A bispecific probe-linker complex and its preparation method and use

Abstract

The present invention discloses a bispecific probe-linker complex and a preparation method and use thereof, wherein the bispecific probe-linker complex comprises a linker, a first probe covalently connected to the linker, and a second probe covalently connected to the linker; the first probe can specifically bind to nucleolin protein, and the second probe can specifically bind to CTLA-4. The bispecific probe-linker complex can bind to tumor cells containing nucleolin protein on the surface through the first probe and simultaneously bind to immune cells with CTLA-4 through the second probe, thereby promoting immunotherapy, and has the characteristics of low immunogenicity, easy synthesis, low side toxicity and high efficiency.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a bispecific probe-linker complex and its preparation and use. Background Art

[0002] Cancer is the second leading cause of death in the world. Malignant tumor cells have the characteristics of unlimited growth, high invasiveness and easy metastasis. Surgery, radiotherapy and chemotherapy can kill tumor cells, but without physical and immune regulation of the patient's body, it cannot change the living environment of tumor cells and prevent the continuous generation of new tumor cells, so cancer will easily recur and metastasize.

[0003] Immunotherapy, which uses the human immune system to attack tumor cells, is one of the latest breakthroughs in the treatment of various cancers. Cytotoxic T lymphocytes play an important role in the immunotherapy of cancer. By using the major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells for precise antigen presentation, T cells can specifically recognize antigens on the surface of tumor cells and be activated, thereby achieving precise killing of tumor cells. In order to avoid the complex antigen presentation process and make full use of the anti-tumor properties of T cells, adoptive T cell immunotherapy has been developed. Among them, CAR-T therapy has good clinical therapeutic effects by targeting cancer cells with high specificity and activating T cells. However, current adoptive T cell immunotherapy requires customized individualized treatment for different patients. The cell engineering involved is complex, time-consuming and expensive, and may also cause severe cytokine release syndrome (CRS) and neurotoxicity, hindering its clinical application. In addition, tumor cells have acquired a variety of mechanisms to evade the immune surveillance of the human immune system and reduce the release of T cell toxic factors through evolution, resulting in patients failing to induce sufficient immune responses even after receiving adoptive T cell immunotherapy or even recurring after cure.

[0004] Bispecific antibodies (BsAb), also known as bifunctional antibodies, have two antigen-binding arms, one of which binds to the target antigen and the other binds to the labeled antigen on the effector cell, which can activate the effector cell. As a result, BsAb causes a direct interaction between the target cell and the effector cell, enabling it to specifically kill tumor cells. Bispecific antibodies with clear targeting do not exist under natural conditions, but are prepared by cell fusion or recombinant DNA technology. At present, there are 7 bispecific antibody drugs on the market worldwide, and a large number of drugs are still in clinical phase II / III studies. A large number of studies have shown that bispecific antibodies can be used to redirect T cells to the periphery of the tumor, and the immune escape ability of tumor cells can be weakened by combining special antibodies, which can induce an immune response in the patient's body with high specificity and efficiency, thereby accurately killing tumor cells. Thanks to the efforts of countless researchers, the structural forms of the "bispecific antibody family" have reached more than 100. Although their molecular structures are different, almost all of them can be divided into two major categories: fragment-based BsAbs and Fc-based BsAbs. Fragment-based BsAbs are composed of two or more antibody fragments, and mainly exert corresponding effector mechanisms through the characteristics of antigen binding. They have the characteristics of small molecular weight, strong permeability, low immunogenicity, short half-life, and faster clearance, and may have more safety advantages in terms of adverse reactions. Fc-based BsAbs have homomeric or heterodimeric Fc domains, which can exert Fc-mediated effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADCP). This type of bispecific antibody generally has a large molecular weight, is easy to purify, has strong solubility and stability, and has a relatively longer serum half-life. However, the preparation process of bispecific antibodies is cumbersome and complicated, and it is difficult to purify. The heavy chain and light chain must be assembled correctly, and their special spatial conformation must be maintained during the preparation process. The production cost is high, and many patients cannot afford the expensive drug costs. What is more serious is that according to clinical application reports, bispecific antibodies have caused cytokine release syndrome and neurotoxicity in some patients, seriously affecting the therapeutic effect of the drug on patients and their health.

[0005] Therefore, there is an urgent need for a tumor treatment drug that can promote immunotherapy, has low immunogenicity, low side effects and is highly effective. Summary of the invention

[0006] The first aspect of the present invention provides a bispecific probe-linker complex that can promote immunotherapy, has low immunogenicity, is easy to synthesize, has low side toxicity and high efficiency, and includes a linker, a first probe covalently linked to the linker, and a second probe covalently linked to the linker; the first probe can specifically bind to nucleolin protein, and the second probe can specifically bind to CTLA-4.

[0007] Furthermore, the first probe is a nucleic acid, and its sequence is Seq_1.

[0008] Furthermore, the second probe is a nucleic acid, and its sequence is Seq_2.

[0009] Furthermore, the linker includes a first linker connected to the first probe, a second linker connected to the second probe, and a third linker connected to both the first linker and the second linker.

[0010] Furthermore, the first linker is cholesterol, distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine or a derivative thereof; and / or, the second linker is cholesterol, distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine or a derivative thereof; and / or, the third linker is a liposome nanoparticle or an extracellular vesicle.

[0011] The second aspect of the present invention provides a method for preparing a bispecific probe-linker complex: S1. covalently linking a first linker to a first probe to prepare a first probe-first linker complex; S2. covalently linking a second linker to a second probe to prepare a second probe-second linker complex; S3. mixing the first probe-first linker complex, the second probe-second linker complex, and a third linker to prepare the probe-linker complex; wherein the first probe can specifically bind to nucleolin protein, and the second probe can specifically bind to CTLA-4.

[0012] Furthermore, the first probe is a nucleic acid whose sequence is Seq_1; and / or, the second probe is a nucleic acid whose sequence is Seq_2; and / or, the first linker is cholesterol, distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine or a derivative thereof; and / or, the second linker is cholesterol, distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine or a derivative thereof; and / or, the third linker is a liposome nanoparticle or an extracellular vesicle.

[0013] Furthermore, the -5' end of the first probe and the second probe are both modified with a first specific group; the first linker and the second linker are both cholesterol, and the cholesterol is also modified with a second specific group; the first probe or the second probe is respectively connected to the cholesterol by forming a covalent bond through a chemical reaction; the first specific group and the second specific group undergo at least one of the following reactions (a) to (g):

[0014] (a) Substitution reaction of NHS ester group and amino group;

[0015] (b) Click chemistry reaction between azide group and diphenylcyclooctyne group;

[0016] (c) Click chemistry reaction of azide group and cyclooctyne group;

[0017] (d) Click chemistry reaction of azide and alkynyl groups;

[0018] (e) addition reaction of maleimido group and furan group;

[0019] (f) SN2 reaction of phosphorothioate groups and chloroacetyl groups;

[0020] (g) Condensation reaction of amino groups and carboxyl groups.

[0021] Furthermore, the first probe is a nucleic acid whose sequence is Seq_1, and one end of the first probe is modified with NH2; the second probe is a nucleic acid whose sequence is Seq_2, and one end of the second probe is modified with NH2; the first linker and the second linker are both cholesterol modified with N-hydroxysuccinimide; the third linker is a liposome nanoparticle; in S1, the feed ratio of the first probe to the first linker is a molar ratio of 1:(1~100); in S2, the feed ratio of the second probe to the second linker is a molar ratio of 1:(1~100); in S3, the feed ratio of the first probe-first linker complex, the second probe-second linker complex, and the third linker is 2.1nmol:0.7nmol:27.2mg.

[0022] Furthermore, the cholesterol is modified with PEG2000-NHS ester group (i.e., cholesterol-PEG2000-NHS). In this configuration, the amino group modified at the -5' end of the first probe and the second probe undergoes a substitution reaction of the NHS ester group and the amino group with cholesterol-PEG2000-NHS in a buffer solution, the first linker and cholesterol, the second linker and cholesterol are covalently linked, the NHS ester-activated cross-linker and the labeled compound react with the primary amine under physiological to weakly alkaline conditions (pH 7.2 to 9) to form a stable amide bond, and the reaction releases N-hydroxysuccinimide (NHS).

[0023] Furthermore, the method for preparing the bispecific probe-linker complex comprises the following steps:

[0024] S1: Dissolve 2.1 nmol of the first probe in 400 ul of buffer; Take 400 ul of the first probe solution, denature at 95°C for 5 min, and renature at 0°C; Mix the first probe and the first connector at a molar ratio of 1:(1-100), and react at room temperature for 6-10 hours to obtain the first probe-first connector complex;

[0025] S2: Dissolve 0.7 nmol of the second probe in 400 ul of buffer. Take all the second probe solution, denature at 95°C for 5 min, and renature at 0°C; mix the second probe and the second connector at a molar ratio of 1: (1-100), react at room temperature for 6-10 hours, and obtain the second probe-second connector complex;

[0026] S3: 16 mg of phospholipid, 8 mg of cholesterol components, and 3.2 mg of functional phospholipid were dissolved in 6 mL of organic solvent, preheated at 50°C for 5 minutes, and the first probe-first connector complex and the second probe-second connector complex were added to the organic solvent to obtain probe and connector mother solutions; the third connector mother solution was mixed with 6 ml of buffer, and heated at 65°C for 1 to 4 hours; ultrasonicated for 3 to 10 minutes to obtain the probe-connector complex;

[0027] Furthermore, in the steps S1 and S2, the buffer used includes, but is not limited to, enzyme-free water or enzyme-free PBS.

[0028] Furthermore, the buffer used is an enzyme-free phosphate buffer (1×PBS), which is mainly composed of sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate, and does not contain DNA / RNA enzymes, Ca 2+ Mg 2+ , pH value is 7.2-7.4, sterilized by high temperature and high pressure.

[0029] Furthermore, in step S3, the organic solvent used includes, but is not limited to, methanol, ethanol, chloroform or a mixture of two or more of these organic solvents.

[0030] Furthermore, the organic solvent used is anhydrous ethanol.

[0031] Furthermore, the first probe-first linker complex and the second probe-second linker complex prepared in steps S1 and S2 are subjected to ultrafiltration, dialysis or other purification operations to remove unreacted linkers and byproducts. This arrangement is performed to avoid affecting the subsequent synthesis and experiments.

[0032] Furthermore, the probe-linker complex prepared in step S3 is subjected to ultrafiltration, dialysis or other purification operations to remove residual organic solvent and reactants, and purify the product bispecific probe-linker complex.

[0033] Preferably, the bispecific probe-linker complex finally prepared is a nanoscale liposome with an average particle size of about 95 nm.

[0034] The third aspect of the present invention provides a use of the above-mentioned bispecific probe-linker complex or the bispecific probe-linker complex prepared by the above-mentioned preparation method, wherein the bispecific probe-linker complex can bind to tumor cells containing nucleolin protein on the surface and immune cells containing CTLA-4 on the surface to form a tumor cell-first probe-linker-second probe-immune cell complex.

[0035] Furthermore, the tumor cells include A549, HepG2, and MCF-7.

[0036] Furthermore, the tumor cells may cause cancer; the cancer may be lung cancer, breast cancer, liver cancer, glioma, prostate cancer, colorectal cancer, pancreatic cancer, ovarian cancer, cervical cancer, rhabdomyosarcoma, neuroblastoma, multiple myeloma, leukemia, acute lymphoblastic leukemia, melanoma, bladder cancer, gastric cancer, head and neck cancer, skin cancer, lymphoma or glioblastoma.

[0037] A fourth aspect of the present invention provides an imaging agent for tumor cell imaging.

[0038] The fifth aspect of the present invention provides a diagnostic reagent for diagnosing cancer, which can be used for early detection, monitoring of treatment effects, and post-prognosis diagnosis of the above-mentioned cancers. The method includes making the test cell specifically contact with the bispecific probe-linker complex, and after sufficient contact, detecting the cell with a detection instrument. The bispecific probe-linker complex is labeled with a fluorescent group. The instrument includes, but is not limited to, a flow cytometer, a qPCR instrument, and an enzyme-linked immunosorbent assay instrument.

[0039] The sixth aspect of the present invention provides a kit comprising the above-mentioned bispecific probe-linker complex.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The above-mentioned bispecific probe-linker complex comprises a linker, a first probe and a second probe simultaneously connected to the linker. The first probe can specifically target the nucleolin protein highly expressed on the surface of cancer cells, and the second probe can specifically target the CTLA-4 highly expressed on the surface of T cells. The bispecific probe-linker complex can specifically aggregate around the tumor and promote the redirection of T cells to tumor cells, promote immunotherapy, and has the characteristics of low immunogenicity, easy synthesis, low side toxicity and high efficiency.

[0042] Nucleolin protein is a multifunctional protein. Its main function is to participate in the biological formation of ribosomes. It can also participate in various processes such as rRNA processing and mRNA stabilization. Recent studies have found that it also plays an important role in the occurrence and development of tumors. Nucleolin is overexpressed on the membranes and cytoplasm of a variety of actively proliferating cancer cells, but is not expressed on the surface of normal cells. Nucleolin, which is highly expressed in malignant tumors, directly or indirectly participates in signal transduction with different mechanisms, affecting the survival, proliferation, metastasis of tumor cells and promoting tumor progression. This has been proven in many tumors such as glioma, neuroblastoma, liver cancer, breast cancer and lung cancer. The high level of expression of nucleolin in a variety of malignant tumors has attracted great attention. Many studies have shown that inhibiting nucleolin can resist the growth of tumor cells. At the same time, nucleolin can bind to gametes with high binding affinity. These are the theoretical basis for nucleolin targeted therapy.

[0043] AS1411 is the earliest discovered and tested nucleolin aptamer. It is a modified guanine nucleoside (G)-rich oligonucleotide (its sequence is Seq_1), which can bind to nucleolin on the cell surface and interfere with nucleolin-related regulatory mechanisms to a certain extent, blocking the proliferation of tumor cells and downregulating tumorigenic genes. ASl411, as a targeting molecule, has significant advantages such as high affinity, strong specificity, and small relative molecular mass, and is an ideal targeted anti-tumor drug carrier.

[0044] Cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152, is an inhibitory surface receptor expressed on activated T cells and regulatory T cells (Treg cells). When CTLA-4 is expressed on activated T cells, this inhibitory effect is intracellular, while when CTLA-4 is expressed on immunosuppressive Treg cells, this inhibitory effect is extracellular. CTLA-4 is involved in regulating the early stages of T cell activation. It binds to co-stimulatory molecules CD80 and CD86 (B7-1 and B7-2) expressed on antigen-presenting cells. At the same time, CD80 and CD86 also bind to the T cell surface receptor CD28, which can promote the mRNA expression of cytokine IL-2, and promote T cell survival and differentiation and immunoglobulin isotope conversion. However, compared with CD28, CTLA-4 has a higher binding affinity for CD80 and CD86. Therefore, the extracellular region signal mediated by CTLA-4 has the effect of inhibiting and shutting down T cell-dependent immune responses. By blocking the CTLA-4 signaling pathway and releasing B7 to interact with the co-stimulatory molecule CD28, the sensitivity of immune cells to tumor cells can be increased and immunity to secondary tumor attacks can be induced. According to research reports, CTLA-4 checkpoint inhibitors have shown long-term and lasting responsiveness and tolerable safety in some clinical trials, which can cause tumor regression. In addition, immune checkpoint inhibitor therapy is combined with other treatment methods, such as other immunosuppressants, radiotherapy, cytotoxic chemotherapy or targeted therapy, to enhance the effect of immunotherapy. Studies have found that CTLA-4 nucleic acid aptamers (whose sequence is Seq_2) have biological functions, bind to CTLA-4 with high affinity, and enhance T cell activity, which can be used in the development of therapeutic drugs.

[0045] The bispecific probe-linker complex proposed in the present invention may contain the above two nucleic acid aptamers and also have the functions of two nucleic acid aptamers, that is, it can specifically bind to nucleolin and CTLA-4 at the same time, thereby releasing B7 to interact with the co-stimulatory molecule CD28, and regulate the immune response, increase the sensitivity of immune cells to tumors, induce immunity to secondary tumor attacks, promote tumor regression, and produce the following net effects:

[0046] 1. Since nucleolin is highly expressed on the surface of cancer cells but not on the surface of normal cells, the complex has the ability to specifically target tumor cells;

[0047] 2. CTLA-4 is highly expressed on the surface of activated T cells and regulatory T cells, so the complex can specifically target T cells and regulatory T cells, and promote T cell activation through CTLA-4 immune checkpoint blockade, and even awaken dormant T cells, thereby promoting the effect of immunotherapy;

[0048] 3. The bispecific probe-linker complex can promote the redirection of T cells to the periphery of the tumor, mediate tumor cell-T cell interaction, and enable tumor cells to directly stimulate T cells by generating immune synapses between the two cells. At the same time, it is beneficial for B7 to interact with the co-stimulatory molecule CD28, thereby enhancing the sensitivity of T cells to tumor cell antigen stimulation, leading to increased T cell immune activation and promoting T cells to kill tumor cells.

[0049] 4. The bispecific probe-linker complex is a nanoscale particle with high permeability and retention effect (EPR effect) of solid tumors. In addition to active targeting capability, it also has the ability to passively target solid tumors, which is beneficial for drugs to converge from the blood circulation system to the surrounding of solid tumors, specifically increase the drug concentration in the tumor microenvironment, improve efficacy, and reduce drug side effects.

[0050] In the present invention, the first probe and the second probe modified with cholesterol at the 5' end anchor cholesterol to the liposome lipid bilayer, resulting in each liposome surface containing multiple first probes and second probes. Therefore, the probability of the bispecific probe-linker complex binding to the target is greatly increased, and the binding force to the target-containing cells is also enhanced, thereby improving the ability of the bispecific probe-linker complex to bind to T cells and redirect T cells.

[0051] The present invention can be used to treat individual diseases or conditions. The bispecific probe-linker complex can enhance immunotherapy by improving the quality and intensity of the immune response, such as the immune response mediated by tumor cells. The bispecific probe-linker complex can be used to guide immune cells expressing CTLA-4 extracellularly to cancer cells expressing nucleolin extracellularly, redirecting the immune response to the periphery of tumor cells. It can be simply by administering the bispecific probe-linker complex or adjusting the dosage or administration time of the probe-linker complex over time. The probe-linker complex can also be used to adjust the intensity of the immune response, or turn on the response, or turn off the response.

[0052] The experimental results show that the bispecific probe-linker complex proposed in the present invention has the advantages of high efficiency, low toxicity, high specificity, good immunostimulating effect and easy synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The particle size diagram of AS1411×CTLA-4-liposome complex.

[0054] Figure 2 Agarose gel electrophoresis of AS1411×CTLA-4-liposome complex.

[0055] FIG3 shows the binding of AS1411×CTLA-4-liposome complexes to cells expressing and not expressing the respective target of interest.

[0056] Figure 4 It was shown that AS1411×CTLA-4-liposome complexes can promote the redirection of immune cells expressing CTLA-4 protein to tumor cells.

[0057] Figure 5 The results showed that the immunotherapeutic effect of AS1411×CTLA-4-liposome complex on A549 tumor cells was concentration-dependent.

[0058] Figure 6 The results showed that the immunotherapeutic effect of AS1411×CTLA-4-liposome complex on MCF-7 tumor cells was concentration-dependent.

[0059] Figure 7 The results showed that the immunotherapeutic effect of AS1411×CTLA-4-liposome complex on HepG2 tumor cells was concentration-dependent.

[0060] Figure 8 It was shown that AS1411×CTLA-4-liposome complexes could promote immunotherapy of HepG2 tumor cells.

[0061] Fig. 9 The cytotoxicity of AS1411×CTLA-4-liposome complexes was shown.

[0062] Fig.10 Shown is a gel electrophoresis image of a bispecific probe-linker complex in which the third linker is an extracellular vesicle.

[0063] Fig.11 It was shown that the bispecific probe-linker complex with the third linker being an extracellular vesicle has a concentration-dependent immunotherapeutic effect on A549 tumor cells. DETAILED DESCRIPTION

[0064] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0065] As used herein, the term "bispecific" refers to having two different antigen binding sites, thereby binding to two target antigens at the same time, mediating another special function while exerting drug targeting, such as one binding to specific antigens on target cells and the other binding to effector cells such as lymphocytes or macrophages, thereby stimulating the advantage of autoimmunity in the process of tumor diagnosis and treatment.

[0066] As used herein, the term "aptamer" refers to any linear or sequentially arranged nucleotides and nucleosides, for example, cDNA, genomic DNA, mRNA, tRNA, oligonucleotides, oligonucleosides and derivatives thereof, which can bind to target molecules with high selectivity and high affinity. Aptamers have a unique three-dimensional structure that tends to form spirals and single-stranded loops, contributing to the selectivity of the target. Aptamers can include modified or derived nucleotides and nucleosides, such as, but not limited to, halogenated nucleotides, such as, but not limited to, 5-bromouracil, and derived nucleotides such as biotin-labeled nucleotides.

[0067] As used herein, the term "Qiaolian body" refers to a substance that can covalently or non-covalently connect an external nucleic acid aptamer to its surface, expose the nucleic acid aptamer to the outside world without affecting the function of the external nucleic acid aptamer. It can comprehensively regulate the distribution of drugs in the body in terms of space, time and dosage. Its goal is to deliver the right amount of different types of nucleic acid molecules to the right location at the right time, thereby increasing the utilization efficiency of nucleic acid molecules, improving efficacy, reducing costs, and reducing toxic side effects. Including but not limited to the following substances: liposomes, extracellular vesicles, cell membrane nanoparticles, polymer nanoparticles, serum albumin nanoparticles and a group consisting of their combination.

[0068] As used herein, the term "liposome" refers to bilayer nanoparticles composed of phospholipids, cholesterols and functional phospholipids. The phospholipids are selected from one or both of soybean lecithin and egg yolk lecithin. The cholesterol components are selected from one or more of cholesterol and its derivatives, cholestane, bile acid and bile acid. The functional phospholipids are selected from one or more of distearoylphosphatidylethanolamine-polyethylene glycol, distearoylphosphatidylethanolamine-polyethylene glycol, distearoylphosphatidylethanolamine-polyethylene glycol and distearoylphosphatidylethanolamine-polyethylene glycol.

[0069] As used herein, the term "probe" refers to a chemical substance that can specifically recognize or label a target molecule and is suitable for direct detection. Probes have the characteristics of strong specificity and high sensitivity, and play a huge role in biomacromolecule research, drug analysis, forensic identification, clinical disease diagnosis and treatment, etc.

[0070] As used herein, the term "linker" refers to a substance that can connect the nucleic acid aptamer and the carrier through chemical or physical action, including but not limited to the following substances: cholesterol, distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, hydrogenated soybean phosphatidylcholine, or modified substances and derivatives thereof.

[0071] As used in this article, the term "extracellular vesicle" is a small membrane vesicle released by cells into the extracellular matrix. It participates in processes such as cell communication, cell migration, angiogenesis and tumor cell growth. It is widely present in various body fluids and cell supernatants and stably carries some important signaling molecules. Extracellular vesicles are assembled from a complex mixture of various lipids, surface and membrane proteins, some of which contribute to tissue targeting, while others ensure minimal nonspecific interactions and can be used as drug carriers.

[0072] As used herein, the term "binding affinity" refers to the strength of the interaction between two or more structures that are capable of reversibly binding to each other, such as a ligand and a receptor. The overall binding affinity of a multivalent ligand depends on its individual binding affinities, the valence states of the ligand and receptor, and the spatial arrangement of the interacting structures. However, the total binding affinity of a multimeric construct is not just the average of the individual binding affinities of the individual aptamers. Factors that affect the affinity of a multimeric structure include the stereochemical fit between the ligand and the receptor, the size of the contact area between them, and the distribution of charged and hydrophobic groups.

[0073] As used herein, the term "target" refers to a structural unit that is bound to an aptamer, typically a component of a biological entity. The target can be a molecule, such as a protein, peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, substrate, metabolite, drug, nutrient or growth factor, etc. The target can be a part of a cell or a cell, such as a T cell, NK cell, monocyte, B cell, tumor cell or a cell that has been pathologically altered (e.g., infected by a virus or other microorganism, or altered due to genetic rearrangement). The target can also be an organ or organelle.

[0074] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0075] Experimental Example 1: Preparation of bispecific probe-linker complex (AS1411×CTLA-4-liposome complex)

[0076] 1. Preparation of the first probe-linker complex (Chelosterol-PEG2000-AS1411)

[0077] S1: Dissolve 2.1 nmol of AS1411 aptamer (sequence Seq_1) in 400ul DEPC (diethylpyrocarbonate) water. Denature 400ul AS1411 aptamer solution at 95℃ for 5min and renature at 0℃. Mix AS1411 aptamer: active ester (NHS-PEG2000-Cholesterol) at a molar ratio of 1:10 and react at room temperature for 10h. Remove unreacted nucleic acid, NHS-PEG2000-Cholesterol and byproducts to obtain the first probe-linker complex, namely Chelosterol-PEG2000-AS1411.

[0078] Among them Seq_1: 5'-TTGGTGGTGGTGGTTGTGGTGGTGGTGG-3.

[0079] 2. Preparation of the Second Probe-Linker Complex (Chelosterol-PEG2000-CTLA-4)

[0080] S1: Dissolve 0.7 nmol of CTLA-4 aptamer (sequence Seq_2) in 400ul DEPC water. Denature 400ul CTLA-4 aptamer solution at 95℃ for 5min and renature at 0℃. Mix CTLA-4 aptamer: active ester (NHS-PEG2000-Cholesterol) at a molar ratio of 1:10 and react at room temperature for 10h. Remove unreacted nucleic acid, NHS-PEG2000-Cholesterol and by-products to obtain the second probe-linker complex, namely Chelosterol-PEG2000-CTLA-4.

[0081] Among them, Seq_2: 5'-TCCCTACGGCGCTAACGATGGTGAAAATGGGCCTAGGGTGGACGGTGCCACCGTGCTACAAC-3'.

[0082] 3. Preparation of bispecific probe-linker complex (AS1411×CTLA-4-liposome complex)

[0083] Prepare liposomes using the dropwise method. Take 16 mg of hydrogenated soybean lecithin, 8 mg of cholesterol, and 3.2 mg of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 and dissolve them in 6 mL of anhydrous ethanol, and preheat at 50°C for 5 minutes; add Chelosterol-PEG2000-AS1411 solution and Chelosterol-PEG2000-CTLA-4 solution to the preheated liposome mother solution to obtain a liposome mother solution containing AS1411 aptamer and CTLA-4 aptamer. Mix 6 ml of enzyme-free PBS with the above liposome mother solution, mix well at 65°C, and heat for 2.5 hours. Ultrasonicate the final solution for 3 minutes and purify it to obtain a bispecific probe-linker complex, which is the AS1411×CTLA-4-liposome complex. The particle size of the synthesized AS1411×CTLA-4-liposome complex was measured using a particle size analyzer, such as Figure 1 As shown, the average particle size of the AS1411×CTLA-4-liposome complex is 95 nm, the particle size range is concentrated, and the uniformity is good.

[0084] Example 2: Gel imaging of bispecific probe-linker complex

[0085] Add 6× loading buffer to the sample at a volume ratio of 1:5, mix well, and run electrophoresis for 40 min at 110 V. Figure 2 As shown in the figure, the total amount of nucleic acid aptamers in the AS1411 and CTLA-4 mixture group and the AS1411×CTLA-4-liposome complex group was consistent. The band brightness of the free nucleic acid aptamer in the AS1411×CTLA-4-liposome complex group in gel imaging was much lower than that in the AS1411 and CTLA-4 mixture group, indicating that a large number of nucleic acid aptamers in the AS1411×CTLA-4-liposome complex group had been loaded onto the liposome surface and the synthesis was successful.

[0086] Example 3: Detection of the targeting ability of the bispecific probe-linker complex to cell surface antigens

[0087] AS1411 nucleic acid aptamers can specifically bind to nucleolin protein, which is highly expressed on the surface of many cancer cells, such as lung adenocarcinoma cells (A549), breast cancer cells (MCF7), liver cancer cells (HepG2), glioma cells (U118, U251, U87), cervical cancer cells (HeLa), melanoma cells (B16), etc. CTLA-4 nucleic acid aptamers can specifically bind to cytotoxic T lymphocyte-associated antigen (CTLA-4), which is highly expressed in activated T cells and also in Jurkat cells. In this experiment, A549 was selected as the cancer cell targeted by AS1411 nucleic acid aptamers, and Jurkat cells were selected as the effector cells targeted by CTLA-4 nucleic acid aptamers.

[0088] The nucleic acid aptamer was labeled with CY5, and then the CY5-labeled nucleic acid-liposome complex was synthesized. 5 Jurkat cells were incubated with AS1411×CTLA-4-liposomes at 4°C for 0.5 h; as controls, 2×10 5 Jurkat cells were incubated in binding buffer or Con-liposome complex (nucleic acid-liposome prepared with CTLA-4 nucleic acid aptamer control sequence) at 4°C for 0.5 h. 5 A549 cells were incubated with AS1411×CTLA-4-liposome complexes at 4°C for 0.5 h; as a control, 2×10 5 A549 cells were incubated in binding buffer solution or CTLA-4-liposome complex at 4°C for 0.5 h. After the incubation, the binding of materials to cells in each group was detected by flow cytometry.

[0089] The results of the study on the combination of materials and cells are shown in Figure 3. Figure 3A The AS1411×CTLA-4-liposome complex has a strong binding affinity to A549 cells, and the displacement of the CTLA-4-liposome complex is four orders of magnitude less than that of the AS1411×CTLA-4-liposome complex on A549 cells according to the flow cytometry results. Figure 3B The AS1411×CTLA-4-liposome complex has a strong binding affinity to Jurkat cells, and the flow cytometry results of the Con-liposome complex on Jurkat cells are two orders of magnitude less than those of the AS1411×CTLA-4-liposome complex. This proves that only the AS1411×CTLA-4-liposome complex can simultaneously target cancer cells expressing nucleolin on the surface and immune cells expressing CTLA-4 on the surface.

[0090] Example 4. T cell redirection ability of the bispecific probe-linker complex

[0091] A549 cells and Jurkat cells were stained with MitoSpy Red and CFSE, respectively; after staining, the cells were washed twice with cold binding buffer. Jurkat cells were incubated with AS1411×CTLA-4-liposome complex (800nM), or the control group PBS group, AS1411 liposome and CTLA-4-liposome complex mixture group, in 1640 complete medium at 4°C for 30 minutes. After the incubation, centrifugation was performed at 1200rpm for 5min to remove unbound materials, and then mixed with A549 cells in 1640 complete medium, and then the cell mixture was added to a 96-well plate and incubated at 4°C for 5 hours. Then the cells were gently washed twice with PBS, fixed with 1% (W / V) paraformaldehyde, 100uL paraformaldehyde solution per well, incubated at room temperature for 15min, aspirated, washed twice with PBS, and then 100ul 1640 complete medium was added to each well, and the cells were imaged using high content. The results are as follows Figure 4 As shown, in the presence of AS1411×CTLA-4-liposome complexes, Jurkat cells were redirected to the periphery of A549 cells, and the number of tightly attached cells increased significantly, indicating that the AS1411×CTLA-4-liposome complexes have the ability to redirect T cells.

[0092] Example 5: In vitro toxicity detection of bispecific probe-linker complex

[0093] 5.1 AS1411×CTLA-4-liposome complex has a concentration-dependent immunotherapeutic effect on A549 cells:

[0094] PBMC cells were extracted from peripheral blood by Ficoll density gradient centrifugation, cultured in RPMI-1640 medium containing 10% FBS and 1% double antibody, and activated with CD3 and HIL-2 cytokines.

[0095] 15×10 3 10 activated PBMC cells were incubated with 0, 0.6uM, 2.4uM, 3.6uM, and 5uM AS1411×CTLA-4-liposome complexes at 4°C for 30min, and then 5×10 3A549 cells were mixed with the above PBMC cells, and the cell mixture was inoculated in a 96-well plate. The 96-well plate after the cells were inoculated was placed in a 5% CO2, 37°C incubator for co-culture for 40 hours. After the culture was completed, the cells were washed twice with PBS to remove the suspended PBMC cells, and then 10% CCK8 solution was added for detection. After incubation in a 5% CO2, 37°C incubator for 1 to 2 hours, the absorbance was measured at 450nm using an enzyme-linked immunosorbent assay. The results are as follows: Figure 5 As shown, the numbers indicate that the immunotherapeutic effect of AS1411×CTLA-4-liposome complex on A549 tumor cells is concentration-dependent. When the concentration of AS1411×CTLA-4-liposome complex reaches 5uM, the activity of A549 cells is 59.1%, that is, the immunotherapeutic effect reaches 40.9%.

[0096] 5.2 AS1411×CTLA-4-liposome complex has a concentration-dependent immunotherapeutic effect on MCF-7 cells:

[0097] The difference from Example 5.1 is that A549 cells were replaced with MCF-7 cells, the concentrations of AS1411×CTLA-4-liposome complexes were 0uM, 0.1uM, 1uM, 10uM, 100uM, 500uM, and 1000uM, respectively, and the rest of the process was the same as Example 5.1. Figure 6 As shown, the quantity indicates that the immunotherapeutic effect of AS1411×CTLA-4-liposome complex on MCF-7 tumor cells is concentration-dependent. When the concentration of AS1411×CTLA-4-liposome complex reaches 1000nM, the MCF-7 cell activity is 72.42%, that is, the immunotherapeutic effect reaches 27.58%.

[0098] 5.3 AS1411×CTLA-4-liposome complex has a concentration-dependent immunotherapeutic effect on HepG2 cells:

[0099] The difference from Example 5.1 is that A549 cells were replaced with HepG2 cells, the concentrations of AS1411×CTLA-4-liposome complexes were 0 nM, 1 nM, 10 nM, 100 nM, 500 nM, and 1000 nM, respectively, and the rest of the process was the same as Example 5.1. Figure 7 As shown, the quantity indicates that the immunotherapeutic effect of AS1411×CTLA-4-liposome complex on HepG2 tumor cells is concentration-dependent. When the concentration of AS1411×CTLA-4-liposome complex reaches 1000nM, the HepG2 cell activity is 58.1%, that is, the immunotherapeutic effect reaches 41.9%.

[0100] Example 6. Detection of the immunotherapy-promoting effect of the bispecific probe-linker complex

[0101] 1.5×10 4 Activated PBMC cells were incubated with 300 nM AS1411×CTLA-4-liposome complex, AS1411-liposome complex, CTLA-4-liposome complex and PBS at 4°C for 30 min, and then 5×10 3 HepG2 cells were mixed with the above PBMC cells, and the cell mixture was inoculated in a 96-well plate. The 96-well plate after the cells were inoculated was placed in a 5% CO2, 37°C incubator for co-culture for 40 hours. After the culture was completed, the cells were washed twice with PBS to remove the suspended PBMC cells, and then 10% CCK8 solution was added for detection. After incubation in a 5% CO2, 37°C incubator for 1 to 2 hours, the absorbance was measured at 450nm using an enzyme-linked immunosorbent assay. The results are as follows: Figure 8 As shown, under the action of 300nM AS1411×CTLA-4-liposome complex, the activated PBMC cells had the greatest killing effect on HepG2 cells, with only 68.24% of the cells still alive; while in the AS1411-liposome complex, 82.40% of the cells were still alive, and in the CTLA-4-liposome complex, 98.94% of the cells were still alive.

[0102] Example 7. Cytotoxicity Detection of Bispecific Probe-Linker Complex

[0103] In order to verify the safety of AS1411×CTLA-4-liposome complex, this experiment will verify the toxicity of AS1411×CTLA-4-liposome complex to cells with high or no nucleolin expression on the surface in the absence of T cells. IOSE is a normal human ovarian epithelial cell that does not express nucleolin protein on its surface. This experiment selected this cell as a negative control cell.

[0104] 8~10×10 3 A549, MCF-7, HepG2 and IOSE (normal human ovarian epithelial cells) were seeded in 96-well plates. After attachment, PBS and 500nM AS1411×CTLA-4-liposome complex were added to each cell group. The 96-well plate was then placed in a 5% CO2, 37°C incubator for 45 hours. After the culture was completed, the cells were washed twice with PBS to remove the suspended PBMC cells, and then 10% CCK8 solution was added for detection. After incubation in a 5% CO2, 37°C incubator for 1 hour, the absorbance was measured at 450nm using an enzyme-linked immunosorbent assay. The results are as follows Fig. 9As shown, the activity of A549 cells was 97.1%, the activity of MCF-7 cells was 106.5%, the activity of HepG2 cells was 95.6%, and the activity of IOSE cells was 100.5%, that is, the AS1411×CTLA-4-liposome complex had no obvious toxicity to the activity of cells at a concentration of 500 nM.

[0105] Example 8: The third linker is a bispecific probe-linker complex of an extracellular vesicle

[0106] 8.1 Preparation of bispecific probe-linker complexes for extracellular vesicles

[0107] The first probe-linker complex (Chelosterol-PEG2000-AS1411) prepared in Example 1 and the second probe-linker complex (Chelosterol-PEG2000-CTLA-4) prepared in Example 1 were mixed with the extracellular vesicles and reacted at 60° C. for 1.5 h.

[0108] The results are as follows Fig.10 As shown in the figure, the corresponding aptamer amounts in the AS1411 group, CTLA-4 group, and AS1411×CTLA-4-EV complex group were consistent. The band brightness of the free aptamer in the AS1411×CTLA-4-EV complex group was much lower than that in the CTLA-4 group in gel imaging, and there were obvious bands in the sample wells of the AS1411×CTLA-4-EV complex group, indicating that a large number of aptamers in the AS1411×CTLA-4-EV complex group had been loaded onto the surface of EVs and synthesized successfully.

[0109] 8.2 The bispecific probe-linker complex of extracellular vesicles has a concentration-dependent immunotherapeutic effect on A549 cells:

[0110] The difference from Example 5.1 is that the concentrations of the bispecific probe-linker complex of the extracellular vesicles are 0 nM, 50 nM, 300 nM, 600 nM, 900 nM, and 1200 nM, respectively, and the rest of the process is the same as Example 5.1. Fig.11 As shown, the quantities indicate that the immunotherapeutic effect of AS1411×CTLA-4-extracellular vesicle complex on MCF-7 tumor cells is concentration-dependent. When the concentration of AS1411×CTLA-4-extracellular vesicle complex reaches 1200nM, the A549 cell activity is 47.2% and the immunotherapeutic effect reaches 52.8%.

[0111] In summary: the bispecific probe-linker complex proposed in the present invention can bind to tumor cells containing nucleolin protein on the surface through the first probe and simultaneously bind to immune cells with CTLA-4 through the second probe, and has the advantages of high efficiency, low toxicity, high specificity, good immunogenic efficacy and easy synthesis.

[0112] Although the present invention has been illustrated and described with specific embodiments, it will be appreciated that many other changes and modifications may be made without departing from the spirit and scope of the present invention. Therefore, it is intended to include all such changes and modifications within the scope of the present invention in the appended claims.

Claims

1. A bispecific probe-linker complex comprising a linker, a first probe covalently linked to the linker, and a second probe covalently linked to the linker; characterized in that: The first probe can specifically bind to nucleolin protein, and the second probe can specifically bind to CTLA-4; The first probe is a nucleic acid, and its sequence is Seq_1, and the second probe is a nucleic acid, and its sequence is Seq_2; The linker includes a first linker connected to the first probe, a second linker connected to the second probe, and a third linker connected to both the first linker and the second linker.

2. The probe-linker complex according to claim 1, wherein the first linker is cholesterol, distearoylphosphatidylethanolamine or dioleoylphosphatidylethanolamine; and / or, the second linker is cholesterol, distearoylphosphatidylethanolamine or dioleoylphosphatidylethanolamine; And / or, the third linker is a liposome nanoparticle or an extracellular vesicle.

3. A method for preparing a bispecific probe-linker complex, characterized in that: S1. covalently linking the first connector to the first probe to prepare a first probe-first connector complex; S2. covalently linking the second connector to the second probe to prepare a second probe-second connector complex; S3. mixing the first probe-first linker complex, the second probe-second linker complex, and the third linker to react, thereby preparing the probe-linker complex; in, The first probe can specifically bind to nucleolin protein, the first probe is a nucleic acid, and its sequence is Seq_1; The second probe can specifically bind to CTLA-4, and the second probe is a nucleic acid, and its sequence is Seq_2.

4. The method for preparing a bispecific probe-linker complex according to claim 3, characterized in that: The first linker is cholesterol, distearoylphosphatidylethanolamine or dioleoylphosphatidylethanolamine; and / or, the second linker is cholesterol, distearoylphosphatidylethanolamine or dioleoylphosphatidylethanolamine; And / or, the third linker is a liposome nanoparticle or an extracellular vesicle.

5. The method for preparing a bispecific probe-linker complex according to claim 3, characterized in that: One end of the first probe is modified with NH2; one end of the second probe is modified with NH2; The first linker and the second linker are both cholesterol modified with N-hydroxysuccinimide; The third linker is a liposome nanoparticle; In S1, the feeding ratio of the first probe to the first connector is a molar ratio of 1: (1-100); In S2, the feeding ratio of the second probe to the second connector is a molar ratio of 1:(1-100); In S3, the feed ratio of the first probe-first linker complex, the second probe-second linker complex, and the third linker is 2.1 nmol: 0.7 nmol: 27.2 mg.

6. Use of the bispecific probe-linker complex according to any one of claims 1-2 or the bispecific probe-linker complex prepared according to the method for preparing a bispecific probe-linker complex according to any one of claims 3-5 in preparing a product for forming a tumor cell-first probe-linker-second probe-immune cell complex, wherein the bispecific probe-linker complex can bind to tumor cells containing nucleolin protein on the surface and immune cells containing CTLA-4 on the surface to form a tumor cell-first probe-linker-second probe-immune cell complex, and the tumor cells are A549, HepG2 or MCF-7.

Images