CN116270702B

CN116270702B - A bispecific probe-linker complex and its preparation method and use

Info

Publication number: CN116270702B
Application number: CN202211313466.8A
Authority: CN
Inventors: 王珊; 任欢欢; 周高雅; 柴志勇; 成昱昊; 周恒雨; 黄婧; 吕品
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2022-10-25
Filing date: 2022-10-25
Publication date: 2025-02-14
Anticipated expiration: 2042-10-25
Also published as: CN116270702A

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

Bispecific probe-connector complex and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicine, and particularly relates to a bispecific probe-connector compound, and preparation and application thereof.

Background

Cancer is the second leading cause of death worldwide, and malignant cells have unlimited growth, high wettability, and susceptibility to metastasis. The operation, the radiotherapy and the chemotherapy can kill the tumor cells, but the living environment of the tumor cells can not be changed and the generation of new tumor cells can be prevented under the condition that the physical condition and the immune condition of a patient body are not regulated, and the cancer is easy to relapse and transfer.

Immunotherapy, which utilizes the human immune system to attack tumor cells, is one of the latest breakthroughs for the treatment of a variety of cancers. Cytotoxic T lymphocytes play an important role in the immunotherapy of cancer. By utilizing Major Histocompatibility Complex (MHC) molecules on the surface of antigen presenting cells for accurate antigen presentation, T cells can specifically recognize antigens on the surface of tumor cells and are activated, thereby realizing accurate killing of the tumor cells. Adoptive T cell immunotherapy was developed in order to avoid complex antigen presentation processes and make full use of the anti-tumor properties of T cells. Among them, CAR-T therapy has a good clinical therapeutic effect by targeting cancer cells with high specificity and activating T cells. However, current adoptive T cell immunotherapy requires custom-tailored personalized treatments for different patients, the involved cellular engineering is complex, time consuming and costly, and may also lead to severe Cytokine Release Syndrome (CRS) and neurotoxicity, impeding its clinical application. In addition, tumor cells have evolved to gain access to a variety of mechanisms that evade immune surveillance of the human immune system and reduce T-cell cytotoxic factor release, resulting in patients failing to elicit a sufficient immune response even after cure even when treated with adoptive T-cell immunotherapy.

Bispecific antibodies (bsabs), also known as bifunctional antibodies, have two antigen-binding arms, one of which binds to a target antigen and the other to a labeled antigen on effector cells, which can activate effector cells. Thus, bsAb causes direct interaction of target cells with effector cells, causing them to specifically kill tumor cells. Bispecific antibodies with defined targeting do not exist under natural conditions but are made by cell fusion or recombinant DNA technology. Currently, 7 bispecific antibody drugs are marketed worldwide, and a large number of drugs remain in clinical phase II/III studies. A large number of researches prove that the bispecific antibody can be utilized to redirect T cells to the periphery of tumor, and the immune escape capacity of the tumor cells can be weakened by matching with the specific antibody, so that the immune response in a patient can be caused with high specificity and high efficiency, and the tumor cells can be killed accurately. In the effort of numerous researchers, the structural form of the "bispecific antibody family" has been as much as 100. Although their molecular structures are different, almost all molecular structures can be divided into two major classes, fragment-based BsAbs and Fc-based BsAbs. The BsAbs based on the fragments consists of two or more antibody fragments, plays a corresponding effect mechanism mainly through the characteristic of antigen binding, has the characteristics of small molecular weight, strong permeability, lower immunogenicity, shorter half-life and higher clearance speed, and possibly has more safety advantages in the aspect of adverse reaction. The Fc-based BsAbs have a homodimeric Fc domain and 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), such bispecific antibodies generally have a relatively large molecular weight, are easy to purify, have high solubility and stability, and have a relatively long serum half-life. However, the preparation process of bispecific antibodies is complicated, difficult to purify, the heavy and light chains must be assembled correctly, and the specific spatial conformation must be maintained during the preparation process, which is costly to produce and difficult for many patients to afford expensive pharmaceutical costs. More seriously, according to clinical application reports, bispecific antibodies cause cytokine release syndrome and neurotoxicity in some patients, and seriously affect the therapeutic effect of the drug on patients and the health of patients.

Therefore, there is an urgent need for a tumor therapeutic agent that can promote immunotherapy, has low immunogenicity, has low side toxicity, and is highly effective.

Disclosure of Invention

In a first aspect, the invention provides a bispecific probe-linker complex that facilitates immunotherapy, has low immunogenicity, is easy to synthesize, has low side toxicity, and is efficient, comprising a linker, a first probe covalently linked to the linker, and a second probe covalently linked to the linker, wherein the first probe specifically binds nucleolin protein and the second probe specifically binds CTLA-4.

Further, the first probe is a nucleic acid, and the sequence of the first probe is seq_1.

Further, the second probe is a nucleic acid, and the sequence of the second probe is seq_2.

Further, 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.

Still further, the first linker is cholesterol, distearoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, or a derivative thereof, and/or the second linker is cholesterol, distearoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, or a derivative thereof, and/or the third linker is a liposome nanoparticle or an extracellular vesicle.

The second aspect of the invention provides a preparation method of a bispecific probe-connector complex, which comprises the steps of S1, covalently connecting a first connector with a first probe to prepare a first probe-first connector complex, S2, covalently connecting a second connector with a second probe to prepare a second probe-second connector complex, S3, mixing and reacting the first probe-first connector complex, the second probe-second connector complex and a third connector to prepare the probe-connector complex, wherein the first probe can specifically bind nucleolin protein, and the second probe can specifically bind CTLA-4.

Still further, the first probe is a nucleic acid having a sequence of seq_1, and/or the second probe is a nucleic acid having a sequence of seq_2, and/or the first linker is cholesterol, distearoyl phosphatidyl ethanolamine, dioleoyl phosphatidyl ethanolamine or a derivative thereof, and/or the second linker is cholesterol, distearoyl phosphatidyl ethanolamine, dioleoyl phosphatidyl ethanolamine or a derivative thereof, and/or the third linker is a liposome nanoparticle or an extracellular vesicle.

Further, the-5' ends of the first probe and the second probe are respectively modified with a first specific group, the first connector and the second connector are cholesterol, the cholesterol also modifies a second specific group, the first probe or the second probe is respectively connected with the cholesterol through covalent bonds formed by chemical reaction, and the first specific group and the second specific group are at least reacted in any one of the following (a) to (g):

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

(b) Click chemistry of azido and diphenylcyclooctyne groups;

(c) Click chemistry of azido and cyclooctynyl groups;

(d) Click chemistry of azido and alkynyl groups;

(e) An addition reaction of a maleimide group and a furan group;

(f) SN2 reaction of phosphorothioate groups with chloroacetyl groups;

(g) Condensation of amino groups with carboxyl groups.

Further, the first probe is nucleic acid, the sequence of the first probe is seq_1, one end of the first probe is modified by NH2, the second probe is nucleic acid, the sequence of the second probe is seq_2, one end of the second probe is modified by NH2, the first connector and the second connector are cholesterol modified by N-hydroxysuccinimide, the third connector is liposome nano particles, the feeding ratio of the first probe to the first connector is 1 (1-100) in the S1, the feeding ratio of the second probe to the second connector is 1 (1-100) in the S2, and the feeding ratio of the first probe to the second connector is 2.1nmol:0.7nmol:27.2mg in the S3.

Further, the cholesterol is modified with PEG2000-NHS ester groups (i.e., cholesterol-PEG 2000-NHS). So arranged, the-5' modified amino group of the first probe and the second probe react with cholesterol-PEG 2000-NHS in buffer solution to generate substitution reaction of NHS ester group and amino group, the first connector is covalently connected with cholesterol, the second connector is covalently connected with cholesterol, the NHS ester activated cross-linking agent and the labeled compound react with primary amine under physiological to weak alkaline conditions (pH 7.2 to 9) to form stable amide bond, and N-hydroxysuccinimide (NHS) is released by the reaction.

Further, the preparation method of the bispecific probe-linker complex comprises the following steps:

S1, dissolving 2.1nmol of a first probe in 400ul of buffer solution, taking 400ul of the first probe solution, carrying out denaturation at 95 ℃ for 5min, and carrying out renaturation at 0 ℃, mixing the first probe and a first connector according to a molar ratio of 1 (1-100), and reacting at room temperature for 6-10 h to obtain a first probe-first connector compound;

S2, dissolving 0.7nmol of the second probe in 400ul of buffer. Mixing the second probe and the second connector according to a molar ratio of 1 (1-100) and reacting for 6-10 hours at room temperature to obtain a second probe-second connector compound;

S3, dissolving 16mg of phospholipid, 8mg of cholesterol components and 3.2mg of functional phospholipid in 6mL of organic solvent, preheating for 5 minutes at 50 ℃, adding the first probe-first connector complex and the second probe-second connector complex into the organic solvent to obtain a probe and connector mother solution, uniformly mixing the third connector mother solution and 6mL of buffer solution, heating for 1-4 hours at 65 ℃, and carrying out ultrasonic treatment for 3-10 minutes to obtain the probe-connector complex;

further, in the steps S1 and S2, buffers used include, but are not limited to, enzyme-free water or enzyme-free PBS.

Furthermore, the buffer solution is enzyme-free Phosphate buffer (1 XPBS) mainly composed of sodium chloride, potassium chloride, disodium hydrogen Phosphate and potassium dihydrogen Phosphate, and does not contain DNA/RNase and Ca ²⁺、Mg²⁺, and the pH value is 7.2-7.4, and the buffer solution is sterilized at high temperature and high pressure.

Further, in the step S3, the organic solvent used includes, but is not limited to, methanol, ethanol, chloroform or a mixture of two or more organic solvents thereof.

Further, the organic solvent used is absolute ethanol.

Further, 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 procedures to remove unreacted linkers and byproducts. So arranged as to avoid affecting the subsequent synthesis and experimentation.

Further, the probe-linker complex obtained in step S3 is subjected to ultrafiltration, dialysis or other purification operations to remove residual organic solvents and reactants, and the product bispecific probe-linker complex is purified.

Preferably, the resulting bispecific probe-linker complex is a nano-sized liposome having an average particle size of about 95nm.

In a third aspect, the present invention provides the use of a bispecific probe-linker complex as described above or a bispecific probe-linker complex prepared by the above method of preparation, which can bind to tumor cells comprising nucleolin protein on the surface, immune cells comprising CTLA-4 on the surface, to form a tumor cell-first probe-linker-second probe-immune cell complex.

Still further, the tumor cells include A549, hepG2, MCF-7.

Still further, the tumor cells may cause cancer, which 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.

In a fourth aspect, the invention provides an imaging agent for tumor cell imaging.

In a fifth aspect, the present invention provides a diagnostic reagent for diagnosing cancer, which can perform early detection, treatment effect monitoring, and post-prognosis diagnosis for the above cancer. The method comprises the steps of specifically contacting a tested cell with a bispecific probe-connector complex, and detecting the cell by a detection instrument after the cell is fully contacted. The bispecific probe-linker complexes are labeled with a fluorophore. Such instruments include, but are not limited to, flow cytometry, qPCR, enzyme-linked immunosorbent assay.

In a sixth aspect the invention provides a kit comprising a bispecific probe-linker complex as described above.

Compared with the prior art, the invention has the beneficial effects that:

The above-described bispecific probe-linker complex comprises a linker, a first probe and a second probe simultaneously linked to the linker. Wherein the first probe can specifically target nucleolin protein with high expression on the surface of cancer cells, and the second probe can specifically target CTLA-4 with high expression on the surface of T cells. The dual-specificity probe-connector complex can specifically gather around a tumor and promote T cells to be redirected to the tumor cells, so that the dual-specificity probe-connector complex can promote immunotherapy and has the characteristics of low immunogenicity, easy synthesis, low side toxicity and high efficiency.

Nucleolin protein is a multifunctional protein, and has the main functions of participating in the formation of ribosomes and simultaneously participating in various processes such as the processing of rRNA and the stabilization of mRNA. Recent studies have found that it also plays an important role in the development of tumorigenesis. Nucleolin is overexpressed on the cell membrane and in the cytoplasm of many cancer cells that proliferate actively, but is not expressed on the normal cell surface. The nucleolin highly expressed in malignant tumor directly or indirectly participates in signal transduction, has different mechanisms, influences the survival, proliferation and metastasis of tumor cells and promotes the progress of tumor, and is proved in various tumors such as glioma, neuroblastoma, liver cancer, breast cancer, lung cancer and the like. High levels of nucleolin expression in a variety of malignancies have attracted great attention. Many studies have shown that inhibition of nucleolin is able to counteract the growth of tumor cells, while nucleolin can bind to gametes of high binding affinity, which are the theoretical basis for nucleolin targeted therapies.

AS1411, which was the nucleolin aptamer discovered and tested the earliest, is a modified guanosine (G) -rich oligonucleotide (sequence of seq_1) that binds to nucleolin on the cell surface and interferes to some extent with nucleolin-related regulatory mechanisms, blocking proliferation of tumor cells and down-regulating tumorigenic genes. ASl411 is used as a targeting molecule, has the remarkable advantages of high affinity, strong specificity, small relative molecular mass and the like, and is an ideal targeting antitumor drug carrier.

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), which is intracellular when CTLA-4 is expressed on activated T cells, and extracellular when CTLA-4 is expressed on immunosuppressive Treg cells. CTLA-4 is involved in regulating the early stages of T cell activation and it binds to costimulatory 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 through CD28 promotes mRNA expression of the cytokine IL-2 and promotes T cell survival and differentiation and immunoglobulin isotope conversion. However, CTLA-4 has a higher binding affinity for CD80 and CD86 than CD28, and therefore, CTLA-4-mediated extracellular domain signaling has the effect of suppressing and shutting down T cell-dependent immune responses. By blocking CTLA-4 signaling pathway, B7 release interacts with co-stimulatory molecule CD28, increasing immune cell sensitivity to tumor cells and inducing immunity to secondary tumor challenge. CTLA-4 checkpoint inhibitors have been reported to show long-lasting reactivity and tolerable safety in some clinical trials, enabling tumor regression. In addition, immune checkpoint inhibitor therapy is used in combination with other therapeutic modalities, such as other immunosuppressants, radiation therapy, cytotoxic chemotherapy or targeted therapy, etc., to enhance the efficacy of the immunotherapy. The research shows that the CTLA-4 aptamer (the sequence of which is seq_2) has biological functions, binds with CTLA-4 with high affinity, enhances the activity of T cells, and can be applied to the development of therapeutic drugs.

The bispecific probe-connector complex provided by the invention can contain the two nucleic acid aptamers, has the functions of the two nucleic acid aptamers, can specifically bind nucleolin and CTLA-4 at the same time, thereby releasing the interaction between B7 and a co-stimulatory molecule CD28, regulating immune response, increasing the sensitivity of immune cells to tumors and inducing immunity to secondary tumor attack, promoting tumor regression, and can generate the following net effects:

1. Because nucleolin is highly expressed on the surface of cancer cells, but not on the surface of normal cells, the complex has the capability of specifically targeting tumor cells;

2. CTLA-4 is highly expressed on the surfaces of activated T cells and regulatory T cells, so that the complex can specifically target the T cells and the regulatory T cells, promote T cell activation, even wake-up sleeping T cells through blocking of CTLA-4 immune check points, and promote the immunotherapeutic effect;

3. The dual-specificity probe-connector complex can promote the redirection of T cells to the periphery of tumors, mediate the interaction of the T cells and the tumor cells, and enable the tumor cells to directly stimulate the T cells by generating immune synapses between the two cells, simultaneously facilitate the interaction of B7 and co-stimulatory molecules CD28, enhance the sensitivity of the T cells to the stimulation of tumor cell antigens, lead to the increase of the immune activation of the T cells and promote the T cells to kill the tumor cells.

4. The bispecific probe-connector compound is nano-sized particles, has high permeability and retention effect (EPR effect) of solid tumors, has the capability of passively targeting the solid tumors besides the active targeting capability, is beneficial to converging drugs from a blood circulation system to the periphery of the solid tumors, and specifically improves the drug concentration in the tumor microenvironment, improves the curative effect and reduces the side effects of the drugs.

According to the invention, the first probe and the second probe with the 5' -end modified with cholesterol are anchored to the liposome lipid bilayer, so that each liposome surface contains a plurality of first probes and second probes, the probability of binding the bispecific probe-connector complex to a target is greatly increased, the binding force to cells containing the target is also enhanced, and the capacity of binding and redirecting the bispecific probe-connector complex to T cells is improved.

The invention may be used to treat a disease or condition in an individual. The bispecific probe-linker complexes can enhance immunotherapy, such as against tumor cell mediated immune responses, by improving the quality and strength of the immune response. Bispecific probe-linker complexes can be used to direct immune cells that express CTLA-4 exogenously to cancer cells that express nucleolin exogenously, redirecting immune responses around tumor cells. The dosage or time of administration of the probe-linker complex can be adjusted simply by administering the bispecific probe-linker complex or over time. The probe-linker complexes may also be used to modulate the intensity of an immune response, or to turn the response on, or to turn the response off.

Experimental results show that the bispecific probe-connector complex provided by the invention has the advantages of high efficiency, low toxicity, high specificity, good immunity-promoting effect, easiness in synthesis and the like.

Drawings

FIG. 1 is a graph showing the particle size of AS1411 XCTLA-4-liposome complex.

FIG. 2 is an agarose gel electrophoresis of AS1411 XCTLA-4-liposome complex.

FIG. 3 shows binding of AS1411 XCTLA-4-liposome complex to cells expressing and not expressing the respective target targets.

FIG. 4 shows that AS1411 XCTLA-4-liposome complex can promote redirection of immune cells expressing CTLA-4 protein to tumor cells.

FIG. 5 shows that AS1411 XCTLA-4-liposome complex is concentration dependent on the pro-immunotherapeutic effect of A549 tumor cells.

FIG. 6 shows that AS1411 XCTLA-4-liposome complex is concentration dependent on the pro-immunotherapeutic effect of MCF-7 tumor cells.

FIG. 7 shows that AS1411 XCTLA-4-liposome complex is concentration dependent on the pro-immunotherapeutic effect of HepG2 tumor cells.

FIG. 8 shows that AS1411 XCTLA-4-liposome complex can promote immunotherapy of HepG2 tumor cells.

FIG. 9 shows cytotoxicity of AS1411 XCTLA-4-liposome complex.

FIG. 10 shows a gel electrophoresis of a bispecific probe-linker complex in which the third linker is an extracellular vesicle.

Figure 11 shows that the bispecific probe-linker complexes with the third linker being an extracellular vesicle are concentration dependent for the pro-immunotherapeutic effect of a549 tumor cells.

Detailed Description

In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto.

The term "bispecific" as used herein refers to the simultaneous possession of two different antigen binding sites, thereby binding to two target antigens simultaneously, while exerting drug targeting, mediating another specific function, such as one binding to a specific antigen on a target cell and the other binding to a lymphocyte or phagocyte equivalent cell, thereby eliciting autoimmune advantages during tumor diagnosis and treatment.

The term "aptamer" as used herein refers to any linear or sequential arrangement of nucleotides and nucleosides, e.g., cdnas, genomes DNA, mRNA, tRNA, oligonucleotides and derivatives thereof, that can bind to a target molecule with high selectivity and high affinity. The aptamer has a unique three-dimensional structure such that it tends to form helices and single stranded loops, contributing to the selectivity of the target. Nucleic acid aptamers may include modified or derivatized nucleotides and nucleosides, such as, but not limited to, halogenated nucleotides, such as, but not limited to, 5-bromouracil, and derivatized nucleotides, such as biotin-labeled nucleotides.

The term "fumet" as used herein refers to a substance that is capable of covalently or non-covalently attaching a foreign aptamer to its surface, exposing the aptamer to the outside without affecting the function of the foreign aptamer. The medicine can comprehensively regulate and control the distribution of the medicine in the organism in space, time and dosage. The target is to deliver a proper amount of different types of nucleic acid molecules to the correct positions at the right time, thereby increasing the utilization efficiency of the nucleic acid molecules, improving the curative effect, reducing the cost and reducing the toxic and side effects. Including but not limited to, liposomes, extracellular vesicles, cell membrane nanoparticles, polymer nanoparticles, serum albumin nanoparticles, and combinations thereof.

The term "liposome" as used herein refers to bilayer nanoparticles composed of phospholipids, cholesterol, and functional phospholipids. Wherein the phospholipid is selected from one or two of soybean lecithin and egg yolk lecithin. The cholesterol component is selected from one or more of cholesterol and its derivatives, cholestane, cholic acid and bile acid. The functional phospholipid is selected from one or more of distearyl phosphatidylethanolamine-polyethylene glycol, distearyl phosphatidylethanolamine-polyethylene glycol and distearyl phosphatidylethanolamine-polyethylene glycol.

The term "probe" as used herein refers to a chemical substance that specifically recognizes or labels a target molecule and is suitable for direct detection. The probe has the characteristics of strong specificity and high sensitivity, and plays a great role in biomacromolecule research, drug analysis, judicial identification, clinical disease diagnosis and treatment and the like.

The term "linker" as used herein refers to a substance that can link the aptamer and the carrier together by chemical or physical action. Including but not limited to cholesterol, distearoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, hydrogenated soybean phosphatidylcholine, or modified and derivatives thereof.

The term "extracellular vesicle" as used herein is a membranous vesicle released from cells into the extracellular matrix, which is involved in cellular communication, cell migration, angiogenesis, and tumor cell growth, 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 non-specific interactions and are useful as drug carriers.

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

The term "target" as used herein refers to a structural unit, typically a component of a biological entity, that binds to an aptamer. The target may be a molecule, such as a protein, peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, substrate, metabolite, drug, nutrient, or growth factor, or the like. The target may be a portion 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 with a virus or other microorganism, or altered due to genetic rearrangement). The target may also be an organ or an organelle.

Unless defined otherwise, all technical and scientific terms used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the present invention.

Experimental example one preparation of bispecific Probe-linker Complex (AS 1411 XCTLA-4-Liposome Complex)

1. Preparation of first Probe-linker Complex (Chelosterol-PEG 2000-AS 1411)

S1 2.1nmol of AS1411 aptamer (sequence seq_1) was dissolved in 400ul DEPC (diethyl pyrocarbonate) water. 400ul of AS1411 aptamer solution was denatured at 95℃for 5min and renatured at 0 ℃. The AS1411 aptamer and active ester (NHS-PEG 2000-Cholesterol) were mixed in a molar ratio of 1:10 and reacted at room temperature for 10 hours. Removing unreacted nucleic acid, NHS-PEG2000-Cholesterol and byproducts to obtain a first probe-linker complex, namely Chelosterol-PEG2000-AS1411.

Wherein seq_1:5' -TTGGTGGTGGTGGTTGTGGTGGTGGTGG-3.

2. Preparation of the second Probe-linker Complex (Chelosterol-PEG 2000-CTLA-4)

S1 0.7nmol of CTLA-4 aptamer (sequence seq_2) was dissolved in 400ul of DEPC water. 400ul of CTLA-4 aptamer solution was denatured at 95℃for 5min and renatured at 0 ℃. Mixing CTLA-4 aptamer and active ester (NHS-PEG 2000-Cholesterol) according to a molar ratio of 1:10, and reacting for 10 hours at room temperature. Removing unreacted nucleic acid, NHS-PEG2000-Cholesterol and byproducts to obtain a second probe-linker complex, namely Chelosterol-PEG2000-CTLA-4.

Wherein, seq_2:5'-TCCCTACGGCGCTAACGATGGTGAAAATGGGCCTAGGGTGGACGGTGCCACCGTGCTACAAC-3'.

3. Preparation of bispecific Probe-linker complexes (AS 1411 XCTLA-4-Liposome complexes)

The liposome is prepared by dripping method. 16mg of hydrogenated soybean phospholipid, 8mg of cholesterol and 3.2mg of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 are taken and dissolved in 6mL of absolute ethyl alcohol, the mixture is preheated for 5 minutes at 50 ℃, chelosterol-PEG2000-AS1411 solution and Chelosterol-PEG2000-CTLA-4 solution are added into preheated liposome mother liquor, and liposome mother liquor containing AS1411 aptamer and CTLA-4 aptamer is obtained. 6ml of enzyme-free PBS was mixed with the above liposome stock solution, and the mixture was heated at 65℃for 2.5 hours. And (3) carrying out ultrasonic treatment on the finally obtained solution for 3 minutes, and purifying to obtain the bispecific probe-connector complex, namely the AS 1411X CTLA-4-liposome complex. The particle size of the synthesized AS1411 XCTLA-4-liposome complex was measured by a particle size analyzer, and AS shown in FIG. 1, the average particle size of the AS1411 XCTLA-4-liposome complex was 95nm, and the particle size range was concentrated and the uniformity was good.

Example two, bispecific Probe-linker Complex gel imaging

Adding 6 times of loading buffer solution (loading buffer) into the sample according to the volume ratio of 1:5, uniformly mixing, and then carrying out electrophoresis for 40min at 110V voltage. As shown in FIG. 2, the total aptamer amounts in the AS1411 and CTLA-4 mixture and AS1411 XCTLA-4-liposome complex were consistent. The free aptamer in the AS1411 XCTLA-4-liposome complex is far lower in band brightness than the AS1411 and CTLA-4 mixture in gel imaging, which indicates that a large amount of aptamer in the AS1411 XCTLA-4-liposome complex is loaded on the surface of the liposome, and the synthesis is successful.

Example three, detection of the ability of bispecific Probe-connector complexes to target cell surface antigens

The AS1411 aptamer can specifically bind to nucleolin protein, and nucleolin protein is highly expressed on the surfaces of various cancer cells, such AS lung adenocarcinoma cells (A549), breast cancer cells (MCF 7), liver cancer cells (HepG 2), glioma cells (U118, U251 and U87), cervical cancer cells (HeLa), melanoma cells (B16) and the like. CTLA-4 nucleic acid aptamer can specifically bind to cytotoxic T lymphocyte-associated antigen (CTLA-4), and the cytotoxic T lymphocyte-associated antigen is highly expressed in activated T cells and also expressed in Jurkat cells. In this experiment, a549 was selected AS the cancer cell targeted by the AS1411 aptamer, and Jurkat cells were selected AS the effector cell targeted by the CTLA-4 aptamer.

The nucleic acid aptamer is labeled with CY5, and then a CY 5-labeled nucleic acid-liposome complex is synthesized. 2X 10 ⁵ Jurkat cells were incubated with AS 1411X CTLA-4-liposomes at 4℃for 0.5h, and AS a control, 2X 10 ⁵ Jurkat cells were incubated in binding buffer or Con-liposome complex (nucleic acid-liposome prepared from CTLA-4 aptamer control sequence) at 4℃for 0.5h, respectively. Meanwhile, 2×10 ⁵ A549 cells were incubated with AS1411×CTLA-4-liposome complex at 4℃for 0.5h, and 2×10 ⁵ A549 cells were incubated in binding buffer or CTLA-4-liposome complex at 4℃for 0.5h AS a control. After incubation, the binding of the material to the cells in each group was detected by flow cytometry.

The results of the material-to-cell binding studies are shown in FIG. 3, where the AS1411 XCTLA-4-liposome complex in FIG. 3A has a strong binding affinity for A549 cells, and the CTLA-4-liposome complex has four orders of magnitude less shifts than the flow results of the AS1411 XCTLA-4-liposome complex on A549 cells. In FIG. 3B, AS1411 XCTLA-4-liposome complex has a stronger binding affinity to Jurkat cells, and Con-liposome complex is two orders of magnitude less shifted than AS1411 XCTLA-4-liposome complex's flow results on Jurkat cells. It was demonstrated that only AS1411 XCTLA-4-liposome complex was able to target both cancer cells expressing nucleolin and immune cells expressing CTLA-4 on their surface.

Example four, T cell redirecting Capacity of bispecific Probe-connector Complex

A549 cells and Jurkat cells were stained with MitoSpy Red and CFSE, respectively, and after staining, the cells were washed twice with cold binding buffer. Jurkat cells were incubated with AS1411 XCTLA-4-liposome complex (800 nM), or a control PBS group, AS1411 liposome, and CTLA-4-liposome complex mixed for 30 min at 4℃in 1640 complete culture. After incubation was completed, unbound material was removed by centrifugation at 1200rpm for 5min, and then mixed with a549 cells in 1640 complete culture, respectively, followed by addition of the cell mixture to a 96-well plate and incubation at 4 ℃ for 5 hours. Cells were then gently washed 2 times with PBS, 1% (W/V) paraformaldehyde fixed, 100uL paraformaldehyde solution per well, incubated at room temperature for 15min, blotted off, washed 2 times with PBS, and 100uL 1640 per well was then used to complete the culture and imaged with high content. The results are shown in FIG. 4, in the presence of AS1411 XCTLA-4-liposome complex, jurkat cells were redirected around A549 cells and the number of tight junctions was significantly increased, indicating that AS1411 XCTLA-4-liposome complex had T cell redirecting ability.

Example five in vitro toxicity detection of bispecific Probe-linker complexes

5.1 AS1411 XCTLA-4-liposome complex has concentration-dependent immunopotentiating effects on A549 cells:

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

15X 10 ³ activated PBMC cells were incubated with 0, 0.6uM, 2.4uM, 3.6uM, 5uM AS1411 XCTLA-4-liposome complexes at 4℃for 30min, respectively, and then 5X 10 ³ A549 cells were mixed with the PBMC cells and the cell mixtures were seeded in 96-well plates. The 96-well plate after the completion of the seeding was placed in an incubator at 37℃with 5% CO ₂ for co-cultivation for 40 hours. After the end of the incubation, the cells were washed twice with PBS to remove suspended PBMC cells, and then detected by adding 10% CCK8 solution. After incubation for 1-2 h in an incubator with 5% CO ₂ and 37 ℃, the absorbance at 450nm is measured by an ELISA. As shown in FIG. 5, the results show that the quantity of AS1411 XCTLA-4-liposome complex is concentration-dependent on the immunopotentiating effect of A549 tumor cells, and when the concentration of the AS1411 XCTLA-4-liposome complex reaches 5uM, the activity of the A549 cells is 59.1%, namely, the immunopotentiating effect reaches 40.9%.

5.2 AS1411 XCTLA-4-liposome complex has concentration-dependent immunopotentiating effects on MCF-7 cells:

The difference from 5.1 was that the A549 cells were changed to MCF-7 cells, and the concentrations of AS1411 XCTLA-4-liposome complex were 0uM, 0.1uM, 1uM, 10uM, 100uM, 500uM, 1000uM, respectively, and the other procedures were the same AS in example 5.1. As shown in FIG. 6, the results show that the AS1411 XCTLA-4-liposome complex has concentration dependency on the immunopotentiation effect of MCF-7 tumor cells, and when the concentration of the AS1411 XCTLA-4-liposome complex reaches 1000nM, the activity of the MCF-7 cells is 72.42%, namely, the immunopotentiation effect reaches 27.58%.

5.3 AS1411 XCTLA-4-liposome complex has concentration-dependent immunopotentiating effects on HepG2 cells:

The difference from 5.1 is that the A549 cells were exchanged for HepG2 cells, and the concentrations of AS1411 XCTLA-4-liposome complex were 0nM, 1nM, 10nM, 100nM, 500nM, 1000nM, respectively, and the rest was the same AS in example 5.1. As shown in FIG. 7, the results show that the AS1411 XCTLA-4-liposome complex has concentration dependency on the immunopotentiation effect of HepG2 tumor cells, and when the concentration of the AS1411 XCTLA-4-liposome complex reaches 1000nM, the activity of the HepG2 cells is 58.1%, namely, the immunopotentiation effect reaches 41.9%.

Example six detection of the Immunoiapeutic Effect of bispecific Probe-linker complexes

1.5X10 ⁴ activated PBMC cells were incubated with 300nM AS1411 XCTLA-4-liposome complex, AS 1411-liposome complex, CTLA-4-liposome complex and PBS at 4℃for 30min, respectively, and then 5X 10 ³ HepG2 cells were mixed with the above PBMC cells, and the cell mixtures were seeded in 96-well plates. The 96-well plate after the completion of the seeding was placed in an incubator at 37℃with 5% CO ₂ for co-cultivation for 40 hours. After the end of the incubation, the cells were washed twice with PBS to remove suspended PBMC cells, and then detected by adding 10% CCK8 solution. After incubation for 1-2 h in an incubator with 5% CO2 at 37 ℃, absorbance at 450nm is measured with an ELISA. As shown in FIG. 8, the killing power of activated PBMC cells against HepG2 cells was maximum with 300nM of AS1411 XCTLA-4-liposome complex, only 68.24% of cells remained viable, while 82.40% of AS 1411-liposome complex and 98.94% of CTLA-4-liposome complex remained viable.

Example seven, cytotoxicity detection of bispecific Probe-linker complexes

To verify the safety of AS1411 XCTLA-4-liposome complex, this experiment will verify the toxicity of AS1411 XCTLA-4-liposome complex to cells that either express high levels of nucleolin or that do not express nucleolin in the absence of T cells. IOSE is a normal human ovarian epithelial cell that does not express nucleolin protein on its surface and was selected for use as a negative control cell in this experiment.

8-10×10 ³ A549, MCF-7, hepG2 and IOSE (human normal ovarian epithelial cells) were seeded in 96-well plates, respectively. After adherence, PBS and 500nM AS1411×CTLA-4-liposome complex were added to each cell group, respectively. The 96-well plates were then CO-cultured in an incubator at 5% CO ₂, 37℃for 45h. After the end of the incubation, the cells were washed twice with PBS to remove suspended PBMC cells, and then detected by adding 10% CCK8 solution. After incubation for 1h in an incubator at 37℃with 5% CO ₂, absorbance was measured at 450nm with an ELISA. As shown in FIG. 9, the A549 cell activity was 97.1%, the MCF-7 cell activity was 106.5%, the HepG2 cell activity was 95.6%, and the IOSE cell activity was 100.5%, i.e., the AS1411 XCTLA-4-liposome complex was not significantly toxic to the cell activity at 500nM concentration.

Example eight bispecific Probe-connector Complex with third connector being extracellular vesicle

8.1 Preparation of bispecific Probe-linker complexes of extracellular vesicles

The first probe-linker complex (Chelosterol-PEG 2000-AS 1411) prepared in example one, and the second probe-linker complex (Chelosterol-PEG 2000-CTLA-4) prepared in example one were mixed with extracellular vesicles and allowed to react at 60℃for 1.5 hours.

As a result, AS shown in FIG. 10, the amounts of the respective nucleic acid aptamers in the AS1411 group, the CTLA-4 group, and the AS1411 XCTLA-4-extracellular vesicle complex group were kept uniform. The free aptamer in the AS1411 XCTLA-4-extracellular vesicle complex group has a strip brightness far lower than that of the CTLA-4 group in gel imaging, and obvious strips are arranged in the sample adding holes of the AS1411 XCTLA-4-extracellular vesicle complex group, which indicates that a large amount of aptamer in the AS1411 XCTLA-4-extracellular vesicle complex group is loaded on the surface of the extracellular vesicle, and the synthesis is successful.

8.2 Bispecific probe-linker complexes of extracellular vesicles are concentration-dependent on a549 cells for pro-immunotherapy:

The difference from example 5.1 is that the concentration of the bispecific probe-linker complexes of extracellular vesicles is 0nM, 50nM, 300nM, 600nM, 900nM, 1200nM, respectively, the rest of the procedure being the same as example 5.1. As shown in FIG. 11, the results show that the quantity of AS1411 XCTLA-4-extracellular vesicle complex is concentration-dependent on the immunopotentiating effect of MCF-7 tumor cells, and when the concentration of the AS1411 XCTLA-4-extracellular vesicle complex reaches 1200nM, the activity of the A549 cells is 47.2%, and the immunopotentiating effect reaches 52.8%.

In summary, the bispecific probe-connector complex provided by the invention can be combined with tumor cells containing nucleolin protein on the surface through the first probe and simultaneously combined with immune cells with CTLA-4 through the second probe, and has the advantages of high efficiency, low toxicity, high specificity, good immunity-promoting effect and easiness in synthesis.

While particular embodiments of the present invention have been illustrated and described, it will be appreciated that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

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, wherein the first probe specifically binds nucleolin protein and the second probe specifically binds CTLA-4;

the first probe is nucleic acid, the sequence of which is seq_1, and the second probe is nucleic acid, the sequence of which is seq_2;

The connector includes a first connector connected to the first probe, a second connector connected to the second probe, and a third connector connected to both the first connector and the second connector.

2. The probe-linker complex according to claim 1, wherein the first linker is cholesterol, distearoyl phosphatidylethanolamine or dioleoyl phosphatidylethanolamine;

And/or the second linker is cholesterol, distearoyl phosphatidylethanolamine or dioleoyl phosphatidylethanolamine;

And/or, the third linker is a liposome nanoparticle or an extracellular vesicle.

3. A method for preparing a bispecific probe-linker complex is characterized in that,

S1, covalently connecting a first connector with a first probe to prepare a first probe-first connector complex;

s2, covalently connecting a second connector with a second probe to prepare a second probe-second connector complex;

S3, mixing and reacting the first probe-first connector complex, the second probe-second connector complex and the third connector to prepare the probe-connector complex;

Wherein the first probe can specifically bind nucleolin protein, the first probe is nucleic acid, and the sequence of the first probe is seq_1;

The second probe can specifically bind to CTLA-4, and the second probe is nucleic acid with a sequence of seq_2.

4. The method of preparing a bispecific probe-linker complex according to claim 3, wherein the first linker is cholesterol, distearoyl phosphatidylethanolamine or dioleoyl phosphatidylethanolamine;

5. The method of preparing a bispecific probe-linker complex according to claim 3, wherein one end of the first probe is modified with NH ₂ and one end of the second probe is modified with NH ₂;

the first connector and the second connector are cholesterol modified by N-hydroxysuccinimide;

The third connector is a liposome nanoparticle;

In the step S1, the feeding ratio of the first probe to the first connector is 1 (1-100);

In the step S2, the feeding ratio of the second probe to the second connector is 1 (1-100);

In the step S3, the first probe-first connector complex, the second probe-second connector complex and the third connector are fed in a ratio of 2.1nmol to 0.7nmol to 27.2mg.

6. Use of a bispecific probe-linker complex according to any one of claims 1-2 or a bispecific probe-linker complex prepared according to the method of preparation of a bispecific probe-linker complex according to any one of claims 3-5 for the preparation of a product for forming a tumor cell-first probe-linker-second probe-immune cell complex that can bind to tumor cells comprising nucleolin protein on the surface, immune cells comprising CTLA-4 on the surface, forming a tumor cell-first probe-linker-second probe-immune cell complex, said tumor cells being a549, hepG2 or MCF-7.