels gradually weakened, while those in the kidneys remained at a higher level, showing the characteristics of rapid uptake and slow clearance, indicating that the kidneys were the main clearance pathway. Figure 8 As shown in b, the SUV peak value of the kidney was the highest at 5 minutes (19.76±8.91), reached the maximum value at 3 minutes, and then gradually decreased to about 3.25±1.61 (60 minutes). The SUV values ​​of the liver and heart remained stable after 30 minutes. The SUV of bones and muscles remained relatively stable, and the overall uptake was low (SUVmean<2). The SUV of the aorta dropped rapidly in the early stage, indicating that the peptide was quickly distributed from the blood to the tissue.

[0113] Static scanning further verifies this trend. Fig. 9As shown in the figure, the whole-body static PET / CT imaging results at 30 and 60 minutes showed that the kidneys were still the main site of radioactivity accumulation, and the SUV value decreased from 4.41±0.58 at 30 minutes to about 2.34±0.04 at 60 minutes, which was consistent with the trend observed in the dynamic scan. The radioactive signals of the heart and large blood vessels were significantly weakened in the static scan, and the SUV values ​​were significantly lower than the dynamic data 30 minutes ago, indicating that the peptides had been basically cleared from the blood circulation at this time point. The SUV value of the liver remained at about 1.0, with no significant changes, while the SUV values ​​of muscles and bones remained at a low level (<1.0), indicating that the overall uptake in systemic tissues was low. The above results prove that the radioactive tracer is rapidly cleared from normal organs, which is consistent with the distribution and metabolism characteristics of small molecule peptides.

[0114] In summary, the bicyclic peptide probe of the present invention can specifically target a variety of tumor cells expressing BCMA, showing significant specificity and affinity. Therefore, in practical applications, the polypeptide probe can be used as a homing peptide, combined with anticancer drugs or imaging agents, suitable for targeted therapy and high-resolution imaging of a variety of tumors, and has the potential to become an integrated molecular probe for tumor diagnosis and treatment.

[0115] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A bicyclic peptide targeting BCMA, comprising a peptide and a molecular scaffold; The polypeptide has an amino acid sequence as shown in SEQ ID No. 1; in, The cysteine ​​residues at positions 5, 9 and 16 in the polypeptide are covalently bonded to the molecular scaffold, so that two polypeptide rings are formed on the molecular scaffold.

2. The BCMA-targeting bicyclic peptide according to claim 1, characterized in that: The molecular scaffold is 1,3,5-tris(bromomethyl)benzene (TBMB) or 1,3,5-triacryloylhexahydro-1,3,5-triazine (TATA); Preferably, it is TBMB; the bromine atom in the TBMB reacts with the sulfhydryl groups on the three cysteine ​​residues in the polypeptide of claim 1 to form two polypeptide rings; 3. An isomer, derivative, mixture, pharmaceutically acceptable salt, hydrate or solvate of the bicyclic peptide targeting BCMA according to claim 1 or 2; The derivative is a divalent or multivalent body formed by the bicyclic peptide.

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

5. A polypeptide targeting BCMA, whose amino acid sequence is shown in SEQ ID No.

1.

6. A biomaterial, characterized in that: The biological material comprises the bicyclic peptide according to claim 1 or 2, or an isomer, derivative, mixture, pharmaceutically acceptable salt, hydrate or solvate of the bicyclic peptide according to claim 3; preferably, the biological material is a vector, an expression cassette, a transposon, a host cell or a transgenic cell line.

7. A drug, characterized in that: The drug comprises a pharmaceutically acceptable excipient and at least one selected from the following components: the bicyclic peptide according to claim 1 or 2, or an isomer, derivative, mixture, pharmaceutically acceptable salt, hydrate or solvate of the bicyclic peptide according to claim 3, or the biomaterial according to claim 6.

8. A conjugate, characterized in that: The conjugate comprises a carrier and at least one selected from the following components: the bicyclic peptide according to claim 1 or 2, or an isomer, derivative, mixture, pharmaceutically acceptable salt, hydrate or solvate of the bicyclic peptide according to claim 3, or the biomaterial according to claim 5; Preferably, the carrier is any one or more of fluorescein, antibodies, polymers, high molecular materials, nanomaterials, liposomes, oily compounds, and inorganic materials.

9. An imaging preparation, characterized in that: The imaging agent comprises an imaging agent and at least one selected from the following components: the bicyclic peptide according to claim 1 or 2, the isomer, derivative, mixture, pharmaceutically acceptable salt, hydrate or solvate of the bicyclic peptide according to claim 3, the biomaterial according to claim 5, the drug according to claim 6, and the conjugate according to claim 7; The imaging agent is at least one of a radionuclide, a radionuclide marker, a fluorescent molecule, a magnetic resonance contrast agent or a molecular imaging agent.

10. Use of the bicyclic peptide according to claim 1 or 2, or the isomer, derivative, mixture, pharmaceutically acceptable salt, hydrate or solvate of the bicyclic peptide according to claim 3, or the biomaterial according to claim 6, or the drug according to claim 7, or the conjugate according to claim 8, or the imaging agent according to claim 9 in at least one of the following aspects: (1) Detect the BCMA expression level of cells; (2) preparing reagents for detecting BCMA expression levels in cells; (3) Preparation of drugs for diagnosing, preventing or treating diseases for which BCMA is a marker; (4) preparing diagnostic reagents, diagnostic kits, or imaging preparations; (5) Prepare products for detecting disease staging or auxiliary staging using BCMA as a marker.

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