Platelet-oncolytic virus complex, its preparation method and its application

Abstract

This disclosure relates to a platelet-oncolytic virus complex, its preparation method, and its applications. The complex comprises platelets and oncolytic viruses, with the oncolytic viruses loaded within the platelets. The platelet-oncolytic virus complex utilizes the close interaction between platelets and circulating tumor cells (CTCs) to adhere to CTCs and directionally infect them with oncolytic viruses. By initiating oncolytic circulation, oncolytic viruses are continuously produced, clearing tumor cells from the circulatory system and metastatic lesions.

Description

Technical Field

[0001] This disclosure pertains to the field of biotherapy and medicine in tumor treatment, specifically relating to a platelet-oncolytic virus complex, its preparation method, and its applications. Background Technology

[0002] Oncolytic viruses are one of the world's leading methods for treating tumors, demonstrating effective anti-tumor effects in numerous preclinical and clinical trials. This technology utilizes the natural host-lysing property of viruses (host lysis cycle), driving the oncolytic cycle through promoters of tumor-specific genes. Therefore, they can specifically replicate within tumor cells, causing tumor cell lysis without affecting normal cells. Oncolytic viruses have the ability to conditionally replicate within tumor cells, directly lysing them and causing the release of soluble antigens, danger signals, and type I interferon, stimulating innate and adaptive immunity, thereby driving anti-tumor immunity. Currently, oncolytic viruses are mainly divided into three major systems: adenoviruses (including adeno-associated viruses), retroviruses, and lentiviruses. Retroviruses and lentiviruses must integrate their reverse-transcribed viral DNA into the host cell's chromosome before entering the oncolytic cycle and exerting their function; therefore, they have potential oncogenic effects (leading to the activation of new cancer genes or accelerating cancer cell growth) and unpredictable biological hazards. Adenoviruses, on the other hand, do not need to integrate into chromosomes during their life cycle; they can function as genetic information while remaining outside the host chromosome. Therefore, its safety is greatly improved, and it is considered a relatively safe oncolytic virus system.

[0003] Based on its anti-tumor mechanism and the characteristics of the virus itself, the main route of administration of oncolytic viruses is intratumoral injection. The main reasons are: (1) Viral membrane receptors are widely present in the human body, which makes the tumor specificity of viral infection low and is very likely to infect and destroy normal cells and tissues; (2) When oncolytic viruses enter the blood, they will neutralize the virus-specific antibodies in the serum and then be removed; (3) Blood will dilute the virus, and the tumor microenvironment will inhibit the effective infiltration of the virus into the tumor tissue.

[0004] However, apart from a few types of tumors such as skin cancer, the vast majority of tumors cannot be treated with intratumoral injection. For example, the treatment of tumors such as intra-organ tumors, deep tissue tumors, small tumors, and multiple metastatic tumors (with dozens or even hundreds of metastatic foci) still requires systemic blood delivery. Therefore, research on intravenous administration of oncolytic viruses is currently a focus and hot topic.

[0005] Circulating tumor cells (CTCs) are a collective term for various types of tumor cells present in peripheral blood. As solid tumors grow, specific changes in the microenvironment surrounding the lesion can induce abnormal activity in some tumor cells. These cells detach from the primary tumor and travel through the bloodstream or lymphatic system to various parts of the body, seeking new sites. These detached tumor cells traveling via blood and lymphatic expressways are called circulating tumor cells (CTCs). This also includes tumor cells that enter the bloodstream due to external factors such as invasion or surgical procedures.

[0006] Mounting experimental evidence suggests that platelets support tumor metastasis. Within the circulatory system, platelets protect circulating tumor cells (CTCs) from immune clearance and promote their retention on endothelial cells, supporting CTC extravasation to metastatic sites. Platelet activation releases growth factors and small molecules that promote tumor growth and invasion. Once tumor cells enter the bloodstream, platelet-tumor cell aggregation occurs, and platelets protect CTCs from NK cell and TNFα-induced cell death. Furthermore, platelets stimulate and facilitate cancer cell adhesion and extravasation, thereby supporting cancer cell metastasis and metastasis formation. On the other hand, tumor cells mediate platelet activation, leading to platelet aggregation and particle release. Activated platelets can also extravasate into the tumor microenvironment via focal adhesion kinase (FAK). Since the first in vivo experiments by Gasic et al. (1968), our understanding of the mechanisms by which platelets and thrombocytosis promote tumor growth and metastasis has greatly increased. Clearly, platelets are not only bystander cells in circulation, but also play an important role in every step of the growth and metastasis of primary tumors. They can enter the tumor microenvironment and interact directly with cancer cells.

[0007] It is generally believed that platelets interact with CTCs and promote tumor metastasis through multiple mechanisms: (1) Platelet-CTC aggregates are more likely to aggregate in microvessels than single CTCs. This aggregation promotes the subsequent extravasation of cancer cells; (2) Platelets aggregate around CTCs and protect them from natural killer (NK) immune-mediated clearance via TGFβ; (3) The escape of CTCSs from NK cells can also be attributed to the physical shielding of platelets.

[0008] In circulation, platelets protect tumor cells (CTCs) from lethal attacks by the immune system and other pro-apoptotic stimuli. Platelets help CTCs attach to endothelial cells and provide signals to establish pre-metastatic niches. They even affect the sensitivity of cancer patients to chemotherapy and other targeted therapies. Methods targeting platelet-tumor cell interactions or reducing platelet counts have been employed, but these methods often interfere with their normal physiological functions and can lead to life-threatening bleeding complications. Therefore, developing methods specifically targeting platelet-tumor cell interactions without interfering with normal platelet function could provide a treatment option for cancer patients. Platelets play important physiological roles in hemostasis, inflammation, and tissue regeneration, and are closely related to wound healing, tumor development, and metastasis. In recent years, platelet-mediated drug release pathways have been developed due to their unique properties, such as rapid replenishment and specific activation / adhesion. As small anucleate cells, 1-3 μm in diameter with a lifespan of approximately 8-10 days, platelets, due to their rapid replenishment and appropriate circulation time, avoid undesirable accumulation in the body, making them a safe carrier for cell therapy. Platelets, as one of the most important components of blood flow, can be collected from donors, engineered in vitro, and then reinfused into the patient's circulation. Alternatively, targeted drug delivery can be achieved directly through platelet-bound prodrugs and nanoparticles circulating in the bloodstream.

[0009] Recently developed membrane coating technologies, or artificial platelets, can mimic the behavior of platelets. Furthermore, dynamic morphological and functional changes during platelet activation are used to trigger drug release. Modification of living cells is one of the most common methods in cell engineering for controlling cellular activities, including cell transport, cell tracking, and protein secretion. The platelet surface is rich in primary amine or thiol residues, which can be chemically linked to nanoparticles or biomolecules. For example, a monoclonal antibody targeting programmed death ligand 1 (aPDL1) binds to the platelet surface as a checkpoint inhibitor delivery platform for postoperative cancer immunotherapy. Platelets can actively target surgical wound sites, releasing aPDL1 in microparticle form. This platelet-based drug delivery strategy has shown strong application potential in postoperative treatment. In addition to postoperative treatment, recent studies have also reported a strategy of combining platelets modified with anti-programmed death protein 1 antibody (aPD-1) with hematopoietic stem cells (HSCs) for the treatment of acute myeloid leukemia. Utilizing the homing ability of HSCs, platelets can reach the bone marrow and, after in situ activation around leukemia cells located in the bone marrow, release aPD-1 as a special drug reservoir. Furthermore, the simultaneous release of pro-inflammatory cytokines during platelet activation can synergistically enhance the efficiency of T cell activation and residual tumor clearance.

[0010] In antitumor therapy, tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL) and programmed cell death protein 1 (PD-1) receptor can be expressed on the platelet surface. Engineered platelets can bind to and neutralize CTCs to reduce tumor metastasis, and can also accumulate in postoperative wounds to enhance tumor immune function and eliminate residual tumor cells. Furthermore, a light-controlled RNA synthesis nanoliposome system has recently been developed that can directly transcribe exogenous RNA within platelets.

[0011] With a deeper understanding of the physicochemical properties and biological functions of platelets, their potential application in engineered drug delivery systems for treating various diseases is increasing. Similar to the basic idea of ​​other cell delivery therapies, therapeutic drugs and nanoparticles can prevent blood clearance by mimicking or utilizing platelets. Furthermore, platelets can be easily separated from the blood, without placing a heavy burden on donors or patients, ensuring higher clinical translational potential. In addition, platelets have a natural selectivity for damaged tissues or the tumor microenvironment. Existing platelet-based drug delivery systems generally load substances such as cytotoxic drugs or mRNA. Due to limited loading capacity, it is difficult to achieve effective concentrations at the tumor site. Moreover, most current platelet-based drug delivery systems destroy the original platelets, utilizing only their phospholipid bilayer membrane for drug or mRNA encapsulation. Therefore, their physiological binding function to CTCs is greatly weakened, retaining only a portion of the platelet homing activity to the tumor microenvironment. Thus, they cannot effectively clear and utilize CTCs. Therefore, obtaining functional platelet carriers will have broader application scenarios.

[0012] The core limitation of existing oncolytic viruses is their inability to effectively and specifically reach the tumor site. As mentioned earlier, with the exception of a few types like skin cancer, most tumors cannot be injected intratumorally. Examples include tumors within organs (involving organ function; inaccurate injection could damage normal organ function), deep-seated tumors (which cannot be precisely guided for injection using non-invasive methods like ultrasound), small tumors (too small and integrated with the surrounding tissue, making them difficult to see, let alone inject precisely), and multiple metastatic tumors (with dozens or even hundreds of foci, making individual injection impossible). Therefore, treatment for these tumors still requires systemic blood delivery. However, intravenous adenovirus infusion (or subcutaneous injection) stimulates the body's immune system; antibodies, macrophages, and neutrophils rapidly clear the virus from the bloodstream. Therefore, it cannot effectively reach the tumor site to exert its oncolytic activity. Most oncolytic viruses can only be injected in situ, which presents the challenge of not being able to clear circulating tumor cells in the bloodstream or multiple, invisible metastatic lesions. Summary of the Invention

[0013] In their previous research, the inventors discovered that after platelets bind to tumor cells (CTCs), they specifically and directionally infect the CTCs with their own contents, including proteins and nucleic acids (primarily RNA). Therefore, the inventors designed an experiment to load oncolytic viruses into platelets. Utilizing the close interaction between platelets and CTCs, the viruses adhered to the CTCs, infecting them with the oncolytic virus as "contents." Because CTCs tend to aggregate towards metastatic lesions, these oncolytic virus-infected CTCs can carry the oncolytic virus into the metastatic lesions before they are dissolved (before the oncolytic cycle begins). Through the initiated oncolytic cycle, oncolytic viruses are continuously produced, clearing metastatic tumors (see below). Figure 1 This method involves modifying platelets to carry viruses that kill tumors (such as adenoviruses). These viruses bind to and infect tumor cells (CTCs). The CTCs then act like Trojan horses carrying tumor-killing oncolytic viruses. After metastasizing to the tumor metastases, they destroy the tumor from within, thereby reducing the risk of metastasis.

[0014] In one aspect, this disclosure provides a platelet-oncolytic virus complex comprising platelets and oncolytic virus, wherein the oncolytic virus is loaded in the platelets.

[0015] In another aspect, this disclosure also provides a method for preparing a platelet-oncolytic virus complex, which includes the following steps:

[0016] (1) Obtaining platelets;

[0017] (2) Obtaining oncolytic viruses;

[0018] (3) Mix platelets and oncolytic virus to obtain a suspension;

[0019] (4) The suspension is incubated to obtain a platelet-oncolytic virus complex.

[0020] In another aspect, this disclosure provides a composition comprising the aforementioned platelet-oncolytic virus complex and a pharmaceutically acceptable carrier.

[0021] In another aspect, this disclosure provides the use of the platelet-oncolytic virus complex or the composition thereof in the preparation of a medicament for killing tumor cells or treating tumors in a subject in need. Attached Figure Description

[0022] Figure 1 Design and working principle roadmap of platelet-loaded oncolytic adenovirus biotherapy system.

[0023] Figure 2This section compares viral loading efficiencies under different conditions. Specifically: A. Residual viral load in the supernatant after platelet loading with oncolytic adenovirus (qPCR). B. Virus load in platelets after oncolytic adenovirus loading by qPCR. C. Comparison of platelet loading efficiencies with oncolytic adenovirus. D. Virus load per platelet calculated using the differential method under different loading conditions.

[0024] Figure 3 A method for optimizing platelet loading of oncolytic adenovirus is shown.

[0025] Figure 4 The image shows the morphology of the platelet-oncolytic adenovirus complex. A. Scanning electron micrographs of the blank platelet group and the platelet-oncolytic adenovirus complex group. B. Transmission electron micrographs of the blank platelet group and the platelet-oncolytic adenovirus complex group. The black dots indicated by the arrows in the images represent oncolytic adenovirus particles.

[0026] Figure 5 The study shows the in vivo survival time of platelets and platelets loaded with oncolytic adenovirus, as detected by flow cytometry. There was no difference in in vivo survival time or biological function between the two. Among them: Figure 5 A(1) shows the characteristics of whole blood under flow cytometry without a fluorescence channel; Figure 5 A(2) shows the characteristics of platelets in flow cytometry without a fluorescence channel; Figure 5 A(3) shows the characteristics of whole blood under flow cytometry detection in the green fluorescent channel; Figure 5 A(4) shows the characteristics of platelets in flow cytometry without fluorescence channel under green fluorescence channel; Figure 5 B(1) shows the survival time and proportion of platelets loaded with oncolytic adenovirus in vivo; Figure 5 B(2) shows the survival time and proportion of normal platelets in the body.

[0027] Figure 6 The distribution of platelet-oncolytic adenovirus complexes in mice is shown. A. In vivo images taken at 4h, 24h, 48h, and 120h after DIR labeling in the platelet group and the platelet-oncolytic adenovirus complex group, administered via tail vein injection. B. In vivo images of organs separated at the last time point (120h). C. Histochemical staining of the spleen and liver in the control group, platelet group, oncolytic adenovirus group, and platelet-oncolytic adenovirus complex group.

[0028] Figure 7 The blood biochemical parameters in mice in the blank group, platelet group, oncolytic adenovirus group, and platelet-oncolytic adenovirus complex group are shown.

[0029] Figure 8 The image shows a bright-field fluorescence overlay of CTCs transfected with a platelet-oncolytic adenovirus complex.

[0030] Figure 9 The study shows the lysis of CTCs induced by platelet-oncolytic adenovirus complexes. Specifically: A. Cell images after 48 hours of transfection with different titers of platelets or platelet-oncolytic adenovirus complexes. B. Normal cell proliferation assay results (MTX method) after 48 hours of transfection with different titers of platelets or platelet-oncolytic adenovirus complexes. C. Tumor cell proliferation assay results (MTX method) after 48 hours of transfection with different titers of platelets or platelet-oncolytic adenovirus complexes.

[0031] Figure 10 This study illustrates the inhibitory effect of platelet-oncolytic adenovirus complex on lung metastases of triple-negative breast cancer in mice. A. Schematic diagram of the in vivo drug administration regimen in mice. B. Image of lung metastases in mice. C. Statistical analysis of lung metastases in mice. Detailed Implementation

[0032] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0033] In this document, the term "oncolytic virus" refers to a recombinant virus that destroys cancer cells, the genes of which are manipulated to specifically replicate within cancer cells. The oncolytic virus can be derived from adenovirus, herpes simplex virus, measles virus, lentivirus, retrovirus, cytomegalovirus, baculovirus, reovirus, adeno-associated virus, myxoma virus, vesicular stomatitis virus, poliovirus, Newcastle disease virus, parvovirus, Coxsackie virus, Seneca Valley virus, Maraba virus, vaccinia virus, or poxvirus. In some embodiments, the oncolytic virus described herein is incorporated into a viral vector.

[0034] In this document, the term "vector" is used to refer to a nucleic acid molecule capable of transferring or transporting nucleic acid molecules. The transferred nucleic acid is typically linked to a nucleic acid molecule, for example, inserted into a vector nucleic acid molecule. A vector may include a sequence that guides autonomous replication in the cell, or may include a sequence sufficient to allow integration into the host cell's DNA. Viral vectors are sometimes referred to as "recombinant viruses" or simply "viruses." The terms "oncolytic virus" and "oncolytic vector" are used interchangeably in this document.

[0035] The term "tumor-specific promoter" refers to a promoter that preferably or specifically functions to initiate gene expression in tumor cells and is inactive or has reduced activity in non-tumor cells or non-cancer cells. Examples of tumor-specific promoters include, but are not limited to, the TERT promoter, survivin promoter, cyclooxygenase (COX-2) promoter, prostate-specific antigen (PSA) promoter, E2F-1 promoter, alpha-fetoprotein (AFP) promoter, cholecystokinase promoter, carcinoembryonic antigen (CEA) promoter, C-erbB2 / neu oncogene promoter, CXCR4 promoter, HE4 promoter, type II hexokinase promoter, L-retin promoter, MUC1 promoter, PSA promoter, survivin promoter, TRP1 promoter, and tyrosinase promoter.

[0036] In this article, the term "platelet-loaded oncolytic virus" refers to the process by which platelets and oncolytic viruses are incubated at a certain temperature, vortexed, or otherwise endocytosed by platelets into the platelet membrane.

[0037] In this article, the term "platelet-oncolytic virus complex" refers to the process by which an oncolytic virus binds to a platelet, enters the platelet, and becomes part of the platelet's contents, thus forming a platelet-oncolytic virus complex.

[0038] In this article, the term "oncolytic circulation" refers to the process by which oncolytic viruses infect tumor cells, replicate within the tumor cells, cause the tumor cells to lyse, and release the oncolytic viruses, which then circulate and infect surrounding tumor cells, thereby completing the infection of tumor cells in metastatic lesions.

[0039] In this document, the term "payload molecule" refers to a molecule that can further enhance the therapeutic efficacy of the virus. Payload molecules applicable to this disclosure include antigen-binding molecules (such as antibodies or their antigen-binding fragments), cytokines, chemokines, soluble receptors, cell surface receptor ligands, dipeptides, enzymes, and nucleic acids (e.g., shRNA, siRNA, antisense RNA, antagomir, ribozymes, aptamers, decoy oligonucleotides, or antagomir). The properties of the payload molecule will vary depending on the disease type and the desired therapeutic outcome.

[0040] The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce allergic reactions or similar adverse reactions when administered to a subject (e.g., a human).

[0041] In one aspect, this disclosure provides a platelet-oncolytic virus complex comprising platelets and oncolytic virus, wherein the oncolytic virus is loaded in the platelets.

[0042] In some embodiments, the oncolytic virus is selected from: adenovirus, herpes simplex virus, measles virus, lentivirus, retrovirus, cytomegalovirus, baculovirus, reovirus, adeno-associated virus, myxoma virus, vesicular stomatitis virus, poliovirus, Newcastle disease virus, parvovirus, Coxsackie virus, Seneca Valley virus, Maraba virus, vaccinia virus, or poxvirus.

[0043] In some embodiments, the herpes simplex virus is selected from herpes simplex virus type I or herpes simplex virus type II.

[0044] In some embodiments, the oncolytic virus contains a tumor-specific promoter selected from: TERT promoter, survivin promoter, cyclooxygenase (COX-2) promoter, prostate-specific antigen (PSA) promoter, E2F-1 promoter, alpha-fetoprotein (AFP) promoter, cholecystokinase promoter, carcinoembryonic antigen (CEA) promoter, C-erbB2 / neu oncogene promoter, CXCR4 promoter, HE4 promoter, type II hexokinase promoter, L-retin promoter, MUC1 promoter, PSA promoter, survivin promoter, TRP1 promoter, and tyrosinase promoter.

[0045] In some implementations, the TERT promoter is the hTERT promoter.

[0046] In some embodiments, the oncolytic virus further comprises at least one polynucleotide encoding a payload molecule that activates or enhances an antitumor immune response.

[0047] In some embodiments, the payload molecule is selected from antigen-binding molecules (such as antibodies or their antigen-binding fragments), bispecific T-cell adaptors (BiTE), cytokines, chemokines, soluble receptors, cell surface receptor ligands, dipeptides, enzymes, and nucleic acids (e.g., shRNA, siRNA, antisense RNA, ribozymes, aptamers, decoy oligonucleotides, or antagomir).

[0048] In some embodiments, the payload molecule is a protein that binds to and activates a cell surface receptor. In some embodiments, the payload molecule comprises an endogenous cell surface ligand, such as the extracellular domain of 41BBL, or the extracellular domains of CD40L and FLT3L.

[0049] In some embodiments, the payload molecule is a protein that binds to and activates an activation receptor (e.g., FcγRI, FcγIIa, FcγIIIa, co-stimulatory receptors, etc.). In some embodiments, the protein is selected from EpCAM, folic acid, A2A, anti-FGF2, anti-FGFR / FGFR2b, anti-SEMA4D, CD137, CD200, CD38, CD44, CSF-1R, endothelin B receptor, ISRE7, LFA-1, NG2 (also known as SPEG4), SMAD, STING, and VCAM1.

[0050] In some embodiments, the cytokines are selected from one or more of IFNγ, IFNα, IFNβ, TNFα, IL-12, IL-2, IL-6, IL-8, IL-15, GM-CSF, IL-21, IL-35, TGFβ, and HSP.

[0051] In some embodiments, the chemokine is selected from one or more of CCL4, CXCL10, CCL5, CXCL13, and XCL1.

[0052] In some implementations, the payload molecule is siRNA.

[0053] In some implementations, the siRNA knocks down proto-oncogenes or immunosuppressive genes.

[0054] In some embodiments, the proto-oncogene is selected from one or more of Ras, Myc, Bcl2, PTEN, and p53.

[0055] In some implementations, the Ras gene is the K-RAS gene.

[0056] In some embodiments, the immunosuppressive gene is selected from one or more of CTLA4, PD-1, TIM-3, and LAG-3.

[0057] In some embodiments, the effective load molecular antibody or its antigen-binding fragment is used.

[0058] In some embodiments, the antibody is selected from one or more of the following: CTLA-4 antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, anti-PDL1 antibody or its antigen-binding fragment, CCR8 antibody or its antigen-binding fragment, and CD47 antibody or its antigen-binding fragment.

[0059] In another aspect, this disclosure also provides a method for preparing a platelet-oncolytic virus complex, which includes the following steps:

[0060] (1) Obtaining platelets;

[0061] (2) Obtaining oncolytic viruses;

[0062] (3) Mix platelets and oncolytic virus to obtain a suspension;

[0063] (4) The suspension is incubated to obtain a platelet-oncolytic virus complex.

[0064] In some embodiments, the suspension is incubated at 4-40°C, preferably 37°C.

[0065] In some embodiments, the suspension is incubated on a shaker at a speed of 150-300 rpm, preferably 220 rpm.

[0066] In some embodiments, the incubation period is 1-10 hours. In some preferred embodiments, the suspension is incubated for 2 hours on a shaker at 37°C and 220 rpm.

[0067] In some embodiments, the suspension is centrifuged before incubation, and then the centrifuged suspension is incubated. In some embodiments, the suspension is centrifuged at 100-300g for 2-20 minutes. In some preferred embodiments, the suspension is centrifuged at 200g for 10 minutes.

[0068] In some embodiments, the suspension is vortexed before incubation, followed by incubation of the centrifuged suspension. In some embodiments, the suspension is vortexed for 2-10 minutes. In some preferred embodiments, the suspension is vortexed for 5 minutes. In another aspect, this disclosure provides a composition comprising the aforementioned platelet-oncolytic virus complex and a pharmaceutically acceptable carrier.

[0069] "Pharmaceutically acceptable carriers" include, but are not limited to, any adjuvants, carriers, excipients, gliding agents, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, surfactants, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents that have been approved by the U.S. Food and Drug Administration for acceptable use in humans and / or domestic animals and are physiologically compatible, including pharmaceutically acceptable cell culture media and / or emulsifiers. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragali gum; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffin wax, silicone, bentonite, silicic acid, and zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginate; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solutions; and any other compatible substances used in pharmaceutical formulations. Unless any conventional media and / or agents are incompatible with the agents disclosed herein, their use in therapeutic compositions should be considered. Complementary active ingredients may also be incorporated into the composition.

[0070] Wetting agents, emulsifiers and lubricants (such as sodium lauryl sulfate and magnesium stearate), as well as colorants, release agents, coating agents, sweeteners, flavorings and aromas, preservatives and antioxidants may also be present in the composition.

[0071] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as palmitic acid ascorbate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0072] In one embodiment, the composition comprising a carrier is suitable for parenteral administration, such as intravascular (intravenous or intra-arterial), intraperitoneal, or intramuscular administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the provisional preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. The use of any conventional media or agent in the pharmaceutical compositions of this disclosure should be considered unless it is incompatible with viral vectors or nucleic acid molecules.

[0073] The compositions disclosed herein may comprise, alone or in combination with one or more other polypeptides or polynucleotides as described herein, a carrier comprising said one or more polypeptides or polynucleotides, infected cells, etc., formulated in a pharmaceutically or physiologically acceptable solution for administration to cells or animals, alone or in combination with one or more other therapeutic modalities. It should also be understood that, if desired, the compositions disclosed herein may also be administered in combination with other agents such as cytokines, growth factors, hormones, small molecules, or various pharmaceutically active agents. In fact, there are no limitations on other components that may be included in the composition, provided that the additional agents do not adversely affect the composition's ability to deliver the intended therapy.

[0074] In the pharmaceutical compositions disclosed herein, pharmaceutically acceptable formulations of excipients and carrier solutions are well known to those skilled in the art, as are the development of suitable dosing and treatment regimens for the use of the specific compositions described herein in various treatment regimens. After formulation, the solution is administered in a manner compatible with the dosage form and at a therapeutically effective amount to produce symptom improvement or amelioration. The formulation is readily administered in various dosage forms, such as injectable solutions, drug-release capsules, etc. Dosage variations may occur depending on the condition of the treated subject. The individual responsible for administration may determine the appropriate dosage for an individual subject in any given situation. Furthermore, for human administration, the formulation meets the general safety and purity standards required for the evaluation and investigation of biological products. Routes of administration will naturally vary depending on the location and nature of the disease being treated and may include, for example, intradermal, transdermal, subcutaneous, parenteral, nasal, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, tracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral administration.

[0075] In some aspects, this disclosure provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of one or more viral vectors or polynucleotides described herein, formulated with one or more pharmaceutically acceptable carriers (additives) and / or diluents (e.g., pharmaceutically acceptable cell culture media). As used herein, “therapeutically effective amount” refers to the amount of the composition or recombinant virus described herein required to achieve the desired physiological and / or biological outcome. The “therapeutically effective amount” of a virus, viral stock, or composition may vary depending on factors such as an individual’s disease state, age, sex, and weight, and the ability of stem cells and progenitor cells to elicit the desired response in an individual. Therapeutically effective amount is also the amount in which the beneficial therapeutic effect outweighs any toxic or harmful effects of the virus or transduced therapeutic cells. The term “therapeutically effective amount” includes the amount that effectively “treats” a subject (e.g., a patient). Therapeutically effective amount can be measured by the total number of plaque-forming units (pfu) (e.g., at least 1 e). 1 Up to at least 1e 20 Especially about 1e 4 To approximately 1e 15More particularly about 1e 6 To approximately 1e 12 (pfu) or the number of viral genomes (e.g., at least 1e) 1 Up to at least 1e 20 Especially about 1e 4 To approximately 1e 15 More particularly about 1e 6 To approximately 1e 12 The effective therapeutic dose will be quantified based on the type of virus administered, the nature of the formulation, the route of administration, the nature and / or severity of the disease to be treated, and / or the overall health and well-being of the subject.

[0076] In another aspect, this disclosure provides the use of the platelet-oncolytic adenovirus complex or the composition thereof in the preparation of a medicament for killing tumor cells or treating tumors in a subject in need.

[0077] In some implementations, the tumor cells are circulating tumor cells (CTCs).

[0078] In some implementations, the tumor is a metastatic tumor.

[0079] In some implementations, the tumor is cancer.

[0080] In one aspect, this disclosure provides a method for killing cancer cells, comprising exposing the cancer cells to the platelet-oncolytic adenovirus complex or a combination thereof under conditions sufficient to cause the oncolytic virus to infect and replicate within the cancer cells, wherein the replication of the oncolytic virus within the cancer cells causes cell death.

[0081] In one aspect, this disclosure provides a method for treating cancer in a subject in need, comprising administering the oncolytic virus or a composition thereof to the subject.

[0082] In some implementations, the subjects are mice, rats, rabbits, cats, dogs, horses, non-human primates, or humans.

[0083] In some embodiments, the oncolytic virus or its composition is administered intravenously, subcutaneously, intratumorally, intramuscularly, or intranasally.

[0084] In some implementations, the tumor is cancer.

[0085] In some implementations, the cancer is selected from lung cancer, breast cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, colorectal cancer, colon cancer, pancreatic cancer, liver cancer, stomach cancer, head and neck cancer, thyroid cancer, malignant glioma, glioblastoma, melanoma, B-cell chronic lymphocytic leukemia, diffuse large B-cell lymphoma (DLBCL), and marginal zone lymphoma (MZL).

[0086] In their preliminary research, the inventors discovered that after platelets bind to tumor-associated tumor cells (CTCs), they directionally infect these CTCs with their own contents, including proteins and nucleic acids (primarily RNA). Therefore, using oncolytic adenovirus as an example, the inventors designed a biotherapy system that loads oncolytic adenovirus onto platelets and utilizes CTCs to eliminate tumor metastases. The specific viral design and working principle are as follows... Figure 1 As shown. Among them, Figure 1 A is a schematic diagram of the DNA sequence design of an oncolytic adenovirus. The viral genome is set under the control of the promoter of a tumor-specific gene (such as the TERT promoter). Figure 1 B illustrates the working principle of a mixture of platelets and oncolytic adenovirus, comprising a platelet-oncolytic adenovirus complex and oncolytic adenovirus. Specifically: (I) it shows that upon contact between platelets and oncolytic adenovirus, the oncolytic adenovirus is loaded into the platelet, forming a platelet-oncolytic adenovirus complex. (II) it shows that oncolytic adenovirus not loaded into platelets, upon entering the bloodstream, elicits an immune response in the host and is cleared by antibodies and immune cells. (III) it shows that under certain exceptional circumstances, platelets may adhere to leukocytes and inject the virus into them; however, due to the very low transcriptional level of the TERT promoter (or other tumor-specific gene promoters) in leukocytes (normal cells), oncolytic circulation is not initiated, thus there is no clearance effect on normal cells. (IV) it shows the use of the close interaction between platelets and CTCs to adhere to CTCs and directionally infect CTCs with oncolytic adenovirus as "contents." Because tumor cells (CTCs) tend to aggregate towards metastatic lesions, these CTCs infected with oncolytic adenovirus can carry the oncolytic virus into the metastatic lesions before they are dissolved (before the oncolytic cycle begins). Through the initiated oncolytic cycle, oncolytic viruses are continuously produced, clearing away metastatic tumor cells.

[0087] The beneficial effects of this disclosure are as follows: 1) The oncolytic virus is encapsulated by platelets. Since platelets are endogenous substances, the oncolytic virus will not be recognized and cleared by the immune system; 2) It utilizes the platelet-specific ability to carry CTCs to efficiently bind to and infect CTCs; 3) It utilizes the effect of CTCs gathering towards metastatic lesions to guide CTCs infected with oncolytic viruses to tumor metastases; 4) After initiating the oncolytic cycle, it can continuously kill tumor cells, unlike cytotoxic drugs or mRNA, which cannot effectively clear tumors due to insufficient concentration. Like snake venom, once it is made, it becomes a source of toxicity and amplifies in a biological geometric progression; 5) The oncolytic cycle of the oncolytic virus is controlled by a promoter specifically expressed by tumor cells (such as the telomerase promoter). Therefore, the virus only replicates and enters the oncolytic cycle within tumor cells, and has almost no effect on normal cells during the process of clearing tumor cells.

[0088] This disclosure uses platelets to load oncolytic viruses. The platelet-oncolytic virus complex is more likely to bind to circulating tumor cells in the circulatory system, thereby achieving the purpose of lysing circulating tumor cells and further circulating to micrometastases, initiating oncolytic circulation, and clearing tumor metastases that are not visible to the naked eye.

[0089] For the purpose of clarity and concise description, the features are described herein as part of some identical or separate embodiments; however, it will be understood that the scope of this disclosure may include some embodiments having a combination of all or some of the features described.

[0090] The present disclosure will now be described in more detail with reference to specific embodiments. However, the embodiments are for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0091] Example

[0092] Example 1: Preparation of platelet-oncolytic adenovirus complex

[0093] 1.1 Preparation of platelet-oncolytic adenovirus complex by incubation method

[0094] Whole blood was collected from Balb / c mice (purchased from Vital River Pharmaceuticals, Beijing). 1-2 ml of blood was drawn from the orbital cavity of each mouse. The plasma was centrifuged at 100g-200g for 3 minutes, and the supernatant rich in platelets was collected. Then, 1-5 ml of thiocyanate buffer (product number: PB180338, Wuhan Pronosei Life Sciences Co., Ltd.) was added, and the plasma was centrifuged at 800g-1200g for 8-10 minutes. The precipitate (platelet pellet) was collected, resuspended in thiocyanate buffer containing 3% sodium citrate, and the platelets were counted. The platelet concentration was adjusted to 1×10⁻⁶. 8 Units / ml. Oncolytic adenovirus (H101, Ankerui, Shanghai Sanwei Biotechnology Co., Ltd.) was collected and the oncolytic adenovirus concentration was adjusted to 1×10⁻⁶.10 pfu / ml, incubated at 37℃: take a concentration of 1×10 8 100 μl of platelets / ml was used to obtain a concentration of 1×10⁻⁶. 10 Prepare a suspension of 200 μl of oncolytic adenovirus at a concentration of pfu / ml, with a virus-to-platelet ratio of 200:1; take a concentration of 1×10 8 100 μl of platelets / ml was used to obtain a concentration of 1×10⁻⁶. 10 Prepare a suspension of 100 μl of oncolytic adenovirus at a concentration of pfu / ml as a virus-to-platelet ratio of 100:1; take a concentration of 1×10 8 100 μl of platelets / ml was used to obtain a concentration of 1×10⁻⁶. 10 Prepare a suspension of oncolytic adenovirus (pfu / ml) at a ratio of 50:1 (virus:platelet) using 50 μl of the virus at a concentration of 1×10⁻⁶ pfu / ml. 8 100 μl of platelets / ml was used to obtain a concentration of 1×10⁻⁶. 10 Prepare a suspension of 25 μl of oncolytic adenovirus at a concentration of pfu / ml as a virus-to-platelet ratio of 25:1; take a concentration of 1×10 8 100 μl of platelets / ml was used to obtain a concentration of 1×10⁻⁶. 10 12.5 μl of pfu / ml oncolytic adenovirus was prepared to form a virus:platelet ratio of 12.5:1; a concentration of 1×10⁻⁶ was taken. 8 100 μl of platelets / ml was used to obtain a concentration of 1×10⁻⁶. 10 6.25 μl of pfu / ml oncolytic adenovirus was prepared into a virus:platelet ratio of 6.25:1; the suspension was placed on a shaker at 37°C and 220 rpm for 2 hours.

[0095] Then centrifuge at 800g-1200g for 8-10 minutes, collect the precipitate and supernatant, and use qPCR (a recognized method for virus detection) to detect the virus content in the supernatant and precipitate. Figure 2 (A and B) are used, where the virus content in the supernatant can be directly detected; when detecting the virus content in the precipitate, it is washed twice with a benchtop solution containing 3% sodium citrate. Figure 2 As shown in Figure B, the viral load in the precipitate was similar at virus-to-platelet ratios of 200:1, 100:1, 50:1, and 25:1. This indicates that the virus-to-platelet ratio was close to saturation at 25:1. Calculations show that a virus-to-platelet ratio of 25:1 during incubation resulted in the highest virus loading efficiency on platelets when platelets were saturated with the virus. Figure 2 (C); When the ratio of virus to platelet incubation is 25:1, each platelet carries approximately 6 viruses (C). Figure 2 (D).

[0096] 1.2. Optimization of the preparation of platelet-oncolytic adenovirus complex

[0097] This embodiment also optimizes the method of loading virus onto platelets. In Example 1.1, the optimal loading ratio of virus to platelets during incubation was calculated to be 25:1. In the optimization experiment, a concentration of 1×10⁻⁶ was used. 8 200 μl of platelets / ml was used to achieve a concentration of 1×10⁻⁶. 10 50 μl of pfu / ml oncolytic adenovirus was prepared into a virus:platelet ratio of 25:1. Then, the efficiency of platelet loading of oncolytic adenovirus was compared using four different methods: centrifugation, Triton X-100 (catalog number: T8200; Solarbio) punching, incubation, and vortexing. The effects of adding protamine sulfate on platelet loading of oncolytic adenovirus were also compared under these four methods: Centrifugation: The suspension was centrifuged at 200g for 10 min, then incubated on a shaker at 37℃ and 220 rpm for 2 hours; Incubation: The suspension was incubated on a shaker at 37℃ and 220 rpm for 2 hours; Triton X-100 punching: Triton X-100 was added to the suspension to a final concentration of 0.5%, then incubated on a shaker at 37℃ and 220 rpm for 2 hours; Vortexing: The suspension was vortexed for 5 min using a Scilogex vortex mixer (USA), which has three speed settings (high, medium, and low), with the medium setting selected. Then, they were placed on a shaker at 37°C and 220 rpm for 2 hours of incubation; each of the four groups was supplemented with protamine under the same conditions, forming a protamine control group. Figure 3 After incubating all groups for two hours, centrifuge at 800g-1200g for 8-10 minutes, collect the centrifuged precipitate, and detect the oncolytic adenovirus content in the precipitate by qPCR.

[0098] Depend on Figure 3As can be seen, no oncolytic adenovirus was detected by qPCR in all protamine groups. Comparing four different methods—centrifugation, Triton X-100 perforation, incubation, and vortexing—it was found that the incubation method is simple to operate, has high loading efficiency, avoids platelet lysis, and ensures efficient virus loading. Vortexing for 5 minutes or centrifugation for 10 minutes before incubation can increase the virus loading content on platelets, with 5 minutes of vortexing increasing the loading by 50%. Triton X-100 is a cell membrane perforator, generally used to evert the cell membrane, making it easier for extracellular substances to enter the cell. However, in this experiment, after perforating platelets with Triton X-100, the amount of oncolytic virus loaded on platelets decreased sharply. This may be because Triton X-100 perforator promotes platelet lysis, leading to a decrease in platelet count and reduced oncolytic virus loading efficiency. Therefore, subsequent experiments in this disclosure all used the vortex incubation method to prepare the platelet oncolytic adenovirus complex.

[0099] Example 2: Characteristics of the platelet-oncolytic adenovirus complex

[0100] The sample used in this embodiment is a product containing a platelet-oncolytic adenovirus complex prepared by the vortex incubation method in Example 1.

[0101] 2.1 Detection of platelet-oncolytic adenovirus complex

[0102] Platelets and platelet-oncolytic adenovirus complex were detected using scanning electron microscopy. Figure 4 A) The results showed that neither aggregated nor dispersed platelets exhibited significant damage due to the loading of oncolytic adenovirus. Transmission electron microscopy was used to examine the structure of oncolytic adenovirus loaded in platelets. Figure 4 B), the black dots indicated by the arrows in the figure are oncolytic adenovirus particles, showing that the oncolytic adenovirus was successfully loaded into platelets.

[0103] 2.2 Survival time of platelets and platelets loaded with oncolytic adenovirus in vivo

[0104] Platelets were extracted from EGFP transgenic mice (purchased from Vital River Pharmaceuticals, Beijing) using the following steps:

[0105] Characteristics of whole blood under flow cytometry without fluorescence channel ( Figure 5 A(1)), characteristics of platelets detected by flow cytometry without a fluorescence channel ( Figure 5 A(2)), characteristics of whole blood under flow cytometry detection in the green fluorescent channel ( Figure 5 A(3)), characteristics of platelets detected by flow cytometry without fluorescence channel under green fluorescence channel (A(3)) Figure 5A(4)). Flow cytometry analysis showed that the extracted green platelets accounted for 98.9% of the total platelets. Figure 5 A(4)).

[0106] Then, platelet-oncolytic adenovirus complex was prepared according to Example 1, 1×10 8 Platelet-oncolytic adenovirus complex and platelets were injected into mice via the tail vein. Peripheral blood samples of 10 μl were collected from the tail vein at 24 h, 48 h, 72 h, and 120 h, and platelet survival was assessed by flow cytometry. Flow cytometry analysis revealed similar circulation times between normal platelets and platelets loaded with oncolytic adenovirus (within the margin of error). The survival time and proportion of oncolytic adenovirus-loaded platelets were as follows: 2.37% at 24 h, 0.83% at 48 h, 0.74% at 72 h, and 0.05% at 120 h. Figure 5 B(1)); The survival time and proportion of normal platelets in the body were as follows: 2.41% at 24h, 0.7% at 48h, 0.56% at 72h, and 0.02% at 120h. Figure 5 B(2)), the platelet count is the sum of the second and third quadrants in the flow cytometry. Both can survive in the body for more than 3 days, and the survival rates are similar. By day 5, both are essentially metabolized and disappear. Platelets loaded with oncolytic adenovirus have the same metabolic cycle as normal platelets in the body. This indicates that there is no difference in their survival time and biological function in the body. Figure 5 B).

[0107] 2.3 Distribution of platelet-oncolytic adenovirus complex in mice

[0108] Platelets and platelet-oncolytic adenovirus complexes were stained with DIR (a fluorescent dye for in vivo imaging) (product code: KM0007, Bio-Rad) in vitro and then injected into Balb / c mice via the tail vein. The distribution of both substances in the mice was detected by in vitro imaging at 4h, 24h, 48h, and 5d. Five days later, the heart, liver, spleen, lung, and kidney were harvested for in vitro imaging to detect the aggregation location and intensity of both substances. Liver and spleen sections were stained with hematoxylin and eosin (HE) to examine for any damage to the aggregated organs. Figure 6 ).

[0109] The results showed that after platelets were loaded with oncolytic adenovirus, their distribution in the body was consistent with that of normal platelets, mainly concentrated in the liver and spleen. HE staining also revealed that intravenous injection of platelets and platelet-oncolytic adenovirus complexes into mice did not damage the liver and spleen. This indicates that the platelet-oncolytic adenovirus complex has no toxic side effects.

[0110] 2.4. Toxicity analysis of platelet-oncolytic adenovirus complex in mice

[0111] In the experiment described in Section 2.2, blood samples were also collected from mice, and their blood biochemical parameters were analyzed. The results showed that after injection of platelets, oncolytic adenovirus, and platelet-oncolytic adenovirus complex, there were no significant changes in platelet count (PLT), platelet volume (MPV), platelet distribution width (PDW), red blood cell count (RBC), white blood cell count (WBC), and lymphocyte count (Lymph) in the mouse blood. Figure 7 ).

[0112] Example 3: In vitro infection of CTCs with platelet-oncolytic adenovirus complex

[0113] Balb / c mice (purchased from Vital Rivers) were subcutaneously inoculated with 4T1 cells expressing red fluorescent protein (RFP) (catalog number: GOY-N0026, Shanghai Guyan Industrial Co., Ltd.) to construct a mouse triple-negative breast cancer lung metastasis model: 1×10⁶ cells were resuspended in 100 μl PBS. 6 Four T1-RFP cells were subcutaneously inoculated into 6-week-old Balb / c mice. After approximately 20 days, spontaneous lung metastases developed in the mice. Blood was collected from the orbital cavity on day 20 post-inoculation. Red blood cells were lysed and cultured. Flow cytometry was used to sort cells expressing red fluorescence, i.e., CTCs. The cultured CTCs were plated, and platelets from EGFP-transgenic mice were used to prepare a platelet-oncolytic adenovirus complex according to the method in Example 1. The transfection of CTCs by the platelet-oncolytic adenovirus complex was observed. Figure 8 Bright-field fluorescence superimposed images of mouse CTCs cells after co-incubation for 0, 6, 12, and 18 hours in the control group (platelets) and the platelet-oncolytic adenovirus complex group (platelets / oncolytic adenovirus). Figure 8 It is evident that the platelet-oncolytic adenovirus complex can carry the EGFP-encoding adenovirus (catalog number: HYFW20210617001, manufactured by Heyuan Biotechnology (Shanghai) Co., Ltd.) from platelets into CTCs within approximately 18 hours, thereby expressing green fluorescence in the CTCs. In contrast, the platelets in the control group did not carry the virus and therefore could not overexpress green fluorescence in the CTCs.

[0114] In this invention, the function of oncolytic adenovirus changes after binding with platelets: it can inject platelet contents into tumor cells.

[0115] Example 4: Platelet-oncolytic adenovirus complex induces CTC death

[0116] Materials used in this embodiment:

[0117] Oncolytic adenovirus (H101, Ankerui, Shanghai Sanwei Biotechnology Co., Ltd.).

[0118] CTCs (circulating tumor cells, tumor cells isolated from tumor-bearing mice in the triple-negative breast cancer lung metastasis model prepared in Example 3);

[0119] Mesenchymal stem cells (isolated and cultured from Balb / c mouse bone marrow);

[0120] Peripheral blood mononuclear cells (prepared from peripheral blood of Balb / c mice);

[0121] T cells (prepared from peripheral blood of Balb / c mice);

[0122] 4T1 (Item No.: CL-0007, Wuhan Pronosai Life Science Technology Co., Ltd.);

[0123] BT549 (Item No.: CL-0041, Wuhan Pronosai Life Science Technology Co., Ltd.);

[0124] MB468 (Item No.: CL-0290B, Wuhan Punosei Life Science Technology Co., Ltd.);

[0125] MCF-7 (Item No.: CL-0149, Wuhan Punosai Life Science Technology Co., Ltd.).

[0126] The effects on CTC lysis when the MOI ratio of the platelet-oncolytic adenovirus complex to CTCs was 10, 5, 2.5, and 0 were as follows: Figure 9 As shown in Figure A, the MTT assay showed no effect on the growth of normal somatic cells (mesenchymal stem cells (MSCs), peripheral blood mononuclear cells (PBMCs), and T cells). Figure 9 B). Platelet-oncolytic adenovirus complex can inhibit the growth of human breast cancer cell lines (4T1, BT549, MB468, MCF-7). Figure 9 C).

[0127] Example 5: Detection of platelet-oncolytic adenovirus complex inhibition of lung metastasis in triple-negative breast cancer in mice

[0128] Balb / c mice aged 6-8 weeks (purchased from Vital River) were subcutaneously inoculated with 1×10⁻⁶ mmol / L. 6 After removing 4T1 tumor cells, the tumor cells were randomly divided into four groups of five each.

[0129] Control group: normal saline was injected into the tail vein;

[0130] Platelet group: Platelets derived from concordant mice were injected into each mouse via the tail vein at a dose of approximately 5 × 10⁻⁶. 7 -1×10 8platelets;

[0131] Oncolytic adenovirus group: Each mouse was injected via tail vein with approximately 2.5 × 10⁻⁶ mmol / L. 8 -5×10 8 PFU oncolytic adenovirus (inject the same viral load as the platelet / oncolytic adenovirus group, based on an average of 5 oncolytic adenoviruses per platelet);

[0132] Platelet / oncolytic adenovirus group: Each mouse was injected via tail vein with approximately 5 × 10⁵ adenoviruses prepared according to the method in Example 1. 7 -1×10 8 Platelet-oncolytic adenovirus complex.

[0133] On days 7, 10, 13, and 16, 100 μl of physiological saline and 100 μl of platelets containing 5 × 10⁻⁶ were injected into the tail vein of mice, respectively. 7 One platelet, one oncolytic adenovirus 100μl contains 2.5×10 8 100 μl of platelet-oncolytic adenovirus complex contains 5 × 10⁶ platelets. 7 Oncolytic adenovirus approximately 2.5 × 10⁻⁶ 8 PFU. Mice were sacrificed after 21 days, and lung metastatic nodules were observed. It was found that the platelet-oncolytic adenovirus complex could significantly reduce lung metastasis. Figure 10 ).

Claims

1. A method for preparing a platelet-oncolytic adenovirus complex, in which the oncolytic adenovirus is loaded in platelets, the oncolytic adenovirus comprising a hTERT promoter, the method comprising the following steps: (1) obtaining platelets; (2) obtaining oncolytic adenovirus; (3) mixing the platelets and the oncolytic adenovirus to obtain a suspension, centrifuging at 200g for 10 min; (4) placing the suspension in a shaking incubator at 37°C at a speed of 220 rpm for 2 h.

2. A method for preparing a platelet-oncolytic adenovirus complex, in which the oncolytic adenovirus is loaded in platelets, the oncolytic adenovirus comprising a hTERT promoter, the method comprising the following steps: (1) obtaining platelets; (2) obtaining oncolytic adenovirus; (3) mixing the platelets and the oncolytic adenovirus to obtain a suspension, vortexing for 5 min; (4) placing the suspension in a shaking incubator at 37°C at a speed of 220 rpm for 2 h.

3. The platelet-oncolytic adenovirus complex prepared by the method of claim 1 or 2.

4. A composition comprising the platelet-oncolytic adenovirus complex of claim 3 and a pharmaceutically acceptable carrier.

5. Use of the platelet-oncolytic adenovirus complex of claim 3 or the composition of claim 4 in the preparation of a medicament for treating breast cancer in a subject in need thereof.

6. Use according to claim 5, wherein, The subject is a mouse, a rat, a rabbit, a cat, a dog, a horse, a non-human primate, or a human.

Images