Polypeptides targeting bcma and uses thereof

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to a polypeptide targeting BCMA and application thereof.The polypeptide of the present application has high affinity to BCMA protein, and can recognize target BCMA positive cells.The polypeptide targeting BCMA of the present application can be combined with anticancer agents or contrast agents to be further designed into tumor-targeting imaging agents, immunotherapy drugs, polypeptide conjugated drugs and the like, thereby providing a new idea for early diagnosis of tumors, dynamic monitoring of immune checkpoints and tumor-targeting treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a polypeptide targeting BCMA and application thereof. BACKGROUND

[0002] Multiple myeloma (MM) is a malignant disease of age-related abnormal proliferation of bone marrow plasma cells and accompanied by excessive production of monoclonal immunoglobulin or light chain (M protein). Its clinical manifestations are bone destruction, lack of specificity, and diagnosis mainly depends on the increase of clonal bone marrow plasma cells in bone marrow biopsy, which is an invasive examination, and improper selection of bone marrow puncture site will lead to false negative results. B cell maturation antigen (BCMA) is significantly and selectively expressed on the surface of MM malignant plasma cells, and is almost only expressed on plasma cells, and is not expressed on normal tissue cells (except part of mature B cells and plasma cells) and CD34 + stem cells. Studies have also found that the expression level of BCMA is similar in different stages (from untreated to relapsed) of MM. The consistent up-regulation and uniqueness of BCMA on the surface of MM cells make BCMA an ideal target antigen for diagnosis and treatment of MM. At present, different types of BCMA targeted drugs, including antibody-drug conjugates (ADC), bispecific antibodies (BsAbs) and chimeric antigen receptor (CAR) T cells, have shown good efficacy and safety in patients with relapsed and refractory MM. However, reviewing the previous studies, the application of targeted drugs can produce various toxic reactions such as cytokine release syndrome, off-target effect, allergic reaction, and some patients do not show clinical benefit, and there is a lack of biomarkers for effective monitoring and evaluation of therapeutic effect, leading to disease progression. Therefore, how to effectively find BCMA high expression lesions at the level of living body by non-invasive method has important value for the screening and efficacy evaluation of BCMA targeted therapy patients.

[0003] Multifocal multiple myeloma (MM), a malignant tumor characterized by abnormal proliferation of plasma cells in the bone marrow, often presents with detectable M protein or urinary Bence Jones protein in blood or urine. However, this is not applicable to diagnosing all types of MM. For some oligosecreting patients, M protein alone cannot accurately assess disease progression and treatment response. Current guidelines both domestically and internationally emphasize the importance of obtaining tumor tissue for pathological examination; however, this is invasive, requiring biopsies to obtain ex vivo tissue, especially bone marrow aspiration, which is often performed at specific sites, making it impossible to pre-screen for viable lesions. Furthermore, the size of the biopsy tissue is limited, potentially leading to false negatives as it cannot represent the entire tumor. In contrast, molecular imaging offers the advantage of being non-invasive, reflecting the expression and distribution of specific molecules at the in vivo level.

[0004] Nuclear medicine possesses inherent advantages in molecular imaging. After radiolabeling specific probes with radionuclides, single-photon computed tomography (SPECT) or positron emission tomography / computed tomography (PET / CT) can detect the in vivo distribution of specific target molecules. Fluorodeoxyglucose (FDEG) 18 F-FDG is a glucose metabolism imaging agent. 18 F-FDG PET / CT is widely used in the diagnosis, staging, treatment guidance, and efficacy monitoring of malignant tumors, but it has significant limitations for multivariate (MM). MM cells often show low expression of glucose transporter 1 (GLUT1) and hexokinase-2 (HK-2), leading to… 18 F-FDG imaging agents have difficulty distinguishing between benign lesions and low-metabolic MM lesions; more than one-third of intramedullary lesions in MM cannot be detected. Other PET / CT imaging agents have also been used in MM research, such as 11C-acetate and choline, and amino acids. However, similar to... 18 Imaging agents such as F-FDG lack tumor specificity and are difficult to reflect the molecular characteristics of multifocal myeloma (MM). Some studies have utilized the interaction between integrins on the surface of MM cells and the stromal environment to perform receptor imaging targeting the α4β1 protein in tumor angiogenesis; however, the background radiation distribution in normal bone marrow is too high (11.6±2.0% ID / g), reducing its value. Whole-body MRI offers excellent soft tissue contrast and spatial resolution, capable of displaying tumor infiltration in the bone marrow. Dynamic contrast-enhanced MRI (DCE) can also visualize bone marrow vascularity; however, bone marrow activation caused by physiological activities or infection can lead to false positives. In summary, previous metabolic and receptor imaging methods have limited value and cannot reflect the specific molecular characteristics of MM. Further research is needed to identify specific markers as imaging targets.

[0005] BCMA is a type III transmembrane glycoprotein consisting of 184 amino acids, also known as CD269, which is a member of the tumor necrosis factor receptor (TNFR) family, encoded by the 2.92 kb TNFRSF17 gene located on the short arm of chromosome 16 (16p13.13). The interaction between BCMA and its ligand promotes the progression of MM by activating signaling pathways such as AKT, MAPK, and NF-kB, enhancing the growth of malignant plasma cells. A large number of studies have confirmed that BCMA is highly selectively expressed on the surface of almost all MM cell lines (80-100%), supporting its as an ideal targeting site for MM diagnosis and treatment. Currently, different categories of BCMA-targeted drugs, including ADC, BsAb, and CAR-T cell therapy, have achieved significant efficacy in relapsed / refractory MM, further improving remission rate and survival. Therefore, non-invasive evaluation of BCMA expression level in vivo is valuable for MM diagnosis, treatment guidance, and efficacy evaluation.

[0006] ImmunoPET is a newly developed specific in vivo imaging method in recent years, which combines the high sensitivity of positron emission tomography (PET / CT) and the specificity of monoclonal antibodies, and can non-invasively evaluate the expression and in vivo biodistribution of targeted markers. Our group has successfully labeled a variety of monoclonal antibodies, including daratumumab, and these imaging probes have shown significant specific accumulation in targeted tumors. However, we found that the main drawback of these immunoPET imaging based on intact monoclonal antibodies is that the peak of tumor radioactivity uptake occurs several days after injection, and the high radioactivity background in the blood after injection leads to low tumor display contrast, which is not conducive to clinical translation. Therefore, whether to image on the same day of injection and obtain better tumor / background contrast is an important challenge and urgent problem for the clinical translation of BCMA-targeted immunoPET imaging.

[0007] One of the main limitations of imaging / diagnostic agents based on intact antibodies is the long blood circulation half-life. In addition, the preparation process of antibodies is complex and costly, leading to high treatment costs. Moreover, as a biological macromolecule, antibodies have poor penetration of solid tumors and strong immunogenicity, and there are many unavoidable side effects in clinical practice. In contrast, polypeptides have better biocompatibility, not only retaining some protein functions such as targeting and selectivity, but also taking advantage of their small molecular weight to reduce immunogenicity and improve tumor penetration. The mature solid-phase synthesis technology also makes the preparation of polypeptides more simple and low-cost. Compared with antibodies, linear polypeptides are also easier to design and modify drugs.

[0008] The polypeptide specifically targeting BCMA can replace the monoclonal antibody and is effectively enriched at the tumor site in blood circulation. Through reasonable structural design, the targeting polypeptide can be combined with different imaging agents, such as nuclide molecules and the like, to realize precise imaging at the tumor site and provide the possibility for early diagnosis of tumors and dynamic monitoring of immunotherapy. Meanwhile, the targeting polypeptide can be combined with small-molecule drugs having anticancer effects to form a polypeptide conjugated drug (PDC), improve tumor permeability and provide a new idea for targeted treatment of tumors. SUMMARY

[0009] The application extracts hot amino acid sites combined by analyzing the crystal structure of the BCMA complex, performs single-point mutation, designs a peptide library with the aid of computer simulation, preliminarily screens candidate peptides by combining docking scoring and binding energy size, and then screens a series of BCMA high-affinity polypeptides by using surface plasmon resonance technology (SPRi).

[0010] The targeting BCMA polypeptide (SEQ ID No. 1 EEYCFYDPYFC) of the application is finally obtained after a large number of experimental verification screening, the polypeptide can specifically and affinely bind to BCMA protein and selectively bind to tumor cells with high expression of BCMA. The application also provides products derived from the polypeptide and capable of specifically binding to BCMA and the use of the polypeptide and the derivatives thereof in tumor treatment, diagnosis and imaging.

[0011] Based on this, the application proposes the following application contents.

[0012] In a first aspect, the application provides a polypeptide specifically targeting BCMA, and the amino acid sequence of the polypeptide is shown in SEQ ID No. 1.

[0013] The polypeptide specifically targeting BCMA has high affinity and specificity for BCMA.

[0014] The amino acid residues of the polypeptide can be L-type, D-type, mirror structure, or a mixture of L-type and D-type, and a sequence change body of the mirror structure, a cyclic peptide structure, a PEG or a fatty acid chain and the like modified change body.

[0015] The polypeptide specifically targeting BCMA can be prepared by using the Fmoc solid-phase polypeptide synthesis method.

[0016] In a second aspect, the application provides an isomer, a derivative, a mixture, a pharmaceutically acceptable salt, a hydrate or a solvate of the polypeptide.

[0017] The derivative is a divalent or multivalent body formed by the polypeptide.

[0018] The divalent or multivalent body can target BCMA.

[0019] Preferably, the divalent or multivalent body is formed by covalent or non-covalent linkage via a linker molecule, or is formed by non-covalent linkage via mixing with a multimer.

[0020] Preferably, the linker is polyethylene glycol (PEG), GSGS, or 8-amino octanoic acid.

[0021] More preferably, the covalently linked linker molecule is at least one of fluorescein isothiocyanate, 6-tert-butoxycarbonylhydrazinonicotinic acid, 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide or N-hydroxysuccinimide, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid.

[0022] More preferably, the non-covalently linked linker molecule includes but is not limited to lipophilic near-infrared dyes such as ICG, IRDye800.

[0023] More preferably, the multimer is at least one of polyethylene glycol (PEG), polyvinyl alcohol (PVA), cyclodextrin, polyamidoamine dendrimer (PAMAM), polylactic acid (PLA), polylactic acid-ethanolamine (PLGA), liposome.

[0024] In a third aspect, the present application provides a nucleic acid encoding the polypeptide.

[0025] In a fourth aspect, the present application provides a biological material comprising the polypeptide, or an isomer, derivative, mixture, pharmaceutically acceptable salt, hydrate or solvate of the polypeptide, or the nucleic acid; preferably, the biological material is a vector, expression cassette, transposon, host cell or transgenic cell line.

[0026] The vector includes but is not limited to a cloning vector, an expression vector, a plasmid vector, and all vectors containing at least one copy of the nucleic acid encoding the targeting BCMA polypeptide of the present application are within the scope of the present application.

[0027] The host cell or transgenic cell line can be a cell or cell line derived from a microorganism, a plant or an animal; a plant cell or plant cell line loses the ability to develop into a complete plant individual.

[0028] In a fifth aspect, the present application provides a medicament comprising a pharmaceutically acceptable adjuvant and at least one component selected from the polypeptide, or an isomer, derivative, mixture, pharmaceutically acceptable salt, hydrate or solvate of the polypeptide, or the nucleic acid, or the biological material.

[0029] Preferably, the active ingredient of the medicament further comprises an agent capable of killing tumor cells.

[0030] More preferably, the agent capable of killing tumor cells is at least one of a chemical drug, a biological drug, a nano-drug, a radioactive drug, a photothermal therapy drug or a photodynamic therapy drug capable of killing tumor cells; or is at least one of an alkylating agent, an antimetabolite drug, an antitumor natural drug, an antitumor antibiotic, a hormone, a metal complex or a tumor radio-targeting marker.

[0031] More preferably, the drug further comprises a carrier conjugated or mixed with the polypeptide or the derivative of the polypeptide.

[0032] The carrier includes, but is not limited to, a carrier for preparing a targeted drug.

[0033] The carrier includes at least one of a nanomaterial, a liposome, an oily compound.

[0034] In a sixth aspect, the present application provides a conjugate comprising a carrier and at least one of the following components: the polypeptide or an isomer, a derivative, a mixture, a pharmaceutically acceptable salt, a hydrate or a solvate of the polypeptide, or the nucleic acid, or the biological material.

[0035] The conjugate is obtained by covalent or non-covalent connection or action of the polypeptide or the derivative of the polypeptide and the carrier.

[0036] Preferably, the carrier is at least one of a fluorescein, an antibody, a polymer, a high molecular material, a nanomaterial, a liposome, an oily compound, an inorganic material.

[0037] Further preferably, the high molecular material is at least one of a polyester, a polyanhydride, a polyamino phospholipid polymer micelle, a polylactic acid-glycolic acid copolymer, a polyethylene glycol, chitosan.

[0038] Further preferably, the inorganic material is at least one of nano-gold, a carbon material, a calcium material, a magnetic material, a mesoporous silica material, a quantum dot.

[0039] Preferably, the carrier is any one or more of a fluorescein, an antibody, a polymer, a high molecular material, a nanomaterial, a liposome, an oily compound, an inorganic material.

[0040] In a seventh aspect, the present application provides an imaging agent comprising an imaging agent and at least one of the following components: the polypeptide or an isomer, a derivative, a mixture, a pharmaceutically acceptable salt, a hydrate or a solvate of the polypeptide, 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 agent.

[0041] Preferably, the fluorescent molecule is at least one of IRDye800CW, Cy7, Cy5.5, Rhodamine or Indocyanine Green ICG, and the radionuclide is at least one of Lu, 131 I, 177 Lu, 64 Cu, 99m Tc, 18 F or 68 Ga.

[0042] Preferably, the polypeptide, or the isomer, derivative, mixture, pharmaceutically acceptable salt, hydrate or solvate of the polypeptide, or the nucleic acid, or the biological material, or the drug, or the conjugate is coupled, conjugated or mixed with the imaging agent.

[0043] Since the polypeptide has the effect of targeting the BCMA protein, it can be coupled with a small molecule drug or a drug-loaded carrier as a homing peptide; or coupled with a variety of imaging molecules such as radionuclides to form a tumor contrast agent, providing more possibilities for tumor treatment and imaging diagnosis.

[0044] In the eighth aspect, the present application provides a reagent or kit comprising the polypeptide, or the isomer, derivative, mixture, pharmaceutically acceptable salt, hydrate or solvate of the polypeptide, or the nucleic acid, or the biological material, or the drug, or the conjugate, or the imaging agent.

[0045] Preferably, the reagent or kit is used for the diagnosis of BCMA-related diseases.

[0046] In the ninth aspect, the present application provides the use of the polypeptide, or the isomer, derivative, mixture, pharmaceutically acceptable salt, hydrate or solvate of the polypeptide, or the nucleic acid, or the biological material, or the drug, or the conjugate, or the imaging agent or the reagent or kit in at least one of the following aspects:

[0047] (1) detecting the expression level of BCMA in cells;

[0048] (2) preparing a reagent for detecting the expression level of BCMA in cells;

[0049] (3) preparing a drug product; the drug product is used for diagnosing, preventing or treating diseases with BCMA as a marker;

[0050] (4) preparing a diagnostic reagent, a diagnostic kit or an imaging agent;

[0051] (5) preparing a product for detecting the stage of diseases with BCMA as a marker or assisting in staging.

[0052] Preferably, the disease is at least one of a tumor and an autoimmune disease.

[0053] More preferably, the tumor comprises at least one of multiple myeloma (MM), diffuse large B cell lymphoma (DLBCL), lung adenocarcinoma, and lung squamous cell carcinoma.

[0054] The BCMA-specific polypeptide of the present application has extremely high affinity for BCMA, and can be used for detecting the expression level of BCMA, diagnosing a BCMA-related tumor, predicting the treatment effect of a BCMA-related tumor, or treating a BCMA-related tumor. In particular, the BCMA-specific molecular imaging probe prepared from the BCMA-specific polypeptide of the present application has the characteristics of significantly improved affinity, significantly reduced non-specific uptake of normal tissues, and significantly improved image quality, and can be used for non-invasive, precise, and efficient detection of the expression of human BCMA, and is therefore particularly suitable for diagnosing a BCMA-related tumor and predicting the treatment effect of a BCMA-related tumor. After being coupled with a suitable radionuclide, it can also be used for precise treatment of a BCMA-related tumor.

[0055] Compared with the prior art, the present application has the following beneficial effects:

[0056] (1) The polypeptide of the present application is a novel specific BCMA-targeting polypeptide reported for the first time, has high selectivity, small molecular weight, high biological safety, low immunogenicity, and high tumor penetration. The polypeptide can be synthesized by a chemical synthesis method, 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, and have strong practicability and application prospect.

[0057] (2) The polypeptide of the present application can be combined with an imaging agent, applied for clinical transformation, used as a molecular probe for detecting the expression of BCMA in tumor cells, used for real-time monitoring of the efficacy of immunotherapy, and can be used as a prediction and companion diagnostic reagent for BCMA immunotherapy. It can also be used as a homing peptide, combined with an anticancer agent to form a polypeptide conjugate drug, and used for targeted therapy and combination therapy of various tumors. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 The figure is a schematic diagram for screening the BP1 polypeptide in the present application.

[0059] Figure 2 The figure is a molecular structure formula and MS mass spectrum diagram of the BP1 in the present application.

[0060] Figure 3The application discloses a method for detecting the affinity of a BP1 polypeptide and a human BCMA protein by using a surface plasmon resonance (SPRi) method.

[0061] Figure 4 The application discloses a method for detecting the specific affinity of a 5-TAMRA-labeled BP1 polypeptide and a negative control polypeptide to a BCMA-positive cell line H929 and a negative cell line K562 by using a flow cytometry analysis method.

[0062] Figure 5 The application discloses a method for detecting the specific affinity of a 5-TAMRA-labeled BP1 polypeptide to a BCMA-positive cell line H929 and a negative cell line K562 by using a laser confocal fluorescence imaging analysis method.

[0063] Figure 6 The application discloses a method for detecting the specific affinity of a 5-TAMRA-labeled BP1 polypeptide to a BCMA-positive cell line H929 and a negative cell line K562 by using a laser confocal fluorescence imaging analysis method. 68 The application discloses a method for detecting the specific affinity of a 5-TAMRA-labeled BP1 polypeptide to a BCMA-positive cell line H929 and a negative cell line K562 by using a laser confocal fluorescence imaging analysis method.

[0064] Figure 7 The application discloses a method for detecting the specific affinity of a 5-TAMRA-labeled BP1 polypeptide to a BCMA-positive cell line H929 and a negative cell line K562 by using a laser confocal fluorescence imaging analysis method. 68 The application discloses a method for detecting the specific affinity of a 5-TAMRA-labeled BP1 polypeptide to a BCMA-positive cell line H929 and a negative cell line K562 by using a laser confocal fluorescence imaging analysis method. 68 The application discloses a method for detecting the specific affinity of a 5-TAMRA-labeled BP1 polypeptide to a BCMA-positive cell line H929 and a negative cell line K562 by using a laser confocal fluorescence imaging analysis method.

[0065] Figure 8 The application discloses a method for detecting the specific affinity of a 5-TAMRA-labeled BP1 polypeptide to a BCMA-positive cell line H929 and a negative cell line K562 by using a laser confocal fluorescence imaging analysis method. 68 The application discloses a method for detecting the specific affinity of a 5-TAMRA-labeled BP1 polypeptide to a BCMA-positive cell line H929 and a negative cell line K562 by using a laser confocal fluorescence imaging analysis method. 68 The application discloses a method for detecting the specific affinity of a 5-TAMRA-labeled BP1 polypeptide to a BCMA-positive cell line H929 and a negative cell line K562 by using a laser confocal fluorescence imaging analysis method. max The application discloses a method for detecting the specific affinity of a 5-TAMRA-labeled BP1 polypeptide to a BCMA-positive cell line H929 and a negative cell line K562 by using a laser confocal fluorescence imaging analysis method.

[0066] Figure 9 The application discloses a method for detecting the specific affinity of a 5-TAMRA-labeled BP1 polypeptide to a BCMA-positive cell line H929 and a negative cell line K562 by using a laser confocal fluorescence imaging analysis method. 68 The application discloses a method for detecting the specific affinity of a 5-TAMRA-labeled BP1 polypeptide to a BCMA-positive cell line H929 and a negative cell line K562 by using a laser confocal fluorescence imaging analysis method. DETAILED DESCRIPTION

[0067] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme of the present application will be described clearly and completely below in combination with the drawings in the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0068] Unless otherwise specified in the examples, all techniques and conditions are conventional or as described in the literature or as described in the product manual. Unless otherwise specified, the reagents and instruments used are conventional products that can be purchased from a regular channel.

[0069] Preparation method of BCMA targeting polypeptide in the examples

[0070] The polypeptide library was synthesized by standard Fmoc solid-phase synthesis method. The C-terminal carboxyl group of the target peptide library was covalently linked to the polymer resin, and then the amino group of this amino acid was used as the starting point to form a peptide bond with the carboxyl group of another amino acid. The solid-phase synthesis sequence was from the C-terminal to the N-terminal, and the above step was repeated to couple single amino acids to the solid-phase resin one by one until the target polypeptide product was obtained. After the reaction was completed, the protecting group was removed, and the peptide chain was separated from the resin by using a cleavage solution to obtain the target peptide library. Then the peptide library was incubated with BCMA protein, and positive candidate polypeptide microspheres were obtained by microchip screening, such as Figure 1 As shown in the figure, and then the positive polypeptide sequence named BP1 (SEQ ID No. 1) was obtained by searching with secondary mass spectrometry sequencing. Mass spectrometry identification was carried out after solid-phase synthesis for subsequent experiments. The structural formula of the polypeptide and the mass spectrometry results are shown in Figure 2 , which indicates the correctness of the synthesized targeting polypeptide.

[0071] Example 2: Detection of the affinity of BP1 polypeptide to human BCMA protein by surface plasmon resonance (SPRi) method

[0072] The BP1 polypeptide solution was spotted on the SPRi chip, incubated overnight under 4°C humidification conditions, washed with 10x PBS for 10 min, 1x PBS for 10 min, deionized water for 10 min, 5% skimmed milk blocking overnight, repeating the above washing steps, and then dried by nitrogen. The Plexera HT surface plasmon resonance imaging system was used for detection.

[0073] The mobile phase passed through 1x PBS, 2x PBS, 27.57 nM, 55.14 nM, 110.28 nM, 220.57 nM and 441.1 nM of BCMA protein sample in sequence, and the SPRi signal was recorded and analyzed. From Figure 3 It can be seen that the SPRi signal of BP1 gradually increases with the increase of protein concentration, and the K D value reaches 10 -7 M, which indicates that the polypeptide of the present application has strong affinity to BCMA and can meet the subsequent in vivo application.

[0074] Example 3: Preparation of 5-TAMRA labeled polypeptide

[0075] ​5-TAMRA conjugate was obtained by solid phase synthesis method, ε-amino caproic acid was coupled on the polypeptide resin synthesized by solid phase. 5-TAMRA was mixed with the peptide beads in the solution of pyridine / N,N dimethylformamide / dichloromethane ratio of 1:5:7, and reacted overnight, avoiding light. After cleavage by cleavage solution, polypeptide 5-TAMRA conjugate was obtained, and identified by MALDI-TOF and purified by HPLC for subsequent experiments.

[0076] Example 4 Flow identification of the affinity of BP1 polypeptide to BCMA protein

[0077] The positive cells selected in the experiment were human myeloma cells H929, and the negative cells were human chronic myeloid leukemia cells K562, which were cultured in RPMI 1640 medium containing 10% FBS. 20 μg / mL 5-TAMRA conjugated BP1 polypeptide and negative control polypeptide (SEQ ID No. 2: ASHESWYGNHC) were respectively incubated with H929 and K562 on ice for 20 min, washed with PBS for 3 times, and resuspended with 500 μL PBS. The fluorescence intensity determination was analyzed by FACS Calibur analyzer (BD Biosciences) and FlowJo software (Tree Star).

[0078] The results are shown in Figure 4 BP1 has obvious displacement with H929 cells and almost no displacement with K562 cells, indicating that the polypeptide BP1 has good binding force with H929 and no binding force with K562, which shows that the BP1 polypeptide has specificity and only recognizes BCMA positive cells.

[0079] Example 5 Laser confocal fluorescence imaging analysis of the affinity of BP1 polypeptide to BCMA protein

[0080] The positive cells selected in the experiment were H929, and the negative cells were K562, which were cultured in RPMI 1640 medium containing 10% FBS. The two kinds of cells were incubated with 10 ug / mL of 5-TAMRA conjugate of BP1 and negative control polypeptide respectively, and then planted on polylysine coated glass slides, cultured in a 37℃, 5% CO2 cell incubator for 30 min, fixed with 2% paraformaldehyde, and washed with PBS for 2 times. Then, the cells were stained with DAPI reagent and incubated at room temperature for 10 min. The fluorescence distribution in the cells was detected by laser scanning confocal microscope (Leica TCSSP8).

[0081] The results are shown in Figure 5As shown, H929 cell membrane has obvious 5-TAMRA red fluorescence signal, while K562 cell membrane has almost no fluorescence. The results show that the target polypeptide can effectively bind to the cell membrane of tumor cells with high expression of BCMA, and cannot bind to tumor cells with low expression. Therefore, it can be seen that the recognition of BP1 polypeptide to BCMA is specific, which is consistent with the SPR data.

[0082] Example 6 PET imaging and biodistribution analysis of polypeptide probe in tumor-bearing mouse model

[0083] All animal experiments were performed according to the protocols approved by the Animal Management and Use Committee of Peking University First Hospital. NCG severe immunodeficient mice (4-6 weeks old, female) lacking T cells, B cells and NK cells were selected for the construction of a multiple myeloma subcutaneous tumor model. The mice were randomly divided into two groups, namely the experimental group and the control group, 5 in each group. NCG immunodeficient mice are products of Jiangsu Jizhuangkang Biotechnology Co., Ltd. 100 μL of Matrigel suspension (Corning, USA) containing 1 × 10 7 H929 and K562 cells were injected subcutaneously into the right axillary of the mice. The health status and tumor volume of the mice were monitored every other day. When the tumor volume reached 1 cm 3 , it could be used for in vivo imaging and biodistribution experiments.

[0084] 0.05M high-purity hydrochloric acid was used to elute 68 Ge- 68 GaCl3 solution was obtained from a Ge-68 generator. 1 mL of Ga-68 solution was taken, 100 microliters of sodium acetate (1M) was added, the cap was covered, and the mixture was mixed. The pH was 4-4.5 measured with 0-6 precision pH paper. The molecule to be labeled was added to the prepared solution. After heating at 90°C for 10 min, elution was performed. After cooling the reaction solution, it was added to the activated Sep-Pak Light C18 column (5 mL of deionized water, 5 mL of ethanol, 5 mL of deionized water, activated according to water-ethanol-water). Impurities were eluted with 3.0 mL of pure water and discarded. A 0.22 μm sterile microporous filter was added, and the product was collected in a sterile vacuum bottle with 0.5 mL of ethanol solution. 5.0 mL of normal saline was added to the system and was ready for use.

[0085] 68The Ga-DOTA-BP1 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.

[0086] The results are as follows Figure 6 As shown, the difference in retention times of the radiochromatographic peaks in the formulation is no greater than 0.5 min. 68 The Ga-DOTA-BP1 peptide-labeled product has a radiochemical purity of nearly 99.9%.

[0087] The tumor volume of the tumor-bearing mouse is about 1 cm. 3 At that time, 11.1 MBq was injected into the tail vein. 68 Ga-DOTA-BP1 68 Ga-DOTA-negative control peptide probe.

[0088] After injection, a micro-PET / CT scanner (Super PET / CT scanner) was used. A one-hour dynamic PET scan was performed in China. Regions of interest (ROIs) were drawn using Avatar 1.0 software and quantitative analysis was performed to obtain the radioactivity concentration in tumors, cardiac blood pools (blood), liver, kidneys, bladder, and muscles at different time points in vivo. The content of the radioactive probe was expressed as the percentage of radioactivity count per gram of tissue relative to the total injected radioactivity count (%ID / g), representing the radioactivity uptake.

[0089] The results are as follows Figure 7 As shown, the maximum intensity projection (MIP) results indicate that in the BCMA-positive H929 tumor model, 68 Ten minutes after Ga-DOTA-BP1 injection, significant radioactivity concentration was observed in the tumor, with high uptake remaining from 10 to 60 minutes. However, in the K562 tumor model negative for BCMA expression, no significant radioactivity concentration was observed. Furthermore, 68Ga-labeled negative control polypeptide also showed no significant uptake in H929 tumor model. By using the corresponding software to draw the region of interest (ROI) and conduct quantitative analysis, the radioactive concentration of tumor at different time points in vivo was obtained. The results are shown in Figure 8 Fig. 6, at each time point from 10 min to 60 min, 68 Ga-DOTA-BP1 showed significantly higher uptake in H929 tumor than in K562 tumor. Compared with 68 Ga-DOTA-negative control polypeptide, 68 Ga-DOTA-BP1 also showed significantly higher uptake in H929 tumor. After injection of 68 Ga-DOTA-BP1 and 68 Ga-DOTA-negative control polypeptide for 20 min, the tumor, blood, heart, liver, spleen, lung, kidney, stomach, small intestine, bladder, muscle, long bone of lower leg, brain and tail were dissected, weighed, and the sample radioactivity was counted using an automatic gamma counter. The uptake of radioactive tracer in different organs / tissues was calculated and the biodistribution was calculated and expressed as %ID / g (mean ± SD). The results of biodistribution are shown in Figure 9 Fig. 6, the uptake of BP1 probe in H929 tumor (3.48 + 0.68 %ID g -1 ) was higher than that in K562 tumor (1.51 + 0.26 %ID g -1 ) and also higher than the uptake of negative polypeptide in H929 tumor (0.98 + 0.5 %ID g -1 , P < 0.05). This is consistent with the results of PET imaging.

[0090] The above results demonstrate that the polypeptide small molecule probe of the present application has rapid targeting of BCMA and good tumor penetration ability, and can realize high sensitivity in vivo imaging of microtumor.

[0091] In summary, the polypeptide of the present application has the property of targeting BCMA-positive tumor cells, and thus in practical application, the polypeptide of the present application can be used as a homing peptide, combined with an anticancer drug or an imaging agent, for targeted treatment and imaging of tumors.

[0092] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A polypeptide targeting BCMA, characterized in that, The amino acid sequence of which is shown in SEQ ID No.

1.

2. A pharmaceutically acceptable salt or solvate of the polypeptide of claim 1.

3. The solvate according to claim 2, characterized in that, The solvate is a hydrate.

4. A nucleic acid encoding the polypeptide of claim 1.

5. A biomaterial, characterized by, The nucleic acid of claim 4.

6. The biomaterial of claim 5, wherein, The biological material is a vector, an expression cassette, a transposon or a host cell.

7. The biomaterial of claim 5, wherein, The biological material is a transgenic cell line.

8. A medicament, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The pharmaceutical composition comprises a pharmaceutically acceptable excipient and at least one component selected from the group consisting of the polypeptide of claim 1, or a pharmaceutically acceptable salt or solvate of the polypeptide of claim 2, or the nucleic acid of claim 4, or the biological material of claim 5.

9. An imaging agent, characterized by, The imaging agent is at least one of a radionuclide, a radionuclide label, a fluorescent molecule or a magnetic resonance contrast agent. The imaging agent is a molecular imaging agent.

10. The imaging agent of claim 9, wherein The pharmaceutical composition comprises the polypeptide of claim 1, or a pharmaceutically acceptable salt or solvate of the polypeptide of claim 2, or the nucleic acid of claim 4, or the biological material of claim 5, or the pharmaceutical of claim 8, or the imaging agent of claim 9 or 10.

11. A reagent or kit characterized in that, 12. Use of the polypeptide of claim 1, or a pharmaceutically acceptable salt or solvate of the polypeptide of claim 2, or the nucleic acid of claim 4, or the biological material of claim 5, or the pharmaceutical of claim 8, or the imaging agent of claim 9 or 10, or the reagent or kit of claim 11 in at least one of the following aspects: (1) preparing a reagent for detecting the expression level of BCMA in a cell; (2) preparing a pharmaceutical product; the pharmaceutical product is used for diagnosing a disease with BCMA as a marker; the disease is multiple myeloma; (3) preparing a diagnostic reagent, a diagnostic kit or an imaging agent; the diagnostic reagent, the diagnostic kit or the imaging agent is used for diagnosing multiple myeloma; (4) preparing a product for detecting the staging or assisting the staging of a disease with BCMA as a marker; the disease is multiple myeloma. ​

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