Fusion protein and its use in the preparation of a drug for preventing and treating nervous system tumors

Abstract

The present application relates to the technical field of medicine, and particularly relates to a fusion protein and application thereof in preparation of a medicine for preventing and treating nervous system tumors. The fusion protein can specifically target lesion cells overexpressing AQP4 through AQP4 antibodies, deliver an effector fragment Apoptin into tumor cell nuclei to play a role, and specifically induce tumor cell apoptosis without damaging normal cells, and can be used for preventing and treating diseases caused by abnormal proliferation of nervous system cells. The present application further provides a medicine, and an active ingredient of the medicine comprises the fusion protein or the biomaterial.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to fusion proteins and their application in the preparation of drugs for the prevention and treatment of nervous system tumors. Background Technology

[0002] Abnormal proliferation of nervous system cells can lead to serious ocular and intracranial lesions. Retinoblastoma (Rb), for example, is the most common primary malignant intraocular tumor in childhood, and its pathogenesis is closely related to the biallelic inactivation of the Rb tumor suppressor gene. This disease not only seriously threatens the lives of affected children, but its location also directly affects visual development, making it a significant cause of childhood blindness. Due to the specific characteristics of this patient group, how to remove the tumor while protecting the ocular structure and salvaging vision has always been a major clinical challenge.

[0003] Currently, clinical intervention for diseases caused by abnormal proliferation of nervous system cells, especially retinoblastoma, has shifted from simple enucleation to comprehensive treatments such as systemic chemotherapy and intra-arterial chemotherapy. However, conventional chemotherapy drugs lack tumor specificity, indiscriminately damaging normal nerve cells while killing tumor cells. Furthermore, the blood-eye barrier limits the effective concentration of drugs at the lesion site; increasing the dosage exacerbates systemic toxicity. Simultaneously, the heterogeneity of tumor cells easily induces multidrug resistance and disease relapse, resulting in poor prognosis.

[0004] Studies have shown that the VP3 protein of chicken anemia virus has a specific apoptosis-inducing effect on tumor cells and abnormally transformed cells from various human tissues, while having no toxic side effects on normal tissue cells. It has been named a specific tumor apoptosis factor (Apoptin). Apoptin promotes tumor cell apoptosis by inhibiting the stress-induced expression of heat shock protein 70 (HP70). HP70 is a heat shock protein that inhibits tumor cell apoptosis. Apoptin binds to the HP70 gene, preventing its transcription and thus inhibiting HP70 expression, thereby inducing tumor cell apoptosis. HP70 expression is only initiated under stress; under normal conditions, cells do not exhibit stress-induced HP70 expression. However, tumor cells upregulate HP70 expression, therefore Apoptin only induces tumor cell apoptosis and does not cause apoptosis in normal cells. Transcription occurs within the cell nucleus; for Apoptin to bind to HP70 and prevent transcription, it must first enter the tumor cell nucleus.

[0005] However, the clinical application of apoptin faces challenges: due to species differences, human cell membranes lack apoptin-associated receptors, preventing apoptin from binding to human cells, including tumor cells. Apoptin must enter tumor cells to induce apoptosis, and this theoretical limitation restricts its application. Furthermore, apoptin, as a viral protein, exhibits low in vitro stability; repeated exposure can trigger antibody responses, which not only reduces its efficacy but may also cause immune damage.

[0006] In summary, there is an urgent need for a novel targeted agent that can efficiently and stably deliver Apoptin to the lesion. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a fusion protein and its application in the preparation of drugs for the prevention and treatment of nervous system tumors.

[0008] This invention provides the application of AQP4 antibody as a delivery vector targeting nervous system cells.

[0009] When nerve cells encounter pathological conditions such as cerebral ischemia / stroke, physical trauma, brain tumors, or neuroinflammation, they activate self-protection or coping mechanisms, leading to a significant overexpression of AQP4 (aquaporin 4) on the cell membrane. This invention uses an AQP4 antibody as a carrier to specifically deliver effector molecules to diseased nerve cells at the lesion site, solving the problems of low cell entry efficiency and ineffective targeting of effector molecules to the lesion.

[0010] In this invention, the AQP4 antibody, used as a delivery carrier, delivers drugs including, but not limited to, protein drugs. Specifically, the functions of these protein drugs include, but are not limited to, inducing apoptosis, inhibiting cell proliferation, or killing tumor cells. The AQP4 antibody of this invention, as a targeted delivery carrier, exhibits excellent specificity, delivering effector molecules to diseased nerve cells without producing toxic side effects on normal nerve tissue, demonstrating excellent safety.

[0011] The present invention describes a drug delivered using the AQP4 antibody as a delivery carrier, which is either mixed with the AQP4 antibody, conjugated with the AQP4 antibody, or conjugated with the AQP4 antibody via a linker. The present invention does not limit this to any particular method.

[0012] In this invention, the AQP4 antibody can be a humanized antibody, a chimeric antibody, or a murine antibody, as long as it has good targeting properties with AQP4, it can be used as a delivery vector. It can be a complete antibody or an antibody fragment retaining binding activity, such as a Fab fragment, an F(ab')2 fragment, or a single-chain antibody scFv. Preferably, the AQP4 antibody is a single-chain antibody.

[0013] This invention provides a fusion protein comprising fragment A and fragment B, wherein:

[0014] Fragment A is an AQP4 antibody;

[0015] Fragment B is the full length of Apoptin or at least one of its fragments.

[0016] In some embodiments, the structure of the fusion protein is: [(fragment A)x-(fragment B)y]z or [(fragment B)y-(fragment A)x]z.

[0017] In the structure of the fusion protein, x represents the number of repetitions of fragment A, y represents the number of repetitions of fragment B, and z represents the number of repetitions of the unit composed of fragments A and B in the fusion protein. x, y, and z are each independently selected from non-zero integers. For example, 1 ≤ x ≤ 10, 1 ≤ y ≤ 10, and 1 ≤ z ≤ 10. The values ​​of x, y, and z can be the same or different, and this invention does not impose any particular restrictions on this. Taking x = 1, y = 1, and z = 1 as an example, the structure of the fusion protein of this invention is either fragment A-fragment B or fragment B-fragment A.

[0018] When the number of repetitions is not 1, the sequences of the two repetitive segments may be the same or different, and this invention does not limit this. For example, when y=2, the two segments B may be the same or different, and this invention does not limit this. When z=2, the segments B in the two repetitive units may be the same or different, and this invention also does not limit this. Taking x=1, y=2 and z=2 as an example, the structure of the fusion protein of this invention is as follows:

[0019] [Fragment A-Fragment B2]2, that is: Fragment A-Fragment B-Fragment B-Fragment A-Fragment B-Fragment B;

[0020] Or [fragment B2-fragment A]2, that is: fragment B-fragment B-fragment A-fragment B-fragment B-fragment A.

[0021] In the fusion protein of this invention, fragment B is the full length of Apoptin or at least one truncated fragment of Apoptin. For example, fragment B may include one truncated fragment of Apoptin, or two truncated fragments of Apoptin, or three truncated fragments of Apoptin, or four truncated fragments of Apoptin; this invention is not limited in this respect. Preferably, fragment B contains two truncated fragments of Apoptin, specifically the hydrophobic amino acid-rich region (LRS) and the nuclear localization signal region (NLS) of Apoptin.

[0022] As a feasible example, the hydrophobic amino acid-rich region of the Apoptin has an amino acid sequence as shown in SEQ ID NO.9; the nuclear localization signal region of the Apoptin has an amino acid sequence as shown in SEQ ID NO.10.

[0023] This invention does not limit the connection order or number of multiple Apoptin truncated fragments in fragment B. Taking fragment B as an example, which consists of the hydrophobic amino acid-rich region (LRS) and the nuclear localization signal region (NLS) of Apoptin, its structure from N-terminus to C-terminus is LRS-NLS or NLS-LRS. Alternatively, if the number of LRS is 2 and the number of NLS is 1, then its structure from N-terminus to C-terminus is LRS-LRS-NLS, or LRS-NLS-LRS, or NLS-LRS-LRS. Alternatively, if the number of LRS is 1 and the number of NLS is 2, then its structure from N-terminus to C-terminus is LRS-NLS-NLS, or NLS-LRS-NLS, or NLS-NLS-LRS. This invention does not limit this.

[0024] This invention fuses AQP4 scFv with the LRS (1-46aa) and NLS1-NLS2 (82-121) positions of Apoptin to prepare a novel recombinant protein, AQP4 scFv-LRS-NLS, which targets and induces apoptosis in Y79 cells. The AQP4 scFv-LRS-NLS recombinant protein exerts both the targeted binding of AQP4 scFv to AQP4 on the surface of Y79 somatic cells and the specific apoptosis-inducing pharmacological effect of Apoptin, while simultaneously reducing immunogenicity and increasing in vivo half-life. This provides a novel genetically engineered product for the treatment of Rb tumors that specifically induces tumor cell apoptosis.

[0025] In this invention, fragment A is a single-chain antibody of AQP4.

[0026] The CDR region of the heavy chain variable region of the single-chain antibody of AQP4 has the amino acid sequences shown in SEQ ID NO. 1~3 in sequence;

[0027] The CDR region of the light chain variable region of the single-chain antibody of AQP4 has the amino acid sequences shown in SEQ ID NO.4~6 in sequence.

[0028] More specifically, the heavy chain variable region of the single-chain antibody of AQP4 has the amino acid sequence shown in SEQ ID NO.7, and the light chain variable region has the amino acid sequence shown in SEQ ID NO.8.

[0029] As a feasible example, the fusion protein of the present invention comprises, from the N-terminus to the C-terminus, the following components in sequence: the heavy chain variable region of the single-chain antibody of AQP4, linker1, the heavy chain variable region of the single-chain antibody of AQP4, linker2, the hydrophobic amino acid-rich region of Apoptin, linker3, and the nuclear localization signal region of Apoptin.

[0030] Optionally, the N-terminus of the fusion protein of the present invention further includes a signal peptide; in the present invention, the signal peptide can be a prokaryotic expression signal peptide or a eukaryotic expression signal peptide, and a suitable signal peptide sequence can be selected according to different expression systems to help the recombinant protein fold correctly and be secreted for expression. In a specific embodiment, the signal peptide has the amino acid sequence shown in SEQ ID NO.11.

[0031] Optionally, the C-terminus of the fusion protein described in this invention further includes a screening tag. In this invention, the screening tag is used to isolate and purify the expressed fusion protein. As a feasible example, the screening tag includes at least one of a His tag, a GST tag, a HA tag, and a Flag tag; this invention does not specifically limit this, but in a specific embodiment, a 6*His tag is used.

[0032] Optionally, linker1 to linker3 are each independently a flexible linker, specifically employing conventional flexible peptide sequences in the art, such as (GGGGS)n, where n is an integer not less than 1, and generally n ranging from 1 to 3 is sufficient. In a specific embodiment of the present invention, n=1. In a specific embodiment, linker1 is (G4S)3; linker2 or linker3 is G3S.

[0033] In one specific embodiment, the fusion protein has the amino acid sequence shown in SEQ ID NO.12.

[0034] Furthermore, the present invention also provides any one or at least one of the following biomaterials:

[0035] I) Nucleic acid encoding the fusion protein as described above;

[0036] II) Expression units containing the nucleic acid described in I);

[0037] III) A vector containing the nucleic acid described in I) or the expression unit described in II);

[0038] IV) Transforming or transfecting a host with the vector described in III);

[0039] The expression products of the host as described in (V) and (IV).

[0040] In this invention, the encoded nucleic acid is DNA or RNA. The nucleic acid molecule described in this invention can not only serve as a template for intracellular expression of fusion proteins, but also exist independently and function as an active pharmaceutical ingredient. In specific embodiments, the nucleic acid is constructed in a recombinant expression vector for transforming host cells to produce the fusion protein; alternatively, the nucleic acid molecule can be used directly as a pharmaceutical preparation without expression, i.e., a DNA drug or an mRNA drug. When preparing DNA or RNA drugs from nucleic acid molecules, the nucleic acid is combined with pharmaceutical excipients or carriers such as lipid nanoparticles and polymers, or with regulatory elements, to achieve efficient in vivo delivery and translation, thereby generating therapeutic proteins in situ on target cells.

[0041] As a feasibility example, the nucleic acid described in this invention has a nucleic acid sequence as shown in SEQ ID NO:13. When used as an expression template, it exists in an expression vector or in the genome of a host cell. When used as a drug formulation, taking DNA drugs as an example, it can be administered via naked DNA injection, gene gun delivery, or liposome delivery, enabling target cells to express the fusion protein and exert a therapeutic effect. Taking mRNA drugs as an example, mRNA can be administered after being encapsulated in lipid nanoparticles.

[0042] In this invention, the expression unit includes the nucleic acid and promoter as described above. As a feasible example, the promoter includes, but is not limited to, constitutive promoters, inducible promoters, tissue-specific promoters, or tumor-specific promoters. For example, using a neurological tumor-specific promoter can further enhance the specific expression of the fusion protein in the lesion region, reduce the impact on normal tissues, and improve treatment safety. Alternatively, if the expression unit of this invention is only used for the in vitro expression of the fusion protein, the promoter can be a conventional constitutive promoter corresponding to a prokaryotic or eukaryotic expression system; this invention does not impose any special limitations on this. The expression unit of this invention can be integrated into the host genome as needed, or exist in the host cell in the form of free nucleic acid.

[0043] The vector described in this invention can be a prokaryotic expression vector or a eukaryotic expression vector, including various types such as plasmid vectors and viral vectors. Conventional vectors in the art can be used in this invention, as long as they can achieve the replication, amplification, or expression of the nucleic acid or expression unit; no particular limitation is made. In a specific embodiment, the vector is pATX2. Other vectors in the art can also be used for the expression of the fusion protein, such as the pET series, pGEX series prokaryotic expression vectors, or the pcDNA series eukaryotic expression vectors, all of which fall within the scope of protection of this invention.

[0044] In this invention, the host is used for the storage or amplification of the nucleic acid encoded by the fusion protein, or for the expression of the fusion protein. It can be a prokaryotic host or a eukaryotic host. As a feasible example, the prokaryotic host includes *Escherichia coli*, and the eukaryotic host includes yeast cells, CHO cells, and HEK293 cells. In a specific embodiment, CHO cells are used to express the fusion protein.

[0045] In this invention, the host expression product includes, but is not limited to, cell lysate containing the fusion protein, supernatant containing the fusion protein, purified fusion protein, and protein derivatives containing the fusion protein. All of these fall within the scope of the host expression product of this invention. As long as they contain the fusion protein of this invention, they can be used in drugs for the prevention and treatment of nervous system tumors.

[0046] The present invention also provides a method for preparing the fusion protein, which includes culturing a host as described above to obtain a culture containing the fusion protein. In this invention, after the fusion protein is prepared, a purification step is further included, specifically comprising: filtering the expression product to obtain a supernatant, precipitating it with saturated ammonium sulfate, dialysis, and then purifying it using an anion exchange chromatography column.

[0047] More specifically, the preparation of the fusion protein includes:

[0048] (1) Construction, fermentation and recovery of AQP4 scFv-LRS-NLS fusion protein mammalian cell line: The artificially synthesized SEQ1 gene fragment was subcloned into a mammalian cell expression vector, transfected into a mammalian cell line for expression, and a high-efficiency expression cell line was screened to express the AQP4 scFv-LRS-NLS fusion protein.

[0049] (2) Preparation of AQP4 scFv-LRS-NLS fusion protein: Select the cell culture supernatant of the high-efficiency expression cell line in (1), add an equal volume of saturated ammonium sulfate, mix well, place at 4℃ for more than 6 hours, centrifuge at 10000g for 30min at 4℃ to collect the precipitate, dissolve the precipitate with an appropriate amount of equilibration buffer, and dialyze with equilibration buffer for 48 hours, changing the buffer 5 times in between; store the sample at -20℃ for later use.

[0050] (3) Purification of AQP4 scFv-LRS-NLS fusion protein sample: Purify the anion exchange chromatography column by equilibrating with equilibration buffer. The sample prepared in (2) above is purified by equilibration column, and eluted with equilibration buffers containing 0.1 mol / L, 0.2 mol / L, 0.5 mol / L and 1 mol / L NaCl respectively. Collect the protein fraction containing a molecular weight of 34.0 kD. Equilibrate the Separose12 chromatography column with equilibration buffer. Load the concentrated 34.0 kD protein fraction at a ratio of 2-5% of the column volume at a flow rate of 1.5 ml / min. Collect in steps. After identification by SDS electrophoresis, the protein fraction with a molecular weight of 34.0 kD and high purity is collected as AQP4 scFv-LRS-NLS fusion protein.

[0051] Furthermore, the present invention also provides a drug comprising the fusion protein as described above, or comprising at least one of the biological materials as described above, or comprising a culture prepared by the preparation method as described above.

[0052] The drug described in this invention is a protein-based drug, specifically including the fusion protein as described above or the culture prepared by the method described above. Such a drug may also include pharmaceutically acceptable excipients. The drug can be prepared into suitable dosage forms, such as injections or lyophilized powder injections, depending on the route of administration and method of delivery; this invention does not impose any particular limitation on these. The drug can be administered to the subject via local or systemic administration.

[0053] Alternatively, the drug described in this invention may be a nucleic acid drug, that is, it contains nucleic acid encoding a fusion protein as described above, or contains an expression unit or carrier containing the nucleic acid; the nucleic acid drug may also contain pharmaceutically acceptable excipients that can be used for nucleic acid delivery, such as nanodelivery carriers, liposomes and other excipients, to achieve effective in vivo delivery of nucleic acid, ensure that the nucleic acid can be stably expressed in target cells to obtain a fusion protein with therapeutic activity, and exert therapeutic and / or preventive effects.

[0054] The medicament described in this invention also includes other active substances or drugs with therapeutic and / or preventive effects. For example, it includes at least one of radiosensitizers, chemotherapy drugs, and immunotherapy drugs for diseases related to abnormal proliferation of nervous system cells. These active substances or drugs, when used in combination with the fusion protein or biomaterial described in this invention, synergistically enhance the prevention and treatment of nervous system tumors, reduce the dosage of single-drug therapy, and reduce the occurrence of adverse reactions.

[0055] Furthermore, the present invention also provides the use of the fusion protein as described above, at least one of the biomaterials, the culture obtained by the preparation method, or the drug as described above in at least one of the following:

[0056] i) Inducing apoptosis in nervous system cells;

[0057] ii) To prepare drugs for the prevention and treatment of diseases related to abnormal proliferation of nervous system cells.

[0058] In this invention, diseases related to abnormal proliferation of nervous system cells can include nervous system tumors caused by abnormal proliferation of nerve cells. As a feasible example, the nervous system tumors include brain tumors and central nervous system tumors, specifically including but not limited to retinoblastoma, glioma, astrocytoma, meningioma, and pituitary adenoma.

[0059] In this invention, the nervous system cells are AQP4-overexpressing nervous system cells, such as cells from nervous system tumors. As a feasible example, the nervous system cells are retinoblastoma Y79 cells.

[0060] In a specific embodiment, the nervous system cells include retinoblastoma cells; the diseases related to abnormal proliferation of nervous system cells include retinoblastoma.

[0061] The present invention also provides a method for inducing apoptosis of nervous system cells, comprising administering the fusion protein as described above, at least one of the biological materials, the culture prepared by the preparation method, or the drug described above.

[0062] The present invention also provides a method for preventing and treating diseases related to abnormal proliferation of nervous system cells, comprising administering the fusion protein as described above, at least one of the biomaterials, the culture prepared by the preparation method, or the drug described above.

[0063] In this invention, the methods of administration include, but are not limited to, intravenous injection, arterial injection, intrathecal injection, local injection, oral administration, transdermal administration, intracavitary administration, and implantation administration. The appropriate administration method can be selected according to the specific type of tumor, the location of the lesion, and the stage of development. For example, intravitreal injection can be selected for retinoblastoma, local intratumoral injection can be selected for tumors in the parenchyma of the central nervous system, and intravenous systemic administration can be selected for multiple metastatic lesions throughout the body.

[0064] In this invention, the objects to which the application is made include, but are not limited to, humans or mammals, such as humans, mice, rats, monkeys, guinea pigs, pigs, cattle, sheep, etc. Specifically, they may be individuals or mammals suffering from diseases related to abnormal proliferation of nervous system cells, or individuals or mammals at risk of developing nervous system tumors.

[0065] The fusion protein described in this invention can specifically target diseased cells overexpressing AQP4 via an AQP4 antibody, delivering the effector fragment Apoptin into the tumor cell nucleus to exert its effect, specifically inducing tumor cell apoptosis without damaging normal cells. It can be used to prevent and treat diseases caused by abnormal proliferation of nervous system cells. This invention also provides a drug, the active ingredient of which includes the fusion protein or the biomaterial. Attached Figure Description

[0066] Figure 1 The purification results of the AQP4 scFv-LRS-NLS fusion protein are shown: 1 is the molecular weight, 2 is the purified sample, and 3 is the sample before purification.

[0067] Figure 2 The reaction of AQP4 scFv-LRS-NLS fusion protein with Y79 cells was shown by immunofluorescence assay. A: AQP4 scFv-LRS-NLS fusion protein; B: cell control.

[0068] Figure 3 This demonstrates the inhibitory effect of AQP4 scFv-LRS-NLS fusion protein on the growth of Y79 cells in vitro;

[0069] Figure 4 This demonstrates the apoptosis-inducing effect of the AQP4 scFv-LRS-NLS fusion protein on Y79 cells;

[0070] Figure 5 This demonstrates the inhibitory effect of AQP4 scFv-LRS-NLS fusion protein on the growth of Y79 cells in vivo;

[0071] Figure 6 The tumor volume is shown under the action of AQP4 scFv-LRS-NLS fusion protein. Detailed Implementation

[0072] This invention provides fusion proteins and their application in the preparation of drugs for the prevention and treatment of nervous system tumors. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0073] Unless otherwise defined in this invention, the scientific and technical terms related to this invention shall have the meanings understood by one of ordinary skill in the art.

[0074] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0075] The term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist simultaneously, or B exists alone. A and B can be singular or plural.

[0076] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0077] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0078] In this invention, the "fusion protein" refers to a single protein formed by linking two or more functional protein fragments from different sources, which can simultaneously retain the biological functions of each original fragment. The fusion protein of this invention includes an AQP4 single-chain antibody fragment capable of targeting and binding to AQP4, and an effector protein fragment capable of inducing tumor cell apoptosis. It can also be linked to a nuclear localization signal sequence that helps the effector protein enter the cell nucleus to exert its function. Through the synergistic effect of these fragments, specific killing of AQP4-overexpressing nervous system tumor cells is achieved.

[0079] In this invention, the term "fragment" refers to an amino acid sequence unit with independent biological function. Each functional fragment in the fusion protein of this invention can independently retain its original biological activity and work synergistically in the fusion protein. Different fragments are linked together by amide bonds.

[0080] In this invention, the "single-chain antibody against AQP4" refers to a small molecule antibody fragment formed by linking the heavy chain variable region and the light chain variable region of an anti-AQP4 antibody with a flexible linker peptide, thus retaining the antibody's specific binding ability to the antigen.

[0081] In this invention, "Apoptin" refers to the VP3 protein derived from Chicken Anemia Virus (CAV), a viral protein with selective apoptosis-inducing function in tumor cells. "Full-length Apoptin" refers to the complete amino acid sequence containing amino acids 1 to 121, which includes the hydrophobic region and nuclear localization signal region necessary for its pro-apoptotic activity. "Fragment of Apoptin" refers to a functional fragment of any length within the full-length sequence, as long as it retains the ability to induce apoptosis by inhibiting the transcription of heat shock protein 70 (HSP70). In a specific embodiment of this invention, a combined fragment containing a hydrophobic amino acid-rich region (LRS) of amino acids 1-46 and a nuclear localization signal region (NLS) of amino acids 82-121 is preferred; this combined fragment is sufficient to mediate the fusion protein's targeted pro-apoptotic function.

[0082] In this invention, the "signal peptide" is a short peptide sequence, typically 15-30 amino acids in length, that guides secretory proteins into the secretory pathway. Located at the N-terminus of the nascent polypeptide chain, it guides the protein through the secretory pathway to cross the cell membrane, facilitating subsequent separation and purification of the protein product. In this invention, a suitable signal peptide can be selected based on the chosen expression system. For example, when selecting a mammalian cell expression system, the signal peptide of a corresponding naturally occurring mammalian secretory protein can be chosen, or an artificially optimized universal signal peptide sequence can be selected, as long as it can mediate the secretory expression of the fusion protein described in this invention. In a specific embodiment of this invention, the signal peptide sequence used has the sequence shown in SEQ ID NO. 11.

[0083] In this invention, "biological material" refers to biological material containing the fusion protein encoding information of the present invention, or biological material capable of expressing the fusion protein of the present invention. Specifically, it may include expression vectors or expression units containing nucleic acids encoded by the fusion protein, or host cells containing the nucleic acids or vectors. As long as such biological material can be used to express the fusion protein of the present invention, it falls within the protection scope of the present invention.

[0084] In this invention, the term "medicine" refers to a composition for the prevention or treatment of diseases related to abnormal proliferation of nervous system cells, comprising an effective dose of the fusion protein of this invention or a biological material capable of expressing the fusion protein, which can be used alone or in combination with other therapeutic active ingredients and pharmaceutical excipients to achieve the prevention and treatment of nervous system tumors.

[0085] In this invention, "prevention and treatment" refers to prevention and / or treatment. Prevention refers to reducing the risk of disease occurrence and delaying disease progression, while treatment refers to inhibiting tumor cell proliferation at the lesion site, inducing tumor cell apoptosis, reducing tumor volume, and prolonging the survival of the affected individual. The fusion protein described in this invention can be administered before the onset of disease to reduce the risk of disease, or administered after the onset of disease to achieve a therapeutic effect; both fall within the scope of "prevention and treatment" as described in this invention.

[0086] In this invention, "inducing apoptosis of nervous system cells" refers to delivering a pro-apoptotic fragment into the nucleus of AQP4-overexpressing nervous system tumor cells, activating the tumor cell apoptosis program, increasing the apoptosis rate of tumor cells, thereby inhibiting tumor cell proliferation and exerting an anti-tumor effect. This process has no significant pro-apoptotic effect on normal nervous system cells that do not express or express low levels of AQP4, and has good tumor cell specificity.

[0087] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0088] The sequences involved in this invention include:

[0089] Heavy chain CDR1: GFTFSNYG (SEQ ID NO.1);

[0090] Heavy chain CDR2: ISSGGDT (SEQ ID NO.2);

[0091] Heavy chain CDR3: TRGNSREPY (SEQ ID NO.3);

[0092] Light chain CDR1: QDINGY (SEQ ID NO.4);

[0093] Light chain CDR2: RAN (SEQ ID NO.5);

[0094] Light chain CDR3: LQSDDFPLT (SEQ ID NO.6);

[0095] SEQ ID NO.7 30-B8-F12-1-A11-E2[VH]

[0096] EVKMVESGGGLVKPGESLKVSCAAS GFTFSNYG MSWVRQTPEKRLEWVAS ISSGGDT YYLESVKGRFTISRENDRNILYLQMSSLRSEDTAMYYC TRGNSREPY WGQGTLVTVSA Note: The underlined area is the CDR area.

[0097] SEQ ID NO.8 30-B8-F12-1-A11-E2[VL]

[0098] MKHLWFFLLLVAAPRWVLSDIKMTQSPSSMYASLGERVTITCKAS QDINGY LSWFQQKPGKSPRTLIY RAN RLIDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYSC LQSDDFPLT FGAGTKLELR Note: The underlined area is the CDR area.

[0099] SEQ ID NO.9: LRS amino acid sequence (46aa), PRT, Chicken anemia virus

[0100] MNALQEDTPPGPSTVFRPPTSSRPLETPHCDEIRIGIAGITITLSL

[0101] SEQ ID NO.10: NLS amino acid sequence (39aa), PRT, Chicken anemia virus

[0102] PPSKKRSCDPSEYRVSELKESLITTTPSRPRTARRRRIRL

[0103] SEQ ID NO.11: Signal peptide, PRT, MKHLWFFLLLVAAPRWVLS

[0104] SEQ ID NO.12 AQP4 scFv-LRS-NLS:

[0105] MKHLWFFLLLVAAPRWVLSDIKMTQSPSSMYASLGERVTITCKAS QDINGY LSWFQQKPGKSPRTLIY R AN RLIDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYSC LQSDDFPLT FGAGTKLELRGGGGSGGGGSGGGGSEVKMVESGGGLVKPGESLKVSCAAS GFTFSNYG MSWVRQTPEKRLEWVAS ISSGGDT YYLESVKGRFTISRENDRNILYLQMSSLRSEDTAMYYC TRGNSREPYWGQGTLVTVSAGGGSMNALQEDTPPGPSTVFRPPTSSRPLETPHCDEIRIGIAGITITLSLGGGSPPSKKRSCDPSEYRVSELKESLITTTPSRPRTARRRIRLGSHHHHHH

[0106] SEQ ID NO.13 AQP4 scFv-LRS-NLS:

[0107]

[0108] SEQ ID NO.14 30-B8-F12-1-A11-E2[VL]:

[0109] GAATTCGCCGCCACCATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCTAGATGGGTGCTGTCCGACATCAAGATGACCCAGTCTCCATCCTCTATGTACGCCAGCCTGGGCGAGAGAGTGACCATTACCTGCAAGGCCAGCCAGGACATCAACGGCTACCTGTCCTGGTTCCAGCAGAAGCCTGGCAAGTCTCCCCGGACACTGATCTACAGAGCCAACAGACTGATCGACGGCGTGCCCTCCAGATTCTCCGGATCTGGATCTGGCCAGGACTACTCCCTGACCATCTCCAGCCTGGAATACGAGGATATGGGCATCTACAGCTGCCTGCAGTCCGACGACTTCCCACTGACATTTGGCGCTGGCACCAAGCTGGAATTGAGA

[0110] SEQ ID NO.15 30-B8-F12-1-A11-E2[VH]:

[0111] GAAGTGAAGATGGTGGAATCTGGCGGCGGACTGGTTAAGCCAGGCGAGTCCCTGAAAGTGTCCTGTGCCGCTTCTGGCTTCACCTTCTCCAACTACGGCATGTCCTGGGTCCGACAGACCCCTGAGAAGAGACTGGAATGGGTCGCCTCCATCTCTTCCGGCGGAGACACCTACTACCTGGAATCCGTGAAGGGCCGCTTTACCATCAGCCGCGAGAACGACCGGAACATCCTGTACCTGCAGATGTCCTCTCTGCGGAGCGAGGACACCGCCATGTACTACTGTACCCGGGGCAACTCCAGAGAGCCTTATTGGGGACAGGGCACCCTGGTCACAGTGTCTGCTGGCTCTCACCACCACCAT CACCAT

[0112] Gene sequence of SEQ ID NO.16 LRS, DNA, Chicken anemia virus.

[0113] AACGCTCTCCAAGAAGATACTCCACCCGGACCATCAACGGTGTTCAGGCCACCAACAAGTTCACGGCCGTTGGAAACCCTCACTGCAGAGAGATCCGGATTGGTATCGCTGGAATTACAATCACTCTATCGCTGTGT

[0114] Gene sequence of SEQ ID NO.17 NLS, DNA, Chicken anemia virus.

[0115] CCCTCGAAGAAGCGATCCTGCGACCCCTCCGAGTACAGGGTAAGCGAGCTAAAAGAAAGCTTGATTACCACTACTCCCAGCCGACCCGAACCGCAAGAAGGCGTATAAGACTGTAAGTCGACTGAGCGGCCGC

[0116] The gene sequence of the linker in SEQ ID NO.18, DNA

[0117] GGAGGTGGAAGT

[0118] Linker amino acid sequence (4aa), PRT, GGGS

[0119] It should be understood that in the various embodiments of this application, the sequence numbers of the above processes do not imply the order of execution. Some or all steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application. The present invention is further illustrated below with reference to embodiments:

[0120] Example 1: Preparation and purification of AQP4 scFv-LRS-NLS fusion protein

[0121] Solution preparation: serum-free 1640 medium.

[0122] Saturated ammonium sulfate: Prepare in units of 1000 ml according to the following proportions: ammonium sulfate 1000g or more

[0123] Equilibration solution: Prepare 20 mmol PB buffer (pH 7.2) in 1000 ml units according to the following proportions: sodium dihydrogen phosphate·2H₂O 3.12 g, disodium hydrogen phosphate·12H₂O 7.16 g, EDTA 0.37 g.

[0124] (1) The AQP4 scFv-LRS-NLS fusion protein gene fragment was directly obtained by artificial synthesis, and the AQP4 scFv-LRS-NLS fusion protein gene was constructed. It was then directionally subcloned into the expression plasmid pATX2 EcoRI and NotI restriction sites. The gene was then subcloned into a mammalian cell expression vector and transfected into mammalian cells using liposomes. The transfection method for CHO cells was as follows: the original culture medium of CHO cells was discarded, and about 5 mL of fresh culture medium was added. Then, 100 μl of DNA / DOTAP mixture was added. The culture flask was gently shaken to distribute the mixture evenly in the culture medium. The cells were cultured in a cell culture incubator at 37°C for 10 h, and then replaced with fresh complete 1640 culture medium. After 48 h, G418 (800 μg / mL) was added for selection. Untransfected cells and empty vector cells were used as negative controls. Cells were cultured in serum-free 1640 culture medium. Each experimental group was tested every 24 hours. h Collect the supernatant, filter it through a 0.45μm filter, and store the supernatant at -20℃; collect the cell culture supernatant, screen for high-efficiency expression cell lines, and express the AQP4 scFv-LRS-NLS fusion protein;

[0125] (2) The purity of the AQP4 scFv-LRS-NLS fusion protein raw material was purified by gel chromatography and examined by SDS-PAGE.

[0126] Specific steps:

[0127] Highly expressed cell lines were cultured in serum-free 1640 medium for 48 h. The supernatant was collected and filtered through a 0.45 μm filter. Saturated ammonium sulfate was slowly added to the culture supernatant at a 1:1 ratio, and the mixture was incubated at 4°C for at least 6 hours. The supernatant was then centrifuged at 10,000 g for 30 min at 4°C to collect the precipitate. The precipitate was dissolved in an appropriate amount of equilibration buffer, and the equilibration buffer was dialyzed for at least 24 hours, with 5 buffer changes in between. The sample was then purified using an anion exchange chromatography column equilibrated with the equilibration buffer or at -20°C. Store for later use. During purification, elute with equilibration buffers containing 0.1 mol / L, 0.2 mol / L, 0.5 mol / L, and 1 mol / L NaCl, respectively. Collect the elution peaks and identify them by SDS-PAGE. Collect the fraction containing the 34.0 kDa protein. Equilibrate the Separose 12 column with equilibration buffer. Load the concentrated 34.0 kDa protein fraction at a bed volume ratio of 2-5% at a flow rate of 1.5 ml / min. Collect in steps. After SDS-PAGE identification, collect the high-purity 34.0 kDa protein fraction, which is the AQP4 scFv-LRS-NLS fusion protein. Results show that the purity of the AQP4 scFv-LRS-NLS fusion protein can reach over 95%. Figure 1 ,).

[0128] Example 2: Binding of AQP4 scFv-LRS-NLS fusion protein to Y79 cells

[0129] The binding of the AQP4 scFv-LRS-NLS fusion protein to the Y79 cell line was examined using indirect immunofluorescence assay. The specific steps are as follows:

[0130] Experimental materials: Human retinoblastoma, preferably Y79 cell line, purchased from Zhejiang Meisen Cell Technology Co., Ltd., cultured in DMEM / F12 medium containing 10% fetal bovine serum at 37℃ and 5% CO2 saturated humidity.

[0131] Experimental methods: A Y79 cell line suspension was prepared at 5 × 10⁴ cells / ml. The suspension was plated onto cell slides, air-dried, and fixed with acetone for 20 min. The results were detected using a standard indirect immunofluorescence assay. The scFv-LRS-NLS fusion protein was found to bind to the Y79 cell line. Figure 2 (A)

[0132] Example 3: Growth inhibitory activity of AQP4 scFv-LRS-NLS fusion protein on Y79 cells

[0133] The inhibitory effect of the AQP4 scFv-LRS-NLS fusion protein on Y79 cells was detected using the CCK8 assay. Specific steps:

[0134] Experimental materials: Human retinoblastoma, preferably Y79 cell line, purchased from Zhejiang Meisen Cell Technology Co., Ltd., cultured in DMEM / F12 medium containing 10% fetal bovine serum at 37℃ and 5% CO2 saturated humidity.

[0135] Experimental methods: A Y79 cell line suspension was prepared and added to a 96-well plate, adjusting the cell count per well to 3 × 10⁶ cells / well. 4 Cells / ml. Cells were cultured in 96-well plates for 24 hours, then AQP4 scFv-LRS-NLS fusion protein was added to final concentrations of 2, 4, 8, 16, 32, and 64 μg / ml, with a final volume of 200 μl. Each concentration was used in triplicate. Zero-adjustment wells (containing only culture medium) and a control group (containing 0.1% DMSO) were also included. After 24 hours of culture, 10 μl of CCK-8 reagent and 100 μl of culture medium were added, mixed, and incubated for another 24 hours. The absorbance (OD) values ​​of each well at 450 nm were then measured using a microplate reader. The average OD value for each concentration was calculated using the average OD value of the three replicates. The cell inhibition rate (IR) for different drug concentrations was calculated using the following formula:

[0136] Cell inhibition rate (%) = (Ac - As) / (Ac - Ab) × 100%.

[0137] As: Experimental wells containing cells, culture medium, CCK-8, fVIIl-LRS-NLS sample;

[0138] Ac: Control well, containing cells + culture medium + CCK-8;

[0139] Ab: Blank wells containing culture medium + CCK-8.

[0140] The half-maximal inhibitory concentration (IC50 value) was calculated using Graphpad Prism software.

[0141] Experimental Results: Treatment of Y79 cell lines with recombinant fVIIl-LRS-NLS protein at final concentrations of 2, 4, 8, 16, 32, and 64 μg / ml for 24 h resulted in cell proliferation inhibition rates of 10.4%, 34.2%, 82.1%, 89.7%, 93.5%, and 94.1%, respectively. Compared with the control group, the recombinant fVIIl-LRS-NLS protein significantly inhibited the proliferation of Y79 cells (P<0.01), exhibiting a concentration-dependent effect; the IC50 after 24 h was 6.5 mg / L. All experimental data are from three different experiments. Figure 3 )

[0142] Example 4: In vitro tumor cell culture-apoptosis experiment using AQP4 scFv-LRS-NLS fusion protein

[0143] The experimental material was human retinoblastoma, with the Y79 cell line being preferred. It was purchased from Zhejiang Meisen Cell Technology Co., Ltd. and cultured in DMEM / F12 medium containing 10% fetal bovine serum at 37°C and saturated humidity of 5% CO2.

[0144] Experimental Methods: Y79 cell lines were seeded into culture flasks. Following the CCK-8 assay results, the concentrations of AQP4scFv-LRS-NLS fusion protein added to the experimental groups were 4 μg / ml and 8 μg / ml, respectively. Culture was terminated after 24 hours, and single-cell suspensions were prepared, with the cell concentration adjusted to 1 × 10⁻⁶ cells / ml. 6 Cells were collected at a concentration of 1 cell per ml. 1 ml of cells was centrifuged at 1000 rpm for 5 min at 4°C. The supernatant was discarded, and 1 ml of cold PBS was added. The cells were gently vortexed to resuspend them. This centrifugation was repeated twice. The cells were resuspended in 400 μL of binding buffer, and 5 μL of Annexin V labeled with fluorescein isothiocyanate (F1TC) was added. The mixture was incubated at 4°C for 15 min in the dark. Then, 10 μL of PI was added, and the mixture was gently mixed and incubated at 4°C for 5 min. Apoptosis was then detected by flow cytometry.

[0145] The experimental results showed that the early and late apoptosis rates and necrosis rates of the experimental group were significantly higher than those of the control group. The early apoptosis rates in the 0, 4, and 8 mg / L AQP4 scFv-LRS-NLS fusion protein experimental groups were 0.64%, 41.96%, and 99.69%, respectively, while the late apoptosis and necrosis rates were 1.92%, 55.52%, and 0.29%, respectively. Figure 4 ).

[0146] Example 5: In vivo antitumor effect of AQP4 scFv-LRS-NLS fusion protein:

[0147] Experimental materials: Male nude mice (18-20g), human retinoblastoma cells, preferably Y79 cell line, purchased from Zhejiang Meisen Cell Technology Co., Ltd., which are, but not limited to, breast cancer cells.

[0148] Experimental method: Y79 cells 1×10 6 Inoculated subcutaneously in the right axilla of nude mice. All animals were fed normally until the tumor reached approximately 150 mm. 3Subsequently, mice were randomly divided into three groups: a 30 μg / ml AQP4 scFv-LRS-NLS fusion protein treatment group, a 50 μg / ml AQP4 scFv-LRS-NLS fusion protein treatment group, and a saline group, with 8 mice per group. In the treatment groups, nude mice received subcutaneous injections of AQP4 scFv-LRS-NLS fusion protein at 30 μg / ml and 50 μg / ml (0.1 mL) per mouse in the right axilla. The saline groups received an equal volume of saline solution. Each group of mice received injections every other day. The mice's general condition and tumor size were observed daily, and their weight was measured every other day. Two weeks after treatment, the mice were euthanized by cervical dislocation, and the tumor tissue was dissected and its volume measured.

[0149] Experimental Results: The general condition of the nude mice was stable after tumor cell inoculation. Mice in all groups were fed normally, and their general physiological condition showed no significant changes within 1 week. After 1 week, compared with the treatment group, mice in the saline group showed reduced food intake, emaciation, weight loss, and dull fur, with significantly enlarged tumor masses; while mice in the treatment group were in good general condition. Two weeks after treatment, the mice were sacrificed, and tumor tissue was collected to measure tumor volume. The tumor inhibition rates in the 30µg / ml and 50µg / ml treatment groups were 60.6% and 62.6%, respectively; the difference in tumor inhibition rate was statistically significant (p<0.01). Figures 5~6 ).

[0150] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fusion protein, comprising fragment A and fragment B, wherein: Fragment A is an AQP4 antibody; Fragment B is the full length of Apoptin or at least one of its fragments.

2. The fusion protein according to claim 1, characterized in that, Its structure is: [(fragment A)x-(fragment B)y]z or [(fragment B)x-(fragment A)y]z; Where x, y, and z are independently selected from non-zero integers.

3. The fusion protein according to claim 1 or 2, characterized in that, Fragment A is a single-chain antibody against AQP4. Its heavy chain variable region CDR region has the amino acid sequences shown in SEQ ID NO. 1~3 in sequence; Its light chain variable region CDR region has the amino acid sequences shown in SEQ ID NO.4~6 in sequence.

4. The fusion protein according to claim 3, characterized in that, The heavy chain variable region of the single-chain antibody of AQP4 has the amino acid sequence shown in SEQ ID NO.7, and the light chain variable region has the amino acid sequence shown in SEQ ID NO.

8.

5. The fusion protein according to claim 1 or 2, characterized in that, Fragment B is the hydrophobic amino acid-rich region and nuclear localization signal region of Apoptin.

6. The fusion protein according to claim 5, characterized in that, The hydrophobic amino acid-rich region has an amino acid sequence as shown in SEQ ID NO. 9; the nuclear localization signal region has an amino acid sequence as shown in SEQ ID NO.

10.

7. The fusion protein according to any one of claims 1 to 6, characterized in that, From the N-terminus to the C-terminus, the following components are included sequentially: the light chain variable region of the AQP4 single-chain antibody, linker1, the heavy chain variable region of the AQP4 single-chain antibody, linker2, the hydrophobic amino acid-rich region of Apoptin, linker3, and the nuclear localization signal region of Apoptin.

8. The fusion protein according to claim 7, characterized in that, Its N-terminus also includes a signal peptide and / or its C-terminus also includes a screening tag.

9. The fusion protein according to claim 7 or 8, characterized in that, The linker1 is (G4S)3; The linker2 or linker3 is G3S; The signal peptide has the amino acid sequence shown in SEQ ID NO.11; The filter label is a 6*His label.

10. The fusion protein according to any one of claims 1 to 9, characterized in that, It has an amino acid sequence as shown in SEQ ID NO.

12.

11. Biomaterials, including any one of the following: I) Nucleic acid encoding the fusion protein according to any one of claims 1 to 10; II) Expression units containing the nucleic acid described in I); III) A vector containing the nucleic acid described in I) or the expression unit described in II); IV) Transforming or transfecting a host with the vector described in III); The expression products of the host as described in (V) and (IV).

12. A method for preparing the fusion protein according to any one of claims 1 to 10, characterized in that, include: Cultivate the host as described in claim 11 to obtain a culture containing the fusion protein.

13. A drug comprising the fusion protein of any one of claims 1 to 10, or comprising at least one of the biological materials of claim 11, or comprising a culture prepared by the method of claim 12.

14. The use of the fusion protein according to any one of claims 1 to 10, at least one of the biomaterials according to claim 11, the culture obtained by the preparation method according to claim 12, or the drug according to claim 13 in at least one of the following: i) Inducing apoptosis in nervous system cells; ii) To prepare drugs for the prevention and treatment of diseases related to abnormal proliferation of nervous system cells.

15. The application according to claim 14, characterized in that, The nervous system cells include retinoblastoma cells; diseases associated with abnormal proliferation of the nervous system cells include retinoblastoma.

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