Her2 antibody capable of being expressed by adeno-associated virus

Abstract

The present disclosure provides a Her2 antibody that can be expressed with an adeno-associated virus. The present disclosure provides a gene expression cassette comprising a coding sequence of an anti-Her2 antibody or an antigen binding fragment thereof, and at least one of a promoter, an intron and a polyadenylation signal, the anti-Her2 antibody or the antigen binding fragment thereof comprises a light chain variable region and a heavy chain; the light chain variable region comprises a sequence having at least 85% identity with SEQ ID NO: 2; the heavy chain has a sequence that is at least 85% identical to SEQ ID NO: 3 or 5. According to the present invention, the problems caused by the direct application of the antibody in the gene therapy in the prior art are avoided through the antibody structure modification, and the efficient transduction can be achieved when the antibody is used for the gene therapy through the antibody structure modification and the design of the gene expression box, the carrier and the like.

Description

Technical Field

[0001] The present disclosure relates to a Her2 antibody that can be expressed by adeno-associated virus, belonging to the fields of gene therapy and antibodies. Background Art

[0002] Breast cancer (BC) is the most common cancer type among women globally, accounting for approximately 11.6% of all cancer cases in 2022 (Reference 1). BC is also the fourth leading cause of cancer death among women, with approximately 666,000 death cases in 2022 (Reference 1). Currently, the diagnosis and treatment level of BC has been improving year by year, and the 5-year survival rate has reached as high as 80%. A mature system including chemotherapy, targeted therapy, radiotherapy, surgery, endocrine therapy, and immunotherapy has been formed (Reference 2). However, the complexity and heterogeneity of BC have led to the failure of patients' survival time and quality of life to meet expectations.

[0003] According to molecular biology and histological classification, BC is divided into hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (Her2)-positive, and triple-negative (Reference 2). Her2 is a transmembrane glycoprotein with a molecular weight of 185 KDa, which easily forms heterodimers with other ErbB family members and causes phosphorylation of tyrosine residues to activate downstream signaling pathways, thereby mediating processes such as gene expression and DNA synthesis, leading to abnormal cell proliferation (Reference 3). The Her2-positive subtype accounts for approximately 20%-30% of BC patients, and this subtype is associated with high tumor invasiveness, high recurrence risk, rapid progression, and poor prognosis (Reference 4). Compared with HR-positive patients, the risk of breast cancer brain metastasis (BCBM) in Her2-positive patients is 2- to 5-fold higher. Although macromolecular monoclonal drugs targeting Her2 have greatly improved the control rate of extracranial lesions, the presence of the blood-brain barrier (BBB) makes it difficult for macromolecular drugs to reach effective drug concentrations in the brain (Reference 5), and the prognosis of BCBM remains unsatisfactory. The median survival times of patients with parenchymal brain metastases and leptomeningeal metastases are 3-23 months and 3-4 months, respectively (Reference 6). Currently, the main treatment methods for BCMB are surgery, whole-brain radiotherapy, and stereotactic radiotherapy. However, multiple surgeries or radiotherapy are likely to cause related complications, resulting in a decrease in the survival time and quality of life of patients (Reference 7). In addition, with the continuous optimization and development of the BC treatment system, the survival time of patients represented by the 5-year survival rate has been significantly extended, and the statistically reported incidence of brain metastases is also increasing (Reference 8). Therefore, the development of effective drugs for BCBM patients is an urgent problem to be solved currently.

[0004] Trastuzumab (TZB) is a monoclonal antibody against the extracellular domain of Her2. Its pharmacological mechanism mainly mediates Her2 degradation, mediates antibody dependent cell mediated cytotoxicity (ADCC), regulates the phosphatidylinositol 3-kinase / protein kinase B signaling pathway, and regulates the mitogen-activated protein kinase signaling pathway (Reference 2). Monotherapy with TZB has brought significant clinical benefits to patients with Her2-positive BC. However, in the treatment of patients with BCBM, the ability of the standard intravenous dose of TZB to cross the BBB is limited, and the ratio of the content of trastuzumab in the cerebrospinal fluid to that in the serum of patients is only 1:420, which is only improved to 1:49 after radiotherapy (Reference 9). Antibody-drug conjugate (ADC) drugs (such as T-DM1, T-DXd) have significant intracranial pharmacological effects. However, with the increase in the survival period of patients, the long-term toxicity of drug use has attracted much attention. Such drugs cause various adverse reactions such as diarrhea and liver injury, which limit the application of the drugs (Reference 8). In addition, the mechanism by which such drugs exert their pharmacological effects in the brain has not been clarified.

[0005] As mentioned above, the BBB limits the pharmacological effects of macromolecular drugs. To achieve an effective drug concentration of TZB in the central nervous system (CNS) of patients with BCBM, in a phase II clinical study, intrathecal administration of TZB was performed on patients with leptomeningeal metastases of Her2-positive BC. The clinical results demonstrated the safety and effectiveness of this regimen (Reference 10). However, such a dosing method greatly reduces the compliance and quality of life of patients and increases the risk of infection.

[0006] Adeno-associated virus (AAV) is a gene vector modified from the non-pathogenic wild-type AAV. It has attracted widespread attention due to its advantages such as diversity, high safety, ability to penetrate the BBB, and long-term gene expression in vivo (Reference 11). A large number of clinical trial results and marketed AAV drugs such as Zolgensma, Upstaza, and Hemgenix have demonstrated the safety and effectiveness of this vector (Reference 11). Taking Upstaza as an example, the drug mediates safe, long-term and stable expression of aromatic L-amino acid decarboxylase through a single intracerebral administration. However, even if an antibody has been proven to be effective as an antibody drug, its effectiveness in transforming it into a gene therapy drug is unknown and unpredictable. On the one hand, traditional monoclonal antibodies often use multi-promoter expression vectors, internal ribosome entry site (IRES) vectors, Furin-2A peptide vectors, etc. to mediate the expression of antibody light and heavy chains. However, the above methods have problems such as unbalanced gene expression, low translation efficiency, and incomplete self-cleavage (reference 12). These problems may lead to the generation of mismatched antibodies, which directly affect the quality and safety of monoclonal antibodies. In order to obtain monoclonal antibodies with correctly assembled light and heavy chains from these expression systems, it is usually necessary to use multi-step in vitro purification technology (difficult to implement in vivo). Therefore, the direct application of existing technologies in gene therapy faces challenges. On the other hand, AAV has different transduction efficiencies for different tissues. Some tissues may be difficult to be efficiently transduced by AAV, resulting in low antibody protein expression and failure to achieve the expected therapeutic effect. At present, methods for the long-term stable expression of AAV-mediated Her2 antibodies in the CNS to treat Her2-positive BCBM still need to be further developed.

[0007] References

[0008] Reference 1: Bray, Freddie et al.Global cancer statistics 2022:GLOBOCANestimates of incidence and mortality worldwide for 36cancers in185countries.CA:a cancer journal for clinicians vol.74,3(2024):229-263.

[0009] Reference 2: Méndez-Valdés G, Gómez-Hevia F, Bragato MC, et al. Antioxidant Protection against Trastuzumab Cardiotoxicity in Breast Cancer Therapy. Antioxidants (Basel). 2023;12(2):457..

[0010] Reference 3: Doherty JK, Bond C, Jardim A, Adelman JP, Clinton GM. The HER-2 / neu receptor tyrosine kinase gene encodes a secreted autoinhibitor. Proc Natl Acad Sci U S A. 1999;96(19):10869-10874.

[0011] Reference 4: Derakhshani A, Rezaei Z, Safarpour H, et al. Overcoming trastuzumab resistance in HER2-positive breast cancer using combination therapy. J Cell Physiol. 2020;235(4):3142-3156.

[0012] Reference 5: Chen Q, Xiong J, Ma Y, Wei J, Liu C, Zhao Y. Systemic treatments for breast cancer brain metastasis. Front Oncol. 2023;12:1086821.

[0013] Reference 6: Jo JC, Kang MJ, Kim JE, et al. Clinical features and outcome of leptomeningeal metastasis in patients with breast cancer: a single center experience. Cancer Chemother Pharmacol. 2013;72(1):201-207.

[0014] Reference 7: Bailleux C, Eberst L, Bachelot T. Treatment strategies for breast cancer brain metastases. Br J Cancer. 2021;124(1):142-155.

[0015] Reference 8: McGranahan TM, Bonm AV, Specht JM, Venur V, Lo SS. Management of Brain Metastases from Human Epidermal Growth Factor Receptor 2 Positive (HER2+) Breast Cancer. Cancers (Basel). 2022;14(20):5136.

[0016] Reference 9: Stemmler HJ, Schmitt M, Willems A, Bernhard H, Harbeck N, Heinemann V. Ratio of trastuzumab levels in serum and cerebrospinal fluid is altered in HER2-positive breast cancer patients with brain metastases and impairment of blood-brain barrier. Anticancer Drugs. 2007;18(1):23-28.

[0017] Reference 10: Oberkampf F, Gutierrez M, Trabelsi Grati O, et al. Phase II study of intrathecal administration of trastuzumab in patients with HER2-positive breast cancer with leptomeningeal metastasis. Neuro Oncol. 2023;25(2):365-374.

[0018] Reference 11: Kang L, Jin S, Wang J, et al. AAV vectors applied to the treatment of CNS disorders: Clinical status and challenges. J Control Release. 2023;355:458-473.

[0019] Reference 12: Li YM, Tian ZW, Xu DH, Wang XY, Wang TY. Construction strategies for developing expression vectors for recombinant monoclonal antibody production in CHO cells. Mol Biol Rep. 2018;45(6):2907-2912. Summary of the Invention

[0020] Problems to be Solved by the Invention

[0021] The present disclosure aims to provide a novel Her2 antibody and a gene expression cassette that can be expressed by AAV. In some specific embodiments, the novel Her2 antibody and the gene expression cassette can achieve single administration through a gene therapy vector and mediate the long-term expression of the Her2 antibody in the CNS of BCBM patients.

[0022] Solutions for Solving the Problems

[0023] In a first aspect of the present disclosure, there is provided a gene expression cassette comprising a coding sequence of an anti-Her2 antibody or an antigen-binding fragment thereof, and optionally at least one of a promoter, an intron, and a polyadenylation signal; the anti-Her2 antibody or an antigen-binding fragment thereof comprises a light chain variable region and a heavy chain; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:2, or a sequence having at least 85% identity with SEQ ID NO:2; the heavy chain comprises the amino acid sequence shown in SEQ ID NO:3 or 5, or a sequence having at least 85% identity with SEQ ID NO:3 or 5.

[0024] In some embodiments, the anti-Her2 antibody or an antigen-binding fragment thereof does not comprise a light chain constant region.

[0025] In some embodiments, the light chain variable region and the heavy chain are linked by a linker.

[0026] In some embodiments, the light chain variable region comprises three complementarity determining regions (CDRs): LCDR1, LCDR2, and LCDR3, and the heavy chain variable region of the heavy chain comprises three complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3; and the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively, or sequences having at least 85% identity to any one of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11; the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, respectively, or sequences having at least 85% identity to any one of SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

[0027] In some embodiments, the anti-Her2 antibody or an antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO:1 or 4, or a sequence having at least 85% identity to SEQ ID NO:1 or 4.

[0028] In some embodiments, the coding sequence of the anti-Her2 antibody or an antigen-binding fragment thereof comprises the nucleotide sequence shown in SEQ ID NO:19 or 21, or a sequence having at least 85% identity to the sequence shown in SEQ ID NO:19 or 21.

[0029] In some embodiments, the promoter comprises a CAG promoter.

[0030] In some preferred embodiments, the CAG promoter comprises the nucleotide sequence shown in SEQ ID NO:15, or a sequence having at least 85% identity to SEQ ID NO:15.

[0031] In some embodiments, the intron comprises a chimeric intron.

[0032] In some preferred embodiments, the chimeric intron comprises the nucleotide sequence shown in SEQ ID NO:16, or a sequence having at least 85% identity to SEQ ID NO:16.

[0033] In some embodiments, upstream of the coding sequence of the anti-Her2 antibody or an antigen-binding fragment thereof, there is a sequence encoding a signal peptide, and the signal peptide comprises a human interleukin-2 signal peptide.

[0034] In some preferred embodiments, the human interleukin-2 signal peptide comprises the amino acid sequence shown in SEQ ID NO:8, or a sequence having at least 85% identity with SEQ ID NO:8.

[0035] In some embodiments, the gene expression cassette further comprises a polyadenylation region.

[0036] In some optional embodiments, the polyadenylation region comprises the polyadenylation signal from SV40.

[0037] In some preferred embodiments, the polyadenylation signal from SV40 comprises the nucleotide sequence shown in SEQ ID NO:17, or a sequence having at least 85% identity with SEQ ID NO:17.

[0038] In some embodiments, the structure of the gene expression cassette is as follows:

[0039] [Promoter] - [Intron] - [Sequence encoding the signal peptide] - [Sequence encoding the anti-Her2 antibody or its antigen-binding fragment] - [Polyadenylation region].

[0040] In some embodiments, the gene expression cassette comprises the nucleotide sequence shown in SEQ ID NO:23 or 24, or a sequence having at least 85% identity with SEQ ID NO:23 or 24.

[0041] In a second aspect of the present disclosure, there is provided a gene delivery vector comprising the gene expression cassette described in the first aspect of the present disclosure.

[0042] In some embodiments, the gene delivery vector is a virus-derived viral vector.

[0043] In some preferred embodiments, the gene delivery vector is a recombinant adeno-associated virus.

[0044] In some more preferred embodiments, the gene delivery vector comprises the nucleotide sequence shown in SEQ ID NO:18 or 20, or a sequence having at least 85% identity with SEQ ID NO:18 or 20.

[0045] In some embodiments, the recombinant adeno-associated virus comprises a capsid protein, and the gene expression cassette is encapsulated within the capsid protein.

[0046] In some optional embodiments, the capsid protein is selected from any one of or variants of the adeno-associated virus serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10.

[0047] In some embodiments, the capsid protein comprises a capsid protein derived from the AAV6 adeno-associated virus serotype or a variant thereof.

[0048] In some embodiments, the capsid protein comprises a targeting peptide as shown in SEQ ID NO:25.

[0049] In some preferred embodiments, the capsid plasmid encoding the capsid protein comprises the nucleotide sequence as shown in SEQ ID NO:29, or a sequence having at least 85% identity with SEQ ID NO:29.

[0050] In a third aspect of the present disclosure, there is provided an anti-Her2 antibody or an antigen-binding fragment thereof, which comprises a light chain variable region and a heavy chain; the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:2, or a sequence having at least 85% identity with SEQ ID NO:2; the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:3 or 5, or a sequence having at least 85% identity with SEQ ID NO:3 or 5.

[0051] In some embodiments, the anti-Her2 antibody or an antigen-binding fragment thereof does not comprise a light chain constant region.

[0052] In some embodiments, the light chain variable region and the heavy chain are linked by a linker.

[0053] In some embodiments, the anti-Her2 antibody or an antigen-binding fragment thereof comprises the amino acid sequence as shown in SEQ ID NO:1 or 4, or a sequence having at least 85% identity with SEQ ID NO:1 or 4.

[0054] In a fourth aspect of the present disclosure, there is provided a pharmaceutical composition, which comprises the gene expression cassette as described in the first aspect of the present disclosure, the gene delivery vector as described in the second aspect of the present disclosure, or the anti-Her2 antibody or an antigen-binding fragment thereof as described in the third aspect of the present disclosure, and, optionally, a pharmaceutically acceptable carrier.

[0055] Use of the gene expression cassette as described in the first aspect of the present disclosure, the gene delivery vector as described in the second aspect of the present disclosure, or the anti-Her2 antibody or an antigen-binding fragment thereof as described in the third aspect of the present disclosure in the preparation of a medicament for treating breast cancer.

[0056] In some preferred embodiments, the breast cancer includes central nervous system metastasis of breast cancer.

[0057] In some preferred embodiments, the breast cancer includes Her2-positive breast cancer.

[0058] In a fifth aspect of the present disclosure, a method for treating breast cancer is provided, which comprises administering to a subject a therapeutically effective amount of a gene expression cassette as described in the first aspect of the present disclosure, a gene delivery vector as described in the second aspect of the present disclosure, an anti-Her2 antibody or an antigen-binding fragment thereof as described in the third aspect of the present disclosure, or a pharmaceutical composition as described in the fourth aspect of the present disclosure.

[0059] In some preferred embodiments, the breast cancer includes central nervous system metastasis of breast cancer.

[0060] In some preferred embodiments, the breast cancer includes Her2-positive breast cancer.

[0061] Effects of the Invention

[0062] In some embodiments of the present disclosure, problems faced in the direct application of antibodies in the prior art to gene therapy, such as unbalanced gene expression, low translation efficiency, incomplete self-cleavage, etc., are circumvented through antibody structure modification. These problems may all lead to the generation of mismatched antibodies, thereby directly affecting the quality and safety of monoclonal antibodies.

[0063] In some embodiments of the present disclosure, through antibody structure modification and the design of gene expression cassettes, vectors, etc., antibodies can be efficiently transduced when used in gene therapy, especially suitable for the central nervous system.

[0064] In some specific embodiments of the present disclosure, the present disclosure provides a novel Her2 antibody that can be expressed by AAV, comprising a binding domain specific to Her2, which can be applied to the gene therapy of BCBM. Due to the limited capacity of the AAV vector (less than 4.7 kb) and the need to minimize the immunogenicity of antibody drugs as much as possible, the present disclosure constructs an scFv-Fc type antibody by tandemly linking single-chain variable region fragments (Single-Chain Fragment Variable, scFv) and the Fc segment. The ADCC effect is one of the main mechanisms of antibody drugs against tumors. The present disclosure screens out an ADCC-enhanced Her2 antibody. In multiple animal models, this AAV-mediated antibody expression strategy was verified, and the results showed that it had significant efficacy in vivo. Through this innovative antibody design and AAV expression technology, it is expected to bring a precise and effective treatment plan for Her2-positive BCBM patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 It is a schematic diagram of the antibody tandem sequence pattern in the shuttle plasmid.

[0066] Figure 2Schematic diagram of SDS-PAGE results of plasmid expression products. In the figure: Expression product of plasmid 1 (left), expression product of plasmid 2 (right), M represents Marker; R (Reduced) represents reduced sample; NR (Non-Reduced) represents non-reduced sample.

[0067] Figure 3 Schematic diagram of in vitro ELISA experiment results. In the figure: The EC50 values of plasmid 1 group, plasmid 2 group, and ARX788 are 35.06 ng / mL, 48.77 ng / mL, and 32.54 ng / mL respectively; The expression product of plasmid 3 is used as a negative control, and the signal intensity is lower than the detection limit.

[0068] Figure 4 Schematic diagram of ADCC experiment results of plasmid expression products. In the figure: The EC50 values of ARX788, the expression product of plasmid 1, and the expression product of plasmid 2 are 5.8 ng / mL, 6.12 ng / mL, and 16.87 ng / mL respectively.

[0069] Figure 5 Schematic diagram of tumor pictures (partial) of subcutaneous pharmacodynamic experiment on the 30th day.

[0070] Figure 6 Schematic diagram of the change curve of tumor volume in subcutaneous pharmacodynamic experiment.

[0071] Figure 7 Schematic diagram of the change curve of mouse body weight in subcutaneous pharmacodynamic experiment.

[0072] Figure 8 Schematic diagram of in vivo imaging pictures (partial) of intracranial orthotopic pharmacodynamic pre-experiment.

[0073] Figure 9 Schematic diagram of the change curve of tumor signal in intracranial orthotopic pharmacodynamic pre-experiment.

[0074] Figure 10 Schematic diagram of the H&E staining results (partial) of the coronal section of mouse brain tissue in intracranial orthotopic pharmacodynamic pre-experiment. In the figure: H&E results of the coronal section of mouse brain (D25), and the tumor area is circled in red.

[0075] Figure 11 Schematic diagram of the change curve of mouse body weight in intracranial orthotopic pharmacodynamic pre-experiment.

[0076] Figure 12 Schematic diagram of the change curve of tumor signal in intracranial orthotopic pharmacodynamic experiment.

[0077] Figure 13 Schematic diagram of the change curve of mouse body weight in intracranial orthotopic pharmacodynamic experiment.

[0078] Figure 14 Schematic diagram of the survival curve of mice in intracranial orthotopic pharmacodynamic experiment.

[0079] Figures 15 to 17 Schematic diagram of the results of the influence of nucleotide sequence optimization on the ability of plasmids to mediate protein expression in human cells. Among them, Figure 15 It shows that all four plasmids can mediate the expression of the target protein in 293T cells. Figure 16 It shows the expression comparison between plasmid 1 and plasmid 1 control. Figure 17 It shows the expression comparison between plasmid 2 and plasmid 2 control.

[0080] Figure 18 Schematic diagram of the results of in vitro verification of the ability of AAV to infect BT474 cells. Detailed implementation manners

[0081] Various exemplary embodiments, features and aspects of the present disclosure will be described in detail below. The word "exemplary" used here means "serving as an example, embodiment or illustration". Any embodiment described as "exemplary" here does not have to be construed as superior or better than other embodiments.

[0082] In addition, in order to better illustrate the present disclosure, numerous specific details are given in the following detailed implementation manners. Those skilled in the art should understand that the present disclosure can be implemented without some specific details. In other instances, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the gist of the present disclosure.

[0083] Unless otherwise stated, the units used in this specification are all international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0084] In this specification, the meaning expressed by using "can" includes the meanings of both performing a certain process and not performing a certain process.

[0085] In this specification, the so-called "some specific / preferred implementation manners", "other specific / preferred implementation manners", "implementation manners", etc. refer to the specific elements (for example, features, structures, properties and / or characteristics) related to the implementation manner described, which are included in at least one of the implementation manners described here, and may or may not exist in other implementation manners. In addition, it should be understood that the elements can be combined in various implementation manners in any suitable way.

[0086] In this specification, the numerical range represented by using "numerical value A~numerical value B" refers to the range including the endpoint numerical values A and B.

[0087] In this specification, the use of "substantially" or "essentially" means that the standard deviation from the theoretical model or theoretical data is within the range of 5%, preferably 3%, more preferably 1%.

[0088] In this specification, "optional" or "optionally" means that the event or situation described next may or may not occur, and this description includes the situation where the event occurs and the situation where the event does not occur.

[0089] In this specification, the terms "comprising" and "having" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product or device that includes a series of steps is not limited to the listed steps or modules, but may optionally further include steps not listed, or may optionally further include other steps inherent to these processes, methods, products or devices.

[0090] In this specification, the term "plurality" means two or more. "And / or" describes the association relationship of associated objects and indicates that three relationships can exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. The character " / " generally represents an "or" relationship between the associated objects before and after.

[0091] According to the present disclosure, the terms "polypeptide", "protein", and "peptide" are used interchangeably herein and refer to a polymeric form of amino acids of any length, which may include encoded and non-encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a similar peptide backbone.

[0092] According to the present disclosure, the terms "nucleic acid molecule", "polynucleotide", "polymeric nucleic acid", and "nucleic acid" are used interchangeably and refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0093] According to the present disclosure, the terms "upstream" and "downstream" are relative terms that define the linear positions of at least two elements in a nucleic acid molecule (whether single-stranded or double-stranded) oriented in the 5' to 3' direction.

[0094] According to the present disclosure, the term "amino acid" may include natural amino acids, unnatural amino acids, amino acid analogs, and all their D and L stereoisomers. According to the present disclosure, the three-letter codes and single-letter codes of amino acids used are as described in J. biol. chem, 243, p3558 (1968). The amino acids, abbreviations, and English short names in the present disclosure are as follows: histidine (His, H); serine (Ser, S); glutamic acid (Glu, E); glutamine (Gln, Q); glycine (Gly, G); threonine (Thr, T); phenylalanine (Phe, F); aspartic acid (Asp, D); tyrosine (Tyr, Y); leucine (Leu, L); isoleucine (Ile, I); arginine (Arg, R); alanine (Ala, A); valine (Val, V); tryptophan (Trp, W); methionine (Met, M); asparagine (Asn, N); cysteine (Cys, C); lysine (Lys, K); proline (Pro, P).

[0095] According to the present disclosure, "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, when the sequences are optimally aligned, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparison is made when the maximum percentage of identity is obtained by aligning the two sequences.

[0096] According to the present disclosure, "administering", "giving", and "treating", when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administering", "giving", and "treating" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes the contact of a reagent with the cells, as well as the contact of the reagent with a fluid that contacts the cells. "Administering", "giving", and "treating" also mean treatment of cells in vitro and ex vivo by a reagent, diagnostic agent, binding composition, or by another cell. "Treating", when applied to humans, veterinary medicine, or research subjects, refers to therapeutic, prophylactic, or preventive measures, research, and diagnostic applications.

[0097] According to the present disclosure, "treatment" means administering to a patient a therapeutic agent, either internally or externally, such as an antibody, gene expression cassette, or gene delivery vector of the present disclosure, to a patient having one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Generally, the therapeutic agent is administered to the treated patient or population in an amount effective to alleviate one or more disease symptoms, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, as well as the ability of the drug to produce the desired effect in the patient. Whether a disease symptom has been alleviated can be evaluated by any clinical test method commonly used by a doctor or other professional healthcare provider to evaluate the severity or progression of the symptom.

[0098] According to the present disclosure, the term "prevention" refers to prophylactic treatment of a subject who does not currently have and has not had in the past a disease but is at risk of developing a disease or who has had a disease in the past and does not currently have the disease but is at risk of disease recurrence. In certain embodiments, the subject has a higher risk of developing a disease or a higher risk of disease recurrence compared to the average healthy member of the subject population.

[0099] According to the present disclosure, an "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or condition of a medical disorder. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the disorder to be treated, the overall health of the patient, the method of administration and dosage, and the severity of side effects. The effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[0100] According to the present disclosure, a "therapeutically effective amount" is an amount sufficient to provide a therapeutic benefit in the treatment of a disorder or sufficient to delay or minimize one or more symptoms associated with the disorder. A therapeutically effective amount refers to the amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of a disorder. The term "therapeutically effective amount" can include amounts that improve overall therapy; reduce or avoid the symptoms, signs, or causes of a disorder; and / or enhance the therapeutic efficacy of another therapeutic agent.

[0101] According to the present disclosure, a "preventively effective amount" is an amount sufficient to prevent a disorder or one or more symptoms associated with the disorder or prevent its recurrence. A preventively effective amount refers to the amount of a therapeutic agent, alone or in combination with other agents, that provides a preventive benefit in preventing a disorder. The term "preventively effective amount" can include amounts that improve overall prevention or enhance the preventive efficacy of another preventive agent.

[0102] According to the present disclosure, the term "subject" refers to a human (i.e., a male or female of any age, e.g., a pediatric subject (e.g., an infant, child, or adolescent) or an adult subject (e.g., a young, middle-aged, or elderly person)) or a non-human animal. In certain embodiments, the non-human animal is a mammal (e.g., a primate (e.g., a cynomolgus monkey or a rhesus monkey), a commercially relevant mammal (e.g., a cow, pig, horse, sheep, goat, cat, or dog), or a bird). The non-human animal can be male or female at any stage of development. The non-human animal can be a transgenic or genetically engineered animal.

[0103] According to the present disclosure, a "vector" refers to a macromolecule or an association of macromolecules that includes a polynucleotide or is associated with the polynucleotide and can be used to mediate delivery of the polynucleotide to a cell. Exemplary vectors include, for example, plasmids, viral vectors (i.e., viruses such as adeno-associated virus), liposomes, and other gene delivery agents.

[0104] According to the present disclosure, the term "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. The term encompasses all subtypes as well as naturally occurring and recombinant forms, unless otherwise required. The term "AAV" includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, "bovine AAV" refers to AAV that infects bovine mammals, etc.

[0105] According to the present disclosure, an "AAV virus" or "AAV viral particle" or "rAAV vector particle" refers to a viral particle composed of at least one AAV capsid protein (usually consisting of all capsid proteins of wild-type AAV) and an encapsidated polynucleotide. If the particle includes a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is generally referred to as a recombinant AAV vector or rAAV. Typically, the heterologous polynucleotide is flanked by AAV inverted terminal repeats (ITRs).

[0106] According to the present disclosure, the terms "gene", "coding sequence", or "coding gene" refer to a nucleotide sequence that encodes a gene product in vitro or in vivo. The term "transgene" refers to a coding sequence or gene delivered into a cell via a vector. The coding sequence or gene can encode a peptide or polypeptide molecule.

[0107] According to the present disclosure, the term "operably linked" refers to the juxtaposition of genetic elements (e.g., a promoter, enhancer, termination signal sequence, polyadenylation sequence, etc.), wherein the elements are in a relationship that permits them to operate in an intended manner. For example, if a promoter aids in initiating the transcription of a coding sequence, the promoter is operably linked to the coding region. There may be intervening residues between the promoter and the coding region as long as this functional relationship is maintained.

[0108] The term "antibody" as used in the present invention refers to an immunoglobulin, which is a four - peptide chain structure composed of two identical heavy chains and two identical light chains linked by inter - chain disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so its antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or called isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain respectively. The same class of Ig can be further divided into different subclasses according to the differences in the amino acid composition of its hinge region and the number and position of the heavy - chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into κ chain or λ chain according to the difference in the constant region. Each of the five classes of Ig can have a κ chain or a λ chain.

[0109] In the present invention, the antibody light chain may further comprise a light - chain constant region, and the light - chain constant region comprises a human - derived κ, λ chain or a variant thereof.

[0110] In the present invention, the antibody heavy chain may further comprise a heavy - chain constant region, and the heavy - chain constant region comprises a human - derived IgG1, IgG2, IgG3, IgG4 or a variant thereof.

[0111] The sequences of approximately 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly and are the variable regions (V regions); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant regions (C regions). The variable region includes 3 hypervariable regions (HVRs) and 4 relatively conserved framework regions (FRs). The 3 hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). Each variable region of the light chain (VL) and the heavy chain (VH) consists of 3 CDR regions and 4 FR regions, and the order arranged from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The CDR amino acid residues in the VL and VH regions of the antibody or antigen-binding fragment conform to the known Kabat numbering rules and the Kabat or AbM or IMGT definition rules (http: / / bioinf.org.uk / abs / ) in terms of quantity and position.

[0112] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and an antibody analogue, which generally includes at least a portion of the antigen-binding region or variable region (such as one or more CDRs) of the parental antibody. The antibody fragment retains at least some of the binding specificities of the parental antibody. Generally, when the activity is expressed based on molarity, the antibody fragment retains at least 10% of the parental binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parental antibody for the target. Examples of antigen-binding fragments include, but are not limited to: Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, linear antibody, single chain antibody fragment (scFv), domain antibody, nanobody, and multispecific antibody (such as diabody). Engineered antibody variants are reviewed in Holliger and Hudson, 2005, Nat. Biotechnol. 23:1126-1136.

[0113] In this specification, "E" is generally used to represent "Exponent", that is, the power of 10. Exemplarily, 2E-02 represents 2×10 -2 ; 1E+4 or 1E4 represents 1×10 4 . Detailed Description of the Invention

[0115] <Gene Expression Cassette>

[0116] In some aspects of the present disclosure, a gene expression cassette is provided, which comprises a coding sequence of an anti-Her2 antibody or an antigen-binding fragment thereof, and, optionally, at least one of a promoter, an intron, and a polyadenylation signal; the anti-Her2 antibody or an antigen-binding fragment thereof comprises a light chain variable region and a heavy chain.

[0117] Coding Sequence of Anti-Her2 Antibody or Its Antigen-Binding Fragment

[0118] In some embodiments, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:2, or a sequence having at least 85% identity with SEQ ID NO:2.

[0119] In some embodiments, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:3 or 5, or a sequence having at least 85% identity with SEQ ID NO:3 or 5.

[0120] In some preferred embodiments, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:3, or a sequence having at least 85% identity with SEQ ID NO:3.

[0121] In some embodiments, the anti-Her2 antibody or an antigen-binding fragment thereof does not comprise a light chain constant region.

[0122] In some embodiments, the light chain variable region and the heavy chain are linked by a linker.

[0123] As used herein, "linker" or "connector" refers to a moiety that connects two molecules or portions, such as two domains of a fusion protein. Typically, the linker is located between or flanks two groups, molecules, or other moieties and is covalently attached to each, thereby connecting the two. In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker can be a polynucleotide. In some embodiments, the linker can be a DNA molecule. In some embodiments, the linker can be an RNA molecule. In some embodiments, the linker can include an aptamer capable of binding a ligand. In some embodiments, the linker is an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker of the present disclosure comprises the amino acid sequence (GGGS)n (SEQ ID NO:30), (GGGGS)n (SEQ ID NO:31), (G)n, (EAAAK)n (SEQ ID NO:32), (GGS)n, (SGGS)n (SEQ ID NO:33), SGSETPGTSESATPES (SEQ ID NO:34), or an (XP)n motif or a combination thereof, where n is independently an integer from 1 to 32, and where X is any amino acid.

[0124] In some specific embodiments, the light chain variable region comprises three complementarity determining regions (CDRs): LCDR1, LCDR2, and LCDR3, and the heavy chain variable region of the heavy chain comprises three complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3; and the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively, or sequences having at least 85% identity to any one of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11; the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, respectively, or sequences having at least 85% identity to any one of SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

[0125] In some specific embodiments, the anti-Her2 antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:1 or 4, or a sequence having at least 85% identity to SEQ ID NO:1 or 4.

[0126] In some preferred embodiments, the anti-Her2 antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:1, or a sequence having at least 85% identity to SEQ ID NO:1.

[0127] In some embodiments of the present disclosure, the coding sequence of the anti-Her2 antibody or its antigen-binding fragment may be a nucleotide sequence that is not codon-optimized or is codon-optimized for encoding the amino acid sequence shown in SEQ ID NO:1 or 4 or a sequence having at least 85% identity to the amino acid sequence shown in SEQ ID NO:1 or 4.

[0128] According to the present disclosure, the term "codon optimization" refers to a polynucleotide sequence modified from its native form. Such modification results in differences in one or more base pairs, with or without an alteration in the corresponding amino acid sequence, which may enhance or inhibit gene expression and / or the cellular response to the modified polynucleotide sequence. A coding sequence is a part of the mRNA sequence that encodes the amino acids for translation. During translation, each of the 61 trinucleotide codons is translated into one of the 20 amino acids, resulting in degeneracy or redundancy in the genetic code. However, different cell types and different animal species utilize tRNAs (each carrying an anticodon) that encode the same amino acid at different frequencies. When a gene sequence contains codons that are not frequently represented by the corresponding tRNAs, the ribosomal translation mechanism may slow down, thus hindering efficient translation. Expression can be improved by "codon optimization" for a particular species, in which the coding sequence is altered to encode the same protein sequence while utilizing codons that are highly represented and / or utilized by highly expressed human proteins (Cid-Arregui et al., 2003; Journal of Virology 77:4928).

[0129] In some specific embodiments, the coding sequence of the codon-optimized anti-Her2 antibody or its antigen-binding fragment comprises a nucleotide sequence as shown in SEQ ID NO:19 or 21 or a sequence having at least 85% identity with the nucleotide sequence shown in SEQ ID NO:19 or 21.

[0130] In some embodiments of the present disclosure, in addition to the sequence encoding the protein product, the gene expression cassette further comprises polynucleotide elements for controlling the expression of the protein product, such as promoters, introns, signal peptides, polyadenylation signals, and the like.

[0131] Promoter

[0132] According to the present disclosure, a promoter can be a native, artificial, or chimeric sequence. A "promoter" encompasses a DNA sequence that guides the binding of RNA polymerase and thereby promotes RNA synthesis. The promoter and the corresponding protein or polypeptide expression can be ubiquitous (meaning having strong activity in a wide range of cells, tissues, and species) or cell type-specific, tissue-specific, or species-specific. A promoter can be "constitutive" (meaning continuous activity) or "inducible" (meaning the promoter can be activated or inactivated by the presence or absence of a biological or abiotic factor).

[0133] In some alternative embodiments, the promoter has a CMV promoter of vertebrate β-actin, β-globin or β-globin regulatory element or other hybrid CMV promoters (referred to as CB and CAG promoters), EF1 promoter, hypoxia response element, ubiquitin promoter, T7 promoter, SV40 promoter, VP16 or VP64 promoter.

[0134] In some preferred embodiments, the promoter comprises a CAG promoter (composed of a human cytomegalovirus enhancer, a chicken β-actin promoter and a rabbit β-globin splice acceptor).

[0135] In some specific embodiments, the CAG promoter comprises the nucleotide sequence shown in SEQ ID NO:15 or a sequence having at least 85% identity thereto.

[0136] Intron

[0137] According to the present disclosure, "intron" includes splice donor / acceptor regions. An intron is a DNA polynucleotide that is transcribed into RNA through intron splicing and removed during mRNA processing. The expression of a gene expression cassette containing an intron is generally higher than that of those gene expression cassettes without an intron.

[0138] In some embodiments, the intron includes a chimeric intron for enhancing gene expression. In some specific embodiments, the chimeric intron comprises the nucleotide sequence shown in SEQ ID NO:16 or a sequence having at least 85% identity thereto.

[0139] Signal Peptide

[0140] In some preferred embodiments, a signal peptide is included upstream of the coding sequence of the anti-Her2 antibody or its antigen-binding fragment.

[0141] According to the present disclosure, a "signal peptide" refers to a polypeptide that is linked to a target protein and can promote the expression or transfer of the target protein. Exemplarily, the signal peptide can be linked to the N-terminus of the target protein and is usually cleaved and removed, so it does not exist in the mature protein secreted by the cell. In some embodiments of the present disclosure, the signal peptide includes a secretion signal peptide, and the secretion signal peptide is respectively upstream of the upstream of the coding sequence of the anti-Her2 antibody or its antigen-binding fragment.

[0142] In some preferred embodiments, the signal peptide includes a human interleukin-2 signal peptide.

[0143] In some specific embodiments, the human interleukin-2 signal peptide comprises the amino acid sequence shown in SEQ ID NO:8 or a sequence having at least 85% identity thereto. In some specific embodiments, the sequence encoding the human interleukin-2 signal peptide comprises the nucleotide sequence shown in SEQ ID NO:7 or a sequence having at least 85% identity thereto.

[0144] Polyadenylation Region

[0145] In some alternative embodiments of the present disclosure, the gene expression cassette further comprises a polyadenylation region (or referred to as a polynucleotide tailing signal or polyadenylation signal).

[0146] As understood in the art, RNA polymerase II transcripts are terminated by cleavage and addition of a polyadenylation region, which can also be referred to as a polynucleotide tailing signal, polyA signal, polyA region or polyA tail. The polyA region contains multiple consecutive adenosine monophosphates, which typically have repeats of the motif AAUAAA. Several effective polyadenylation sites have been identified, including those from SV40, bovine growth hormone, human growth hormone and rabbit β-globin. The most effective polyA signal for expressing a transgene in mammalian cells can depend on the cell type and species of interest as well as the particular vector used. In some embodiments of the present disclosure, the gene expression cassette includes the polyadenylation signal from SV40 (SV40 polyA signal).

[0147] In some specific embodiments, the polyadenylation signal from SV40 comprises the nucleotide sequence shown in SEQ ID NO:17 or a sequence having at least 85% identity thereto.

[0148] In some exemplary embodiments, the gene expression cassette from the 5'-end to the 3'-end includes:

[0149] [Promoter]-[Intron]-[Sequence encoding the signal peptide]-[Sequence encoding the anti-Her2 antibody or its antigen-binding fragment]-[Polyadenylation region].

[0150] The above-mentioned promoter, intron, signal peptide, polyadenylation region, and the protein coding sequence after codon optimization cooperate with each other to overcome problems such as unbalanced gene expression, low translation efficiency, and incomplete self-cleavage of the antibody protein coding sequence. At the same time, through antibody structure modification and the design of gene expression cassettes, the antibody can be efficiently transduced when used for gene therapy. As demonstrated in the examples, the gene expression cassette or AAV containing the gene expression cassette has an enhanced ability to mediate protein expression in human cells, an enhanced ADCC effect, and good tumor suppression effects in vivo.

[0151] <Gene delivery vector>

[0152] In some aspects of the present disclosure, the gene expression cassette of the present disclosure is used to deliver a gene (encoding an antibody or an antigen-binding fragment thereof) to an animal cell, for example, to determine the effect of the gene on cell viability and / or function, treat cell disorders, etc. Therefore, in some aspects of the present disclosure, a gene delivery vector is provided, which comprises the gene expression cassette of the present disclosure. In some preferred embodiments, the gene delivery vector is used to express a transgene (encoding an antibody or an antigen-binding fragment thereof) in mammalian cells.

[0153] The gene delivery vectors of the present disclosure encompass any convenient gene delivery vector for delivering a polynucleotide sequence to mammalian cells. For example, the vector can include single-stranded nucleic acid or double-stranded nucleic acid, such as single-stranded DNA or double-stranded DNA. For example, the gene delivery vector can be DNA, such as naked DNA, such as a plasmid or a minicircle, etc. The vector can include single-stranded RNA or double-stranded RNA, including modified forms of RNA. In another example, the gene delivery vector can be RNA, such as mRNA or modified mRNA.

[0154] As another example, the gene delivery vector can be a viral vector derived from a virus, such as an adenovirus, an adeno-associated virus (AAV), a lentivirus, a herpesvirus, an alphavirus, or a retrovirus, such as Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), foamy virus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV) or a lentivirus. Although examples covering the use of adeno-associated virus are described in more detail below, it is expected that those of ordinary skill in the art will recognize that similar knowledge and skills in the art can also be applied to non-AAV gene delivery vectors.

[0155] In some embodiments, the gene delivery vector is a recombinant adeno-associated virus (rAAV). In such embodiments, the gene expression cassette is flanked at the 5' and 3' ends by functional AAV inverted terminal repeat (ITR) sequences. A "functional AAV ITR sequence" refers to an ITR sequence that is expected to be used for rescuing, replicating, and packaging AAV viral particles. Thus, the AAV ITRs for the gene delivery vectors of the present disclosure do not need to have a wild-type nucleotide sequence and can be altered by insertions, deletions, or substitutions of nucleotides, or the AAV ITRs can be derived from any of several AAV serotypes, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10. Preferred AAV vectors have a wild-type Rep gene and Cap gene that are entirely or partially deleted, but retain functional flanking ITR sequences. In certain embodiments, the AAV viral vector is an AAV variant. In some embodiments, the AAV variant is an AAV viral vector that includes a variant AAV capsid (or referred to as an AAV capsid protein variant).

[0156] In some embodiments, the gene expression cassette is encapsulated within an AAV capsid, which can be derived from any adeno-associated virus serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, etc., and any of the adeno-associated virus serotypes can serve as a gene delivery vector. For example, the AAV capsid can be a wild-type capsid or a native capsid. However, like the ITRs, the capsid does not need to have a wild-type nucleotide sequence, but rather can be altered relative to the wild-type sequence by insertions, deletions, or substitutions of nucleotides in the VP1, VP2, or VP3 sequences as long as the capsid is capable of transducing mammalian cells. In other words, the AAV capsid can be a variant AAV capsid that includes one or more amino acid substitutions, deletions, or insertions relative to the parental capsid protein from which it is derived or an AAV capsid protein.

[0157] The AAV capsid is an icosahedron composed of 60 VP capsid protein monomers, including 5 VP1 monomers, 5 VP2 monomers, and 50 VP3 monomers. The VP1, VP2, and VP3 monomers are all transcribed and translated from the Cap gene of AAV. Among them, VP1 is the longest, containing approximately 735 amino acids. VP2 and VP3 are "truncated forms" of VP1 and do not contain the amino acids at the N-terminal part of the VP1 protein. By convention, the modification sites of the capsid proteins are named according to the amino acid sequence of the VP1 protein. In this specification, the description of the insertion sites of the targeting peptides in VP2 and VP3 is based on their corresponding amino acid positions in the respective VP1. For example, the 454 insertion site of AAV6 refers to inserting a short peptide after the 454th amino acid of the VP1 monomer (containing 736 amino acids) of AAV6, so that AAV6 VP1, VP2, and VP3 all contain this short peptide.

[0158] In some exemplary embodiments, the capsid protein is an AAV6 capsid protein or a variant thereof.

[0159] In some embodiments, the AAV capsid protein variant can be an AAV capsid protein variant containing a targeting peptide that is targeted to a specific protein. In some alternative embodiments, the AAV capsid protein variant is an AAV6 capsid protein variant.

[0160] In some specific embodiments, the AAV6 capsid protein variant includes the capsid proteins of AAV6.2 or AAV6.3 as disclosed in WO2024 / 234803, which are incorporated herein by reference.

[0161] In some preferred embodiments, the AAV6 capsid protein variant includes the capsid protein of AAV6.2 as disclosed in WO2024 / 234803.

[0162] Specifically, in the capsid protein of AAV6.2, the targeting peptide (GRGDSP, SEQ ID NO:25) is inserted at the position of amino acid 454 of AAV6 VP1, so that the position corresponding to amino acid 454 of AAV6 VP1 in AAV6 VP2, and the position corresponding to amino acid 454 of AAV6 VP1 in AAV6 VP3 are both inserted with the targeting peptide.

[0163] The amino acid sequence of AAV6 VP1 is shown as follows (SEQ ID NO:26):

[0164] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNLQSSSTDPATGDVHVMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL

[0165] (Total 736 amino acids)

[0166] The amino acid sequence of AAV6 VP2 is shown below (SEQ ID NO:27):

[0167] TAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNLQSSSTDPATGDVHVMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL

[0168] (Corresponding to AAV6 VP1 amino acids 138 - 736, a total of 599 amino acids)

[0169] The amino acid sequence of AAV6 VP3 is shown below (SEQ ID NO:28):

[0170] MASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKR LNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNLQSSSTDPATGDVHVMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL

[0171] (Corresponding to AAV6 VP1 amino acids 203 - 736, a total of 534 amino acids)

[0172] In some preferred embodiments, the rAAV is replication - defective because the AAV vector cannot independently further replicate and package its genome. For example, when cells are transduced with rAAV viral particles, the gene is expressed in the transduced cells. However, due to the fact that the transduced cells lack the AAV rep gene, cap gene, and helper function genes, the rAAV cannot replicate.

[0173] Standard methods can be used to generate gene delivery vectors (e.g., rAAV viral particles) that encapsidate the gene expression cassettes of the present disclosure. For example, in the case of rAAV viral particles, the AAV expression vectors according to the present disclosure can be introduced into producer cells, and then an AAV helper construct is introduced, wherein the helper construct contains AAV coding regions capable of being expressed in the producer cells and the AAV coding regions can complement AAV helper functions that are absent in the AAV vector. Then, helper virus and / or additional vectors are introduced into the producer cells, wherein the helper virus and / or additional vectors provide helper functions capable of supporting efficient rAAV virus production. Then, the producer cells are cultured to produce rAAV. These steps are performed using standard methods. Replication-deficient AAV viral particles that encapsidate the recombinant AAV vectors of the present disclosure are prepared using AAV packaging cells and packaging techniques known in the art.

[0174] Viral particles at any concentration suitable for efficient transduction of mammalian cells can be prepared for contacting mammalian cells in vitro or in vivo. Similarly, any total amount of viral particles suitable for providing appropriate cell transduction to confer a desired effect or treat a disease can be administered to a mammal.

[0175] The viral vector can be formulated into a pharmaceutical composition comprising any suitable unit dose of the vector, and the pharmaceutical composition can be administered to a subject to effect a change in the subject or treat a disease of the subject.

[0176] In some cases, the multiplicity of infection (MOI) can be used to measure the unit dose of the pharmaceutical composition. MOI refers to the ratio or multiple of the vector or viral genome to the cells into which the nucleic acid can be delivered.

[0177] In preparing rAAV compositions, any host cell can be employed for generating rAAV viral particles, including but not limited to, for example, mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast. The host cell can also be a packaging cell or a producer cell, in which the AAV rep gene and cap gene are stably maintained in the host cell, and the AAV vector genome is stably maintained and packaged in the producer cell. Exemplary packaging and producer cells are derived from SF-9, 293, A549, or HeLa cells. The AAV vector is purified and formulated using standard techniques known in the art.

[0178] In some specific embodiments, a plasmid containing a capsid protein (capsid plasmid) contains the nucleotide sequence shown in SEQ ID NO:29 (RC6.2 plasmid) or a sequence having at least 85% identity thereto.

[0179] Nucleotide sequence of plasmid RC6.2 (SEQ ID NO:29):

[0180] AGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAA

[0181] AAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAA

[0182] AGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAA

[0183] AGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCAC

[0184] CACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGG

[0185] CGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTA

[0186] ACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAA

[0187] TTCGAGCTCGGTACCCCTAGAGGTCCTGTATTAGAGGTCACGTGAGTGTTTTGCGACATTTTGCGACACCATGTGG

[0188] TCACGCTGGGTATTTAAGCCCGAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCAGCCAC

[0189] CACGCCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCCGGCATTTCTGACAG

[0190] CTTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGATTGAGCA

[0191] GGCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCCCGG

[0192] AGGCCCTTTTCTTTGTGCAATTTGAGAAGGGAGAGAGCTACTTCCACATGCACGTGCTCGTGGAAACCACCGGGG

[0193] TGAAATCCATGGTTTTGGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGGGATCG

[0194] AGCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCGGAGGCGGGAACAAGGTGGTGGA

[0195] TGAGTGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGGCGTGGACTAATATGGAACA

[0196] GTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAACGGTTGGTGGCGCAGCATCTGACGCACGTGTCGCAGAC

[0197] GCAGGAGCAGAACAAAGAGAATCAGAATCCCAATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAGGT

[0198] ACATGGAGCTGGTCGGGTGGCTCGTGGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGGCC

[0199] TCATACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGCGGGAAAGATTA

[0200] TGAGCCTGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTT

[0201] ATAAAATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAAAAGTT

[0202] CGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTACCGGGAAGACCAACATCGCGGAGGCCATAGCCC

[0203] ACACTGTGCCCTTCTACGGGTGCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGG

[0204] TGATCTGGTGGGAGGAGGGCAAGATGACGGCCAAGGTCGTGGAGTCCGCCAAGGCCATTCTCGGCGGCAGCAAG

[0205] GTGCGCGTGGACCAAAAGTGCAAGTCGTCCGCCCAGATCGATCCCACCCCCGTGATCGTCACCTCCAACACCAAC

[0206] ATGTGCGCCGTGATTGACGGGAACAGCACCACCTTCGAGCACCAGCAGCCGTTGCAGGACCGGATGTTCAAATTT

[0207] GAACTCACCCGCCGTCTGGAGCATGACTTTGGCAAGGTGACAAAGCAGGAAGTCAAAGAGTTCTTCCGCTGGGC

[0208] GCAGGATCACGTGACCGAGGTGGCGCATGAGTTCTACGTCAGAAAGGGTGGAGCCAACAAGAGACCCGCCCCCG

[0209] ATGACGCGGATAAAAGCGAGCCCAAGCGGGCCTGCCCCTCAGTCGCGGATCCATCGACGTCAGACGCGGAAGGA

[0210] GCTCCGGTGGACTTTGCCGACAGGTACCAAAACAAATGTTCTCGTCACGCGGGCATGCTTCAGATGCTGTTTCCC

[0211] TGCAAAACATGCGAGAGAATGAATCAGAATTTCAACATTTGCTTCACGCACGGGACCAGAGACTGTTCAGAATGT

[0212] TTCCCCGGCGTGTCAGAATCTCAACCGGTCGTCAGAAAGAGGACGTATCGGAAACTCTGTGCCATTCATCATCTG

[0213] CTGGGGCGGGCTCCCGAGATTGCTTGCTCGGCCTGCGATCTGGTCAACGTGGATCTGGATGACTGTGTTTCTGAG

[0214] CAATAAATGACTTAAACCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGC

[0215] ATTCGCGAGTGGTGGGACTTGAAACCTGGAGCCCCGAAACCCAAAGCCAACCAGCAAAAGCAGGACGACGGCC

[0216] GGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCG

[0217] GCGGATGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCG

[0218] GTATAACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAG

[0219] CAGTCTTCCAGGCCAAGAAGAGGGTTCTCGAACCTTTTGGTCTGGTTGAGGAAGGTGCTAAGACGGCTCCTGGA

[0220] AAGAAACGTCCGGTAGAGCAGTCGCCACAAGAGCCAGACTCCTCCTCGGGCATTGGCAAGACAGGCCAGCAGC

[0221] CCGCTAAAAAGAGACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGTCCCCGACCCACAACCTCTCGGAGAA

[0222] CCTCCAGCAACCCCCGCTGCTGTGGGACCTACTACAATGGCTTCAGGCGGTGGCGCACCAATGGCAGACAATAAC

[0223] GAAGGCGCCGACGGAGTGGGTAATGCCTCAGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATC

[0224] ACCACCAGCACCCGAACATGGGCCTTGCCCACCTATAACAACCACCTCTACAAGCAAATCTCCAGTGCTTCAACG

[0225] GGGGCCAGCAACGACAACCACTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGATTTCAACAGATTCCACTGC

[0226] CATTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAATTGGGGATTCCGGCCCAAGAGACTCAACTTCAAG

[0227] CTCTTCAACATCCAAGTCAAGGAGGTCACGACGAATGATGGCGTCACGACCATCGCTAATAACCTTACCAGCACG

[0228] GTTCAAGTCTTCTCGGACTCGGAGTACCAGTTGCCGTACGTCCTCGGCTCTGCGCACCAGGGCTGCCTCCCTCCG

[0229] TTCCCGGCGGACGTGTTCATGATTCCGCAGTACGGCTACCTAACGCTCAACAATGGCAGCCAGGCAGTGGGACGG

[0230] TCATCCTTTTACTGCCTGGAATATTTCCCATCGCAGATGCTGAGAACGGGCAATAACTTTACCTTCAGCTACACCTT

[0231] CGAGGACGTGCCTTTCCACAGCAGCTACGCGCACAGCCAGAGCCTGGACCGGCTGATGAATCCTCTCATCGACCA

[0232] GTACCTGTATTACCTGAACAGAACTCAGAATCAGTCCGGAGGTCGTGGCGACAGCCCGAGTGCCCAAAACAAGG

[0233] ACTTGCTGTTTAGCCGGGGGTCTCCAGCTGGCATGTCTGTTCAGCCCAAAAACTGGCTACCTGGACCCTGTTACC

[0234] GGCAGCAGCGCGTTTCTAAAACAAAAACAGACAACAACAACAGCAACTTTACCTGGACTGGTGCTTCAAAATAT

[0235] AACCTTAATGGGCGTGAATCTATAATCAACCCTGGCACTGCTATGGCCTCACACAAAGACGACAAAGACAAGTTC

[0236] TTTCCCATGAGCGGTGTCATGATTTTTGGAAAGGAGAGCGCCGGAGCTTCAAACACTGCATTGGACAATGTCATG

[0237] ATCACAGACGAAGAGGAAATCAAAGCCACTAACCCCGTGGCCACCGAAAGATTTGGGACTGTGGCAGTCAATCT

[0238] CCAGAGCAGCAGCACAGACCCTGCGACCGGAGATGTGCATGTTATGGGAGCCTTACCTGGAATGGTGTGGCAAG

[0239] ACAGAGACGTATACCTGCAGGGTCCTATTTGGGCCAAAATTCCTCACACGGATGGACACTTTCACCCGTCTCCTCT

[0240] CATGGGCGGCTTTGGACTTAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACGCCTGTTCCTGCGAATCCTCCG

[0241] GCAGAGTTTTCGGCTACAAAGTTTGCTTCATTCATCACCCAGTATTCCACAGGACAAGTGAGCGTGGAGATTGAAT

[0242] GGGAGCTGCAGAAAGAAAACAGCAAACGCTGGAATCCCGAAGTGCAGTATACATCTAACTATGCAAAATCTGCCA

[0243] ACGTTGATTTCACTGTGGACAACAATGGACTTTATACTGAGCCTCGCCCCATTGGCACCCGTTACCTCACCCGTCC

[0244] CCTGTAATTGTCTGTTAATCAATAAACCGGTTGATTCGTTTCAGTTGAACTTTGGTCTCGCGGCCGCTCGATAAGCT

[0245] TTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATC

[0246] CGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAAC

[0247] TCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGG

[0248] CCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGT

[0249] CGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGC

[0250] AGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTC

[0251] CATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACT

[0252] ATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATAC

[0253] CTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGG

[0254] TCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCG

[0255] TCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAG

[0256] GTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATC

[0257] TGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGT

[0258] AGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTT

[0259] TCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATC

[0260] TTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAG

[0261] TTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGT

[0262] CGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTC

[0263] ACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATC

[0264] CGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTT

[0265] GTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATC

[0266] AAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGT

[0267] AAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATG

[0268] CTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCG

[0269] GCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGC

[0270] GAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGC

[0271] ATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGG

[0272] CGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGA

[0273] GCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACC

[0274] TGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGC

[0275] GTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATG

[0276] CCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCA

[0277] TCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGC

[0278] ATCAGGAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAAT

[0279] AGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGAT

[0280] <Drug composition>

[0281] In some aspects of the present disclosure, a drug composition is provided, which comprises the gene expression cassette provided by the present disclosure, a gene delivery vector and / or an anti-Her2 antibody or an antigen-binding fragment thereof, and, optionally, a pharmaceutically acceptable carrier.

[0282] In some embodiments, the drug composition according to the present disclosure contains the above gene expression cassette, the above gene delivery vector and / or the above anti-Her2 antibody or an antigen-binding fragment thereof as active ingredients.

[0283] In some specific embodiments, the drug composition according to the present disclosure contains a gene delivery vector as an active ingredient, and the gene delivery vector is a recombinant adeno-associated virus.

[0284] The individual dose is generally not less than the amount required to produce a measurable effect on the subject and can be determined based on the pharmacokinetics and pharmacology of the absorption, distribution, metabolism, and excretion (“ADME”) of the drug composition or its by-products and thus based on the disposition of the composition within the subject. This includes considering the route of administration and the dose. The effective dose and / or dosage regimen can be readily determined empirically based on preclinical assays, based on safety and escalation and dose range trials, the individual clinician-patient relationship, and in vitro and in vivo assays.

[0285] As used herein, the term “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, and the like that are compatible with drug administration.

[0286] Drug compositions are generally formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intraarterial, subcutaneous, intraperitoneal, intrathecal, intramuscular, or by injection or infusion. Thus, delivery can be systemic or local.

[0287] In some specific embodiments, the pharmaceutical compositions of the present disclosure are designed, engineered, or adapted for administration to primates (e.g., non-human primates and human subjects) by intravenous injection.

[0288] Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the series of books Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY).

[0289] <Use and Method>

[0290] In some aspects of the present disclosure, methods for treating or preventing a disease (e.g., breast cancer) in a subject in need thereof are provided, the methods comprising administering to the subject in need thereof an effective amount of a gene expression cassette, gene delivery vector, anti-Her2 antibody or antigen-binding fragment thereof, or pharmaceutical composition of the present disclosure.

[0291] In other aspects of the present disclosure, use of the gene expression cassette, gene delivery vector, or anti-Her2 antibody or antigen-binding fragment thereof of the present disclosure in the preparation of a medicament for treating a disease (e.g., breast cancer) is further provided.

[0292] In some specific embodiments, the gene expression cassette, gene delivery vector, anti-Her2 antibody or antigen-binding fragment thereof, or pharmaceutical composition of the present disclosure can at least partially ameliorate breast cancer.

[0293] In some embodiments, the gene expression cassette, gene delivery vector, anti-Her2 antibody or antigen-binding fragment thereof, or pharmaceutical composition of the present disclosure is used for preventing or treating breast cancer. In some optional embodiments, the breast cancer includes central nervous system metastases of breast cancer (e.g., breast cancer brain metastases), Her2-positive breast, etc.

[0294] In some specific embodiments, the gene expression cassette, gene delivery vector, anti-Her2 antibody or its antigen-binding fragment, or pharmaceutical composition is administered locally or directly to the tissue or organ of interest, for example, by injection into the brain. Specifically, the gene expression cassette, gene delivery vector, anti-Her2 antibody or its antigen-binding fragment, or pharmaceutical composition can be administered by in-situ administration and / or inoculation administration. In some more specific embodiments, the gene expression cassette, gene delivery vector, anti-Her2 antibody or its antigen-binding fragment, or pharmaceutical composition is administered by in-situ administration (for example, for brain metastases of breast cancer, in-situ brain administration is used). In some embodiments, the method includes a single administration; in other embodiments, multiple administrations can be performed over time when the attending clinician deems it appropriate.

[0295] The subject can be a mammal, including, for example, a human subject in need of treatment for a specific disease (such as breast cancer).

[0296] Examples

[0297] The embodiments of the present disclosure will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be regarded as limiting the scope of the present disclosure. For those not specified in the examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained commercially.

[0298] Example 1. Construction of AAV shuttle plasmid

[0299] The present disclosure designed a shuttle plasmid XCG-002-P2-002 (hereinafter referred to as plasmid 2) that can express scFv-Fc type. On this basis, to achieve an enhanced ADCC effect, mutations F379L / R428P / Y436L / V441I / P532L were introduced into the Fc segment to form XCG-002-P2-001 (hereinafter referred to as plasmid 1). In addition, XCG-002-P2-003 (hereinafter referred to as plasmid 3) was synthesized, which expresses an isotype control antibody. The vector construction was entrusted to Jiangsu GenScript Prosperous Biotechnology Co., Ltd. The schematic diagram of the antibody tandem sequence pattern in the shuttle plasmid is as Figure 1 shown, and the antibody coding sequence in the plasmid has been optimized.

[0300] Plasmid 1 (nucleotide sequence of shuttle plasmid, SEQ ID NO:18):

[0301]

[0302]

[0303] Among them, the single-underlined part is the signal peptide sequence, the double-underlined part is the antibody coding sequence, the dotted-underlined part is the CAG promoter, the wavy-line part is the chimeric intron, which is used to enhance the gene expression level, and the italic part is the SV40 polyA signal. Antibody coding sequence (SEQ ID NO:19):

[0304] GACATCCAGATGACCCAGTCCCCTAGCAGCCTGAGCGCCAGCGTGGGCGATAGAGTGACCATCA

[0305] CATGTAGAGCCAGCCAGGATGTGAACACCGCCGTGGCCTGGTACCAGCAGAAACCTGGCAAGG

[0306] CACCAAAGCTGCTGATCTACAGCGCCTCATTCCTGTACAGCGGCGTGCCTAGCAGATTCAGCGGC

[0307] AGCCGGAGCGGCACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCTGAGGATTTCGCCACAT

[0308] ACTACTGCCAGCAACACTACACCACCCCACCTACATTCGGCCAGGGCACCAAGGTGGAAATCAA

[0309] GCGGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGAGGAGGCGGCAGCGGCGGCGGCGGAT

[0310] CTGGCGGCGGCGGCAGTGAAGTGCAGCTGGTTGAGAGCGGAGGCGGCCTGGTGCAGCCCGGCG

[0311] GCTCCCTTAGACTGTCCTGCGCCGCTAGCGGATTTAACATCAAGGACACGTACATCCACTGGGTG

[0312] CGGCAGGCGCCTGGAAAGGGCCTCGAGTGGGTCGCCAGAATCTACCCCACCAATGGCTACACCA

[0313] GATACGCCGACAGCGTGAAGGGCAGATTCACCATTTCTGCTGATACCAGCAAGAACACCGCCTA

[0314] CCTGCAGATGAACTCTCTGCGGGCCGAGGACACCGCCGTATACTACTGTAGCAGGTGGGGAGGC

[0315] GATGGCTTTTACGCCATGGACTATTGGGGCCAGGGAACACTGGTGACCGTGTCTAGCGCCTCTAC

[0316] AAAGGGCCCTTCTGTGTTCCCTCTGGCCCCTAGTTCTAAAAGCACCAGCGGCGGAACCGCCGCC

[0317] CTGGGTTGTCTGGTAAAGGACTACTTCCCCGAACCCGTGACCGTGTCCTGGAACAGCGGCGCCC

[0318] TCACAAGCGGAGTTCACACATTCCCAGCCGTGCTGCAAAGCAGCGGCCTGTACAGCCTGAGCTC

[0319] CGTTGTGACAGTGCCCAGCAGCTCCCTGGGCACACAGACCTACATCTGCAACGTTAATCACAAG

[0320] CCTAGCAACACAAAAGTGGACAAGAAGGTGGAACCTAAGAGCTGCGACAAGACACACACTTGT

[0321] CCTCCGTGCCCCGCTCCTGAGCTGCTGGGTGGGCCTAGCGTGTTTCTGCTGCCCCCCAAGCCTAA

[0322] GGATACCCTGATGATTAGTAGAACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCTCACGAG

[0323] GACCCCGAGGTGAAATTCAATTGGTATGTGGACGGCGTGGAAGTGCATAACGCCAAGACCAAAC

[0324] CTCCTGAAGAGCAGTACAACTCTACACTGAGAGTGGTGAGCATCCTGACGGTGCTGCACCAGGA

[0325] CTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCTAACAAGGCCCTGCCTGCTCCAATCGAG

[0326] AAGACCATCTCTAAGGCTAAGGGACAACCTAGAGAGCCTCAGGTGTACACCCTGCCACCTAGCC

[0327] GGGACGAGCTGACCAAAAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCTTCTGA

[0328] CATCGCCGTGGAATGGGAGAGCAATGGCCAACCCGAGAACAACTACAAGACCACACCTCTGGT

[0329] GCTGGACTCTGATGGATCTTTTTTCCTGTATAGCAAACTGACCGTGGATAAGAGCCGCTGGCAGC

[0330] AGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGA

[0331] GCCTGAGCCTGAGTCCTGGCtaa

[0332] Gene expression cassette (SEQ ID NO:23):

[0333]

[0334] Among them, the single-underlined part is the signal peptide sequence, the double-underlined part is the antibody coding sequence, the dotted-underlined part is the CAG promoter, the wavy-line part is the chimeric intron used to enhance gene expression level, and the italic part is the SV40 polyA signal. Plasmid 2 (shuttle plasmid nucleotide sequence, SEQ ID NO:20):

[0335]

[0336]

[0337] Among them, the single-underlined part is the signal peptide sequence, the double-underlined part is the antibody coding sequence, the dotted-underlined part is the CAG promoter, the wavy-line part is the chimeric intron, which is used to enhance the gene expression level, and the italic part is the SV40 polyA signal. Antibody coding sequence (SEQ ID NO:21)

[0338] GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGAGATAGAGTGACTATCA

[0339] CATGCAGAGCCAGCCAGGACGTGAACACCGCTGTGGCCTGGTACCAGCAGAAGCCTGGCAAGG

[0340] CCCCTAAGCTGCTGATCTACTCAGCCTCTTTTCTGTACAGCGGCGTGCCTAGCCGGTTCAGCGGA

[0341] TCTAGAAGCGGCACTGATTTCACACTGACCATCTCCTCTCTGCAGCCCGAGGACTTCGCCACCTA

[0342] CTATTGCCAGCAGCACTACACAACACCCCCTACATTCGGCCAGGGCACCAAGGTGGAAATCAAG

[0343] CGGGGCGGAGGCGGCAGCGGCGGCGGCGGGAGCGGAGGCGGCGGCAGCGGGGGCGGCGGCA

[0344] GCGGCGGAGGCGGAAGCGAGGTGCAGCTGGTCGAGTCTGGCGGCGGACTGGTGCAGCCTGGC

[0345] GGCAGTCTGAGACTGAGCTGCGCCGCTAGCGGCTTCAATATCAAGGACACCTACATCCACTGGG

[0346] TTAGACAGGCTCCTGGCAAAGGCCTGGAATGGGTGGCCAGAATCTACCCCACCAATGGCTACAC

[0347] CAGATACGCCGACTCTGTGAAGGGCAGGTTCACCATCTCCGCTGATACATCTAAAAACACAGCCT

[0348] ACCTGCAGATGAACAGCCTGCGCGCCGAGGACACAGCCGTGTACTACTGCAGCCGGTGGGGCG

[0349] GCGACGGCTTCTACGCCATGGACTACTGGGGCCAGGGTACCCTGGTGACCGTTAGCTCCGCCAG

[0350] CACCAAAGGCCCCAGCGTGTTCCCTCTGGCTCCTTCCTCTAAGAGCACCTCTGGAGGCACCGCC

[0351] GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCTGTGACGGTGAGCTGGAACTCTGGCG

[0352] CCCTGACATCCGGCGTGCATACATTCCCAGCCGTGCTGCAGAGCAGCGGGCTGTACAGCCTGAG

[0353] TAGCGTGGTGACCGTGCCCTCATCTAGCCTCGGCACCCAGACCTACATCTGCAACGTGAACCAC

[0354] AAGCCCAGCAATACCAAGGTCGACAAGAAGGTGGAACCGAAGAGCTGCGACAAGACCCACAC

[0355] ATGTCCTCCTTGTCCTGCCCCCGAGCTCCTGGGCGGACCTAGCGTTTTCTTGTTTCCCCCTAAGCC

[0356] TAAAGATACCCTGATGATTAGCAGAACCCCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAC

[0357] GAGGACCCTGAAGTGAAATTTAACTGGTATGTGGACGGCGTGGAGGTGCACAACGCCAAGACA

[0358] AAACCCAGAGAGGAACAGTACAACTCAACATATAGAGTGGTCTCCGTTCTCACAGTGCTGCACC

[0359] AGGATTGGCTGAACGGCAAGGAATACAAGTGCAAGGTTTCTAACAAGGCCCTGCCTGCTCCTAT

[0360] CGAGAAGACCATCAGCAAGGCAAAGGGCCAGCCACGGGAACCCCAGGTCTATACCCTGCCACC

[0361] TAGCAGAGATGAGCTGACCAAAAACCAGGTGAGCCTTACATGCCTGGTGAAGGGCTTCTACCCT

[0362] AGCGATATCGCCGTGGAATGGGAAAGCAACGGACAACCTGAGAACAATTACAAGACCACCCCTC

[0363] CAGTGCTGGACAGCGACGGAAGCTTCTTCCTGTACAGCAAGCTGACCGTTGATAAGTCCCGGTG

[0364] GCAACAAGGCAATGTGTTTAGCTGTTCTGTGATGCACGAGGCGCTGCACAACCACTACACACAA

[0365] AAAAGCCTGTCTCTGTCCCCTGGTta g

[0366] Gene expression cassette (SEQ ID NO:24):

[0367]

[0368] Among them, the single-underlined part is the signal peptide sequence, the double-underlined part is the antibody coding sequence, the dotted-underlined part is the CAG promoter, the wavy line part is the chimeric intron, which is used to enhance the gene expression level, and the italic part is the SV40 polyA signal. Plasmid 3 (shuttle plasmid nucleotide sequence, SEQ ID NO:22):

[0369]

[0370]

[0371] Among them, the single-underlined part is the signal peptide sequence, the double-underlined part is the control antibody coding sequence, the dotted-underlined part is the CAG promoter, the wavy line part is the chimeric intron, which is used to enhance the gene expression level, and the italic part is the SV40 polyA signal. Amino acid sequence of the expression product of plasmid 1 (SEQ ID NO:1):

[0372]

[0373] Among them, the single-underlined part is the light chain variable region, the double-underlined part is the heavy chain, and the bold + italic part is the linker.

[0374] Amino acid sequence of the light chain variable region (SEQ ID NO:2):

[0375] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSG

[0376] TDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR

[0377] Amino acid sequence of the heavy chain (SEQ ID NO:3):

[0378] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKG

[0379] RFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLA

[0380] PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT

[0381] YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLLPPKPKDTLMISRTPEVTCVVV

[0382] DVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSILTVLHQDWLNGKEYKCKVSNKALP

[0383] APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLV

[0384] LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0385] Amino acid sequence of the expression product of plasmid 2 (SEQ ID NO: 4):

[0386]

[0387] Among them, the single-underlined part is the light chain variable region, the double-underlined part is the heavy chain, and the part shown in bold + italic is the linker.

[0388] Amino acid sequence of the light chain variable region (SEQ ID NO: 2):

[0389] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSG

[0390] TDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR

[0391] Amino acid sequence of the heavy chain (SEQ ID NO: 5):

[0392] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKG

[0393] RFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLA

[0394] PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT

[0395] YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV

[0396] DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

[0397] PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP

[0398] VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0399] Amino acid sequence of the expression product of plasmid 3 (CH3 domain of human IgG1 antibody) (SEQ ID NO: 6):

[0400] SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

[0401] NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL

[0402] PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ

[0403] QGNVFSCSVMHEALHNHYTQKSLSLSPG

[0404] Nucleotide sequence encoding the signal peptide (human interleukin-2 signal peptide) (SEQ ID NO: 7):

[0405] ATGTACCGGATGCAGCTGCTGTCTTGTATCGCCCTGTCCCTGGCCCTGGTGACCAACTCC

[0406] Amino acid sequence of the signal peptide (human interleukin-2 signal peptide) (SEQ ID NO: 8): MYRMQLLSCIALSLALVTNS CDR regions of the expression product of plasmid 1 (kabat method):

[0407] CDRL1 (SEQ ID NO: 9): RASQDVNTAVA

[0408] CDRL2 (SEQ ID NO: 10): SASFLYS

[0409] CDRL3 (SEQ ID NO:11): QQHYTTPPT

[0410] CDRH1 (SEQ ID NO:12): DTYIH

[0411] CDRH2 (SEQ ID NO:13): RIYPTNGYTRYADSVKG

[0412] CDRH3 (SEQ ID NO:14): WGGDGFYAMDY

[0413] CDR regions of the expression product of plasmid 2 (kabat method):

[0414] CDRL1 (SEQ ID NO:9): RASQDVNTAVA

[0415] CDRL2 (SEQ ID NO:10): SASFLYS

[0416] CDRL3 (SEQ ID NO:11): QQHYTTPPT

[0417] CDRH1 (SEQ ID NO:12): DTYIH

[0418] CDRH2 (SEQ ID NO:13): RIYPTNGYTRYADSVKG

[0419] CDRH3 (SEQ ID NO:14): WGGDGFYAMDY

[0420] Elements involved in the above plasmid:

[0421] CAG promoter (SEQ ID NO:15):

[0422] gacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctg

[0423] gctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggactatttac

[0424] ggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtac

[0425] atgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatgggtcgaggtgagccccacgttctgcttcactctccccatctcccc

[0426] cccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggcgcgcgccaggcggggcggggcgggg

[0427] cgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcg

[0428] gcggccctataaaaagcgaagcgcgcggcgggcg

[0429] Chimeric intron (SEQ ID NO:16):

[0430] ggagtcgctgcgttgccttcgccccgtgccccgctccgcgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggc

[0431] gggacggcccttctcctccgggctgtaattagcgcttggtttaatgacggctcgtttcttttctgtggctgcgtgaaagccttaaagggctccgggagggcccttt

[0432] gtgcgggggggagcggctcggggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgcggcccgcgctgcccggcggctgtgagcgctgcggg

[0433] cgcggcgcggggctttgtgcgctccgcgtgtgcgcgaggggagcgcggccgggggcggtgccccgcggtgcgggggggctgcgaggggaacaaag

[0434] gctgcgtgcggggtgtgtgcgtgggggggtgagcagggggtgtgggcgcggcggtcgggctgtaacccccccctgcacccccctccccgagttgctgag

[0435] cacggcccggcttcgggtgcggggctccgtgcggggcgtggcgcggggctcgccgtgccgggcggggggtggcggcaggtgggggtgccgggcgg

[0436] ggcggggccgcctcgggccggggagggctcgggggaggggcgcggcggccccggagcgccggcggctgtcgaggcgcggcgagccgcagccatt

[0437] gccttttatggtaatcgtgcgagagggcgcagggacttcctttgtcccaaatctggcggagccgaaatctgggaggcgccgccgcaccccctctagcgggc

[0438] gcgggcgaagcggtgcggcgccggcaggaaggaaatgggcggggagggccttcgtgcgtcgccgcgccgccgtccccttctccatctccagcctcggg

[0439] gctgccgcagggggacggctgccttcgggggggacggggcagggcggggttcggcttctggcgtgtgaccggcggctctagagcctctgctaaccatgtt

[0440] catgccttcttctttttcctacag

[0441] SV40 polyA signal (SEQ ID NO:17):

[0442] tatttgtgaaatttgtgatgctattgctttatttgtaaccatctagctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaa

[0443] caacaacaattgcattcattttatgtttcaggttcagggggagatgtgggaggttttttaaagcgg

[0444] Example 2, Plasmid Expression Verification

[0445] By transforming the plasmids prepared in Example 1 into competent Escherichia coli cells, picking monoclonal colonies and then expanding the culture, plasmid extraction was performed to obtain Plasmid 1, Plasmid 2 and Plasmid 3; the three amplified plasmids were respectively transfected into suspension Chinese hamster ovary cells (ExpiCHO-S) (Gibco) using liposomes. Prepare 70 mL of 3×10^7 ExpiCHO-S cells for transfection; use 1 mL of Opti-MEM (Gibco) and 100 μg of plasmid, and mix well according to the ratio of liposome:plasmid = 3:1, and incubate at room temperature for 5 min to form a transfection complex. The transfection complex was added dropwise to the cells to be transfected and cultured in suspension in a cell culture incubator; 8 mL, 9 mL and 10 mL of feeding medium (Gibco) were supplemented respectively 1 day, 3 days and 5 days after transfection; the culture supernatant was collected 8 days after transfection, and the supernatant was purified by one-step affinity chromatography and then subjected to two-step purification; finally, the expression product was analyzed by SDS-PAGE. The plasmid expression verification was entrusted to Zhejiang Newma Biopharmaceutical Co., Ltd.

[0446] Experimental Results: Antibody Expression and Molecular Weight Identification (SDS-PAGE)

[0447] The SDS-PAGE results showed that the purities of the expression products of Plasmid 1 and Plasmid 2 reached 95% and 98% respectively after purification (as Figure 2 shown). The above results indicate that Plasmid 1 and Plasmid 2 can express proteins with the correct molecular weight under conventional experimental conditions.

[0448] In addition, by comparing Plasmid 1 and Plasmid 2 with the plasmid whose antibody coding sequence was not optimized, it was found that the optimization significantly improved the antibody protein expression ability. The specific steps are as follows:

[0449] A. Cell Culture and Transfection: After culturing 293T (Wuhan Punosai) cells to the third generation, plate them at 2×10^5 cells / well (6-well plate); when the confluence reaches about 60%, prepare the transfection reagent for cell transfection as follows. After adding the mixture, continue to culture for 48 hours; collect the cell supernatant.

[0450]

[0451] Among them, NA means not applicable, that is, no plasmid was added.

[0452] Note: The plasmid 1 control and the plasmid 2 control were synthesized by Shanghai Jiman Biotechnology, without sequence optimization, and the expression products correspond to the expression products of plasmid 1 and plasmid 2. Lipo8000 was purchased from Beijing Biotium; DMEM medium was purchased from gibco.

[0453]

[0454]

[0455] B. Protein sample concentration detection: Centrifuge the supernatant at 12,000 rpm for 10 min at 4°C; use a BCA kit (Beijing Biosynthesis) to detect the protein concentration; pipette 0, 2, 4, 6, 8, 12, 16, 20 μl of 5 mg / mL BSA protein standard into a 96-well plate (Corning), and add PBS buffer (Gibco) to a final volume of 20 μl. Take 20 μL of the centrifuged supernatant and add it to the 96-well plate; add 200 μL of working solution (BCA reagent: Cu reagent = 50:1) to each well, and react in the dark at 37°C for 15 min. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance at 562 nm, draw a standard curve, and calculate the corresponding protein sample concentration.

[0456] C. Sample preparation: Pipette 160 μL of the remaining supernatant from step B, add 40 μL of 5× SDS loading buffer (Beijing Biosynthesis), and incubate in a 100°C water bath for 10 minutes.

[0457] D. SDS-PAGE immunoblot analysis: Use a precast gel (Beijing Lamboid) for electrophoresis. The protein sample loading amount is uniformly 50 μg. Add protein molecular weight standards (Thermo Fisher) to the two side lanes, and perform electrophoresis at a constant voltage of 160 V for 60 minutes; take out the 1× transfer buffer (Beijing Biosynthesis) pre-cooled in the refrigerator, soak the transfer equipment, soak the PVDF membrane (Merck) with the PVDF membrane activation solution (Beijing Biosynthesis) for 30 s, and then transfer at 400 mA for 25 minutes in an ice bath; after the transfer is completed, immerse the PVDF membrane completely in the blocking solution (Beijing Biosynthesis), place it on a low-speed shaker (Beijing Lamboid) at room temperature for 20 minutes; after blocking, add the corresponding 1:2000 diluted anti-human fab (Jackson ImmunoResearch) antibody, and incubate at room temperature for 2 h; rinse 3 times with the washing solution (Beijing Biosynthesis); add the ECL luminescent solution (Beijing Biosynthesis) and develop immediately. Save the picture data and perform gray-scale analysis using Image J.

[0458] Results:

[0459] The results showed ( Figure 15 ), all four plasmids could mediate the expression of the target protein in 293T cells. The expression level in the plasmid 1 group was significantly higher than that in the plasmid 1 control group ( Figure 16 ), and the expression level in the plasmid 2 group was significantly higher than that in the plasmid 2 control group ( Figure 17 ), indicating that the nucleotide sequence optimization improved the ability of the plasmid to mediate protein expression in human cells.

[0460] Example 3. Identification of antibody-antigen binding ability

[0461] Add 100 μL of Human HER2-Tag Free (0.5 g / ml) (Acro) coating solution to each well of a high-binding 96-well plate, seal the plate with a sealing film, and place it in a refrigerator (2 - 8 °C) overnight (10 - 18 h); discard the liquid in the plate, add 300 μL of washing solution (PBST) to each well and wash the plate three times, then pat it dry on paper; pre-dilute the test samples uniformly to 10.0 μg / ml with the blocking solution. Then, starting from a concentration of 10 μg / ml, perform 3-fold serial dilutions with the blocking solution, and the concentration gradient range is (0.169 - 10000 ng / ml); add 100 μL of the diluted test samples to the corresponding wells in duplicate, seal the plate with a sealing film, and let it stand for 2 h ± 15 min; discard the liquid in the plate, add 300 μL of washing solution to each well and wash the plate three times, then pat it dry on paper; add 100 μL of anti-human IgG-Fc-HRP (Miaodi) detection antibody solution (1:500, 0.4 g / ml) to each well, seal the plate with a sealing film, and let it stand for 1 h ± 15 min; discard the liquid in the plate, add approximately 300 μL of washing solution to each well and wash the plate five times, then pat it dry on paper; add 100 μL of TMB chromogenic solution (Seracare) to each well, and when the reaction reaches a certain degree, add 100 μL of sulfuric acid termination solution, shake the plate to mix well; finally, use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance at 450 nm - 650 nm. This value is positively correlated with the amount of anti-Her2 antibody in the sample. The identification of antibody-antigen binding ability was entrusted to Zhejiang Newma Biopharmaceutical Co., Ltd.

[0462] Experimental results: Effective expression of Her2 antibody (ELISA)

[0463] The ELISA experimental results showed that the expression products of plasmid 1 and plasmid 2 had the ability to bind to Her2 protein (as Figure 3 shown). The median effect concentration (EC50) was 35.06 ng / mL and 48.77 ng / mL respectively. ARX788 (Zhejiang Newma Biopharmaceutical Co., Ltd.) is an antibody-drug conjugate targeting Her2, and as a positive control, EC50 = 32.54 ng / mL. The expression product of plasmid 3 was used as a negative control, and the signal intensity was below the detection limit. The above results indicate that the expression products of plasmid 1 and plasmid 2 are functional proteins targeting Her2.

[0464] Example 4. Identification of ADCC effect

[0465] This disclosure uses the ADCC FcyRIII 158V Jurkat Effector cell line (hereinafter referred to as Jurkat or Jurkat-158V) (a gift from Zhejiang NewCode Biopharma Co., Ltd.) as the effector cell. The Jurkat cells stably express the FcγRIIIa receptor and firefly luciferase driven by a transcription factor. The biological activity of the antibody in the ADCC mechanism is quantified by the luciferase produced by the activation of the transcription factor, while the luciferase activity in the effector cells is quantified by bioluminescence readings.

[0466] In addition, the antibody to be tested (10×) and the target cell Human Breast Cancer Cells (BT474) (a gift from Zhejiang NewCode Biopharma Co., Ltd.) were prepared. First, 40 μL of a total of 1E4 BT474 cells were added to the corresponding wells, and then 50 μL of a total of 2E5 Jurkat cells were added; 10 μL of each drug was taken from the 10× drug storage plate and added to the corresponding cell wells (final concentration 1×), and the control group was added with the same volume of complete culture medium. After thorough mixing, the cells were incubated at 37 °C for 24 hours; the GMOne-Step Luciferase Reporter Gene Detection Kit (Giman Biotechnology Co., Ltd.) was taken out and restored to room temperature. 100 μL of GMOne-Step was added to each well, shaken for 2 minutes, and then left to stand in the dark for 30 minutes. The signal intensity was detected with an enzyme-linked immunosorbent assay reader, and the signal intensity was positively correlated with the ADCC effect.

[0467] Experimental results: The Her2 antibody has an ADCC effect

[0468] The results showed that under the conditions of this disclosure, when using Jurkat as the effector cell, ARX788 (positive control), the expression product of plasmid 1, and the expression product of plasmid 2 all showed dose-dependent ADCC activity against BT474 cells (as Figure 4 shown). The EC50 values of ARX788, the expression product of plasmid 1, and the expression product of plasmid 2 were 5.8 ng / mL, 6.12 ng / mL, and 16.87 ng / mL, respectively. It is worth noting that at the same concentration, the signal intensity of the expression product of plasmid 1 was greater than that of the expression product of plasmid 2, indicating that the Fc segment modification application in plasmid 1 achieved an enhanced ADCC effect.

[0469] Example 5: AAV virus packaging and titer determination

[0470] The AAV virus packaging was carried out according to the common three-plasmid transfection method. The capsid gene plasmid of AAV6.2 type (RC6.2 disclosed in WO2024 / 234803, the specific sequence of RC6.2 is shown in SEQ ID NO:29) or the wild-type capsid gene plasmid of AAV2 (Weizhen, Shandong) or the wild-type capsid gene plasmid of AAV6 (Weizhen, Shandong), the Helper plasmid (Weizhen, Shandong) and the shuttle plasmid (Table 1) were co-transfected into HEK293 cells. The AAV virus particles were separated and purified from HEK293T cells by iodixanol density gradient. The titer of AAV was determined by the common qPCR method. pAAV-CAG-RFP (Weizhen, Shandong) was used as the shuttle plasmid to package AAV2-RFP, AAV6-RFP, and AAV6.2-RFP respectively. Plasmid 1, Plasmid 2, and Plasmid 3 were packaged as XG0201, XG0202, and XG0203 respectively. The packaging and titer determination of the AAV virus were completed by Weizhen Biotechnology Co., Ltd., Shandong.

[0471] Experimental results: Production and packaging of AAV

[0472] The AAV packaging situation is shown in Table 1.

[0473] Table 1 AAV packaging situation

[0474]

[0475] Example 6. In vitro verification of the ability of AAV to infect cells

[0476] After culturing BT474 (gifted by Zhejiang NewCode Biopharmaceuticals Co., Ltd.) cells to the third generation, they were plated at 1E4 / well (96-well plate); when the confluence reached about 60%, the following AAV working solution was prepared for cell transduction. Cultivation was continued for 72 hours; the fluorescence intensity at 579 nm - 605 nm was detected by an enzyme-labeled instrument.

[0477] Group Setting AAV Dose (vg / well) Complete Medium (μL / well) Blank Group (Medium) NA 200 AAV2-RFP 1E9 200 AAV6-RFP 1E9 200 AAV6.2-RFP 1E9 200

[0478] Among them, NA means not applicable, that is, AAV was not added.

[0479] Experimental results: The transduction efficiency of the modified capsid in BT474 was significantly improved

[0480] After 72 hours of in vitro transduction, the results detected by the enzyme-labeled instrument showed that the transduction efficiency of AAV6.2-RFP in BT474 cells was significantly higher than that of the AAV2-RFP and AAV6-RFP groups, and the AAV2-RFP and AAV6-RFP groups could hardly transduce BT474 cells (see Figure 18 ).

[0481] Example 7. In vivo pharmacodynamic experiment in mice

[0482] (1) Pharmacodynamic experiment of subcutaneous transplanted tumor mice with BT474 cells

[0483] In this disclosure, 6-8-week-old female BALB / c nude mice (Jiangsu Jicui Yakang Biotechnology Co., Ltd.) were used. BT474 cells (Nanjing Pengbo Biotechnology Co., Ltd.) were cultured in vitro in RPMI 1640 medium (Gibco) containing 15% fetal bovine serum (Gibco) and 1% penicillin-streptomycin at 37 °C in an environment of 5% CO 2 . They were routinely digested and passaged with trypsin (Gibco) twice a week. When the cells reached the required amount, the cells were collected, counted, and inoculated. 0.72 mg estrogen tablets (Innovative Research of America, IRA) were implanted subcutaneously 72 h before the inoculation of tumor cells. On the day of inoculation of tumor cells, the mice were randomly grouped according to their body weight. BT474 cells were collected, resuspended in RPMI 1640 medium without fetal bovine serum to 13.3E6 / 0.1 mL, and then an equal volume of Matrigel was added and mixed well. The test substances were mixed with the cell suspension according to Table 2, and a suspension of 1E7 BT474 cells and the test substance was inoculated subcutaneously above the right scapula of the mice (day 0). Then, the tumor volume of the mice was monitored using a vernier caliper. To explore the correlation between tumor volume and Her2 antibody expression level, the mice were sacrificed at the end point of the experiment (day 30) to collect tumor tissues for ELISA experiments (the method was the same as in Example 3). This experiment was commissioned to Nanjing Pengbo Biotechnology Co., Ltd.

[0484] Table 2 Inoculation and administration plan

[0485]

[0486] Among them, NA means not applicable, that is, AAV was not added.

[0487] Experimental results: XG0201 significantly inhibited the growth of subcutaneous tumors in mice

[0488] The results of the subcutaneous pharmacodynamic experiment showed (see Table 3) (as Figures 5 - 6 ), on the 27th day after inoculation, the average tumor volume of the mice in the control group (saline group) reached 1478.55 mm 3 . Compared with the control group, after administering XG0203 at 1E11 vg / mouse to the tumor-bearing mice, at the end point of the experiment, the average tumor volume of the mice was 1585.19 mm 3 , and the Tumor Growth Inhibition (TGI) value was -7.21%; after administering XG0201 at 1E11 vg / mouse to the tumor-bearing mice, at the end point of the experiment, the average tumor volume of the mice was 589.65 mm 3, the TGI value was 60.12%; after administering XG0202 at 1E11 vg / mouse to tumor-bearing mice, at the end of the experiment, the average tumor volume of the mice was 982.86 mm 3 , and the TGI value was 33.53%. Compared with the control group, XG0201 at 1E11 vg / mouse showed a significant effect on inhibiting tumor growth (p value < 0.05).

[0489] In the present disclosure, the tolerance of animals to the test substance was indirectly indicated by changes in body weight. In all the mice in the present disclosure, there was no obvious decrease in body weight, nor any case of mouse death. The experimental results showed that the tumor-bearing mice showed good tolerance to the test substance at the experimental dose (such as Figure 7 ).

[0490] The results of ELISA experiments on tumor tissue samples showed that there was a certain correlation between the size of subcutaneous transplanted tumors and the concentration of Her2 antibody in tumor samples (see Table 4).

[0491] Table 3 Tumor growth inhibition in subcutaneous models

[0492]

[0493] Note: TGI (%) = 100% × (1 - (tumor volume change in the experimental group) / (tumor volume change in the control group)); NA means not applicable, that is, AAV was not added.

[0494] Table 4 Detection of Her2 antibody content in tumor tissue samples

[0495]

[0496] Note: BQL is below the detection limit

[0497] (2) In vivo pharmacodynamic pre-experiment of BT474-luc cell mouse brain orthotopic model

[0498] In the present disclosure, 6-8-week-old female BALB / c nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used. BT474-luc (Nanjing Pengbo Biotechnology Co., Ltd.) cells were cultured in 1640 medium (Gibco) containing 15% fetal bovine serum (Gibco) and 1% double antibody at 37 °C and 5% CO 2In vitro culture was carried out under the [specific environment]. Passage was performed by routine digestion with trypsin (Gibco) twice a week. When the required amount of cells was reached, the cells were collected, counted, and inoculated. 0.72 mg of estrogen tablets (IRA) were implanted subcutaneously 72 h before inoculation of tumor cells. BT474-luc cells were collected on the day of tumor cell inoculation. The inoculation protocol is shown in Table 5. After anesthetizing the animals by inhaling isoflurane, brain in situ inoculation was performed using a brain stereotaxic instrument (day 0). Inoculation location: AP (from bregma backward): -2 mm; ML (lateral to the sagittal suture): 1.5 mm; DV (depth of penetration into the brain): -3.5 mm. After cell inoculation, D-luciferin potassium salt was intraperitoneally injected at a dose of 150 mg / kg twice a week. Luc imaging was performed in the auto mode in the prone position within 15 minutes after injecting D-luciferin potassium salt. At the experimental endpoint (day 25), the mice were sacrificed, and the brain tissues were fixed. Coronal sections were made starting from the injection center, and the growth of tumors in the mouse brain tissues was identified by H&E staining. This experiment was commissioned to Nanjing Pengbo Biotechnology Co., Ltd.

[0499] Table 5 Inoculation and administration protocol

[0500]

[0501]

[0502] Among them, NA indicates not applicable, that is, AAV was not added.

[0503] Experimental results: XG0201 significantly inhibited the growth of intracranial tumors in mice

[0504] The results of the in situ pharmacodynamic pre-experiment showed (see Table 6 and Figure 8 ), on the 25th day after inoculation, the average tumor signal of the mice in the control group (saline group) reached 3.05E8. Compared with the control group, after giving XG0203 at 6E10 vg / mouse to the tumor-bearing mice, at the experimental endpoint, the average tumor signal of the mice was 2.83E8, and the TGI value was 9%; after giving XG0201 at 6E10 vg / mouse to the tumor-bearing mice, at the experimental endpoint, the average tumor signal of the mice was 9.4E6 ( Figure 9 ), and the TGI value was 97%; compared with the control group, XG0201 at 6E10 vg / mouse showed a significant effect of inhibiting tumor growth (p value < 0.05), while even though XG0202 showed a certain tumor inhibitory effect in the pharmacodynamic experiment of subcutaneous transplanted tumor mice, in this experiment, 6E10 vg / mouse did not show an effect of inhibiting tumor growth ( Figure 9 ). The H&E results of the mouse brain coronal sections were consistent with the in vivo imaging results, and XG0201 significantly inhibited the growth of intracranial tumors ( Figure 10 ).

[0505] The present disclosure indirectly indicates the tolerance of animals to the test substance by changes in body weight. In the present disclosure, no significant decrease in body weight was observed in all mice, nor was there any case of mouse death. The experimental results show that the tumor-bearing mice showed good tolerance to the test substance at the experimental dose( Figure 11 ).

[0506] Table 6 In-situ model tumor growth inhibition

[0507]

[0508] Note: TGI(%) = 100% × (1 - (tumor signal change in the experimental group) / (tumor signal change in the control group)); NA indicates not applicable, that is, AAV was not added.

[0509] (3) In-vivo efficacy test of BT474-Luc cell mouse brain in-situ model

[0510] The present disclosure uses 6-8 week-old female BALB / c nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.). BT474-luc cells are cultured in vitro in 1640 medium (Gibco) containing 15% fetal bovine serum (Gibco) and 1% double antibody at 37 °C and 5% CO 2 environment. Trypsin (Gibco) is used for routine digestion and passage twice a week. When the cells reach the required amount, the cells are collected, counted and inoculated. 0.72 mg estrogen tablets (IRA) are implanted subcutaneously 72 h before tumor cell inoculation. On the day of tumor cell inoculation, BT474-luc cells are collected. The inoculation protocol is shown in Table 7. After the animals are anesthetized by inhaling isoflurane, 1E5 BT474-luc cells / mouse are inoculated in-situ into the brain using a brain stereotaxic apparatus (day 0). Inoculation location: AP (from bregma backward): -2 mm; ML (lateral to the sagittal suture): 1.5 mm; DV (depth into the brain): -3.5 mm. After cell inoculation, the animals are randomly grouped according to IVIS in-vivo imaging data (tumor grows to 1E6 Total flux) and body weight, and the drug is administered according to Table 7. Administration location: AP (from bregma backward): -2 mm; ML (lateral to the sagittal suture): 1.5 mm; DV (depth into the brain): -3.5 mm (administer 5 μL) and -2.5 mm (administer 5 μL). After drug administration, 150 mg / kg dose of D-luciferin potassium salt is injected intraperitoneally twice a week, and luc imaging is performed in the auto mode in the prone position within 15 minutes after injecting D-luciferin potassium salt. Imaging is no longer performed after day 25, and the mice are continued to be raised. When the body weight of the mice drops by 30%, they are euthanized and the death time is recorded, and the survival curve is plotted.

[0511] Table 7 Drug administration protocol

[0512]

[0513] Experimental results: XG0201 significantly inhibits the growth of intracranial tumors in mice and prolongs the survival period of mice in a breast cancer brain metastasis model

[0514] The results of in-situ pharmacodynamic experiments showed that, compared with the control group (XG0203), after administration of XG201 at 6E10 vg / mouse, on the 25th day after inoculation, there was a significant tumor inhibitory effect (see Table 8, Figure 12 ), the statistical analysis of the original data showed that TGI = 72%, and the survival period of the mice in this group was significantly prolonged (p = 0.0037), and the median survival period increased by 47.5% (see Table 9, Figure 14 ).

[0515] In the present disclosure, the tolerance of animals to the test substance is indirectly indicated by body weight changes. Within three weeks after administration, the body weights of all mice in the present disclosure did not show obvious decreases ( Figure 13 ), and there was no case of mouse death. The experimental results showed that the tumor-bearing mice showed good tolerance to the test substance at the experimental dose.

[0516] Table 8 Tumor growth inhibition in in-situ model

[0517]

[0518] Note: TGI (%) = 100% × (1 - (tumor signal change in experimental group) / (tumor signal change in control group))

[0519] Table 9 Survival curve analysis

[0520]

[0521] In summary, the AAV of the present disclosure can mediate the expression of Her2 antibody. In-vivo experiments have proved that XG0201 inhibits the growth of subcutaneous tumors and in-situ tumors, and significantly prolongs the survival period of mice in a breast cancer brain metastasis model, showing good application prospects.

[0522] It should be noted that although the technical solutions of the present disclosure are introduced by specific examples, those skilled in the art can understand that the present disclosure should not be limited thereto.

[0523] The above have described the embodiments of the present disclosure. The above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art in the technical field without departing from the scope and spirit of the described embodiments. The selection of the terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other ordinary skill in the art in the technical field to understand the disclosed embodiments.

Claims

1. A gene expression cassette comprising a coding sequence of an anti-Her2 antibody or an antigen-binding fragment thereof, and, optionally, at least one of a promoter, an intron, and a polyadenylation signal; the anti-Her2 antibody or the antigen-binding fragment thereof comprises a light chain variable region and a heavy chain; The light chain variable region comprises the amino acid sequence shown in SEQ ID NO:2, or a sequence having at least 85% identity with SEQ ID NO:2; The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 3 or 5, or a sequence having at least 85% identity with SEQ ID NO: 3 or 5.

2. The gene expression cassette according to claim 1, wherein The anti-Her2 antibody or antigen-binding fragment thereof does not comprise a light chain constant region.

3. The gene expression cassette according to claim 1 or 2, wherein The light chain variable region and the heavy chain are connected via a linker.

4. The gene expression cassette according to any one of claims 1 to 3, wherein The light chain variable region comprises three complementarity determining regions (CDRs): LCDR1, LCDR2 and LCDR3, and the heavy chain variable region of the heavy chain comprises three complementarity determining regions (CDRs): HCDR1, HCDR2 and HCDR3; and, The LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively, or a sequence having at least 85% identity with any one of SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively, or a sequence that is at least 85% identical to any one of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO:

14.

5. The gene expression cassette according to any one of claims 1 to 4, wherein The anti-Her2 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 1 or 4, or a sequence having at least 85% identity with SEQ ID NO: 1 or 4.

6. The gene expression cassette according to any one of claims 1 to 5, wherein The coding sequence of the anti-Her2 antibody or antigen-binding fragment thereof comprises the nucleotide sequence shown in SEQ ID NO: 19 or 21, or a sequence having at least 85% identity with the sequence shown in SEQ ID NO: 19 or 21.

7. The gene expression cassette according to any one of claims 1 to 6, wherein The promoter comprises a CAG promoter; Preferably, the CAG promoter comprises the nucleotide sequence as shown in SEQ ID NO:15, or a sequence having at least 85% identity with SEQ ID NO:

15.

8. The gene expression cassette according to any one of claims 1 to 7, wherein The intron comprises a chimeric intron; Preferably, the chimeric intron comprises the nucleotide sequence as shown in SEQ ID NO:16, or a sequence having at least 85% identity with SEQ ID NO:

16.

9. The gene expression cassette according to any one of claims 1 to 8, wherein The coding sequence of the anti-Her2 antibody or antigen-binding fragment thereof comprises a sequence encoding a signal peptide upstream, wherein the signal peptide comprises a human interleukin-2 signal peptide; Preferably, the human interleukin-2 signal peptide comprises the amino acid sequence shown in SEQ ID NO: 8, or a sequence having at least 85% identity with SEQ ID NO:

8.

10. The gene expression cassette according to any one of claims 1 to 9, wherein: The gene expression cassette further comprises a polyadenylation region; Optionally, the polyadenylation region comprises a polyadenylation signal from SV40; Preferably, the polyadenylation signal from SV40 comprises the nucleotide sequence shown in SEQ ID NO:17, or a sequence having at least 85% identity with SEQ ID NO:

17.

11. The gene expression cassette according to any one of claims 1 to 10, wherein: The structure of the gene expression cassette is as follows: [Promoter]-[Intron]-[Sequence encoding signal peptide]-[Coding sequence of anti-Her2 antibody or antigen-binding fragment thereof]-[Polyadenylation region].

12. The gene expression cassette according to any one of claims 1 to 11, wherein The gene expression cassette comprises a nucleotide sequence as shown in SEQ ID NO: 23 or 24, or a sequence having at least 85% identity with SEQ ID NO: 23 or 24.

13. A gene delivery vector comprising the gene expression cassette according to any one of claims 1 to 12.

14. The gene delivery vector according to claim 13, wherein The gene delivery vector is a viral vector derived from a virus; Preferably, the gene delivery vector is a recombinant adeno-associated virus; More preferably, the gene delivery vector comprises the nucleotide sequence shown in SEQ ID NO: 18 or 20, or a sequence having at least 85% identity to SEQ ID NO: 18 or 20.

15. The gene delivery vector according to claim 14, wherein The recombinant adeno-associated virus comprises a capsid protein, and the gene expression cassette is encapsidated within the capsid protein; Optionally, the capsid protein is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10 adeno-associated virus serotypes or variants thereof.

16. The gene delivery vector according to claim 15, wherein The capsid protein includes a capsid protein derived from the AAV6 adeno-associated virus serotype or a variant thereof.

17. The gene delivery vector according to claim 16, wherein The capsid protein comprises a targeting peptide as shown in SEQ ID NO: 25; Preferably, the capsid plasmid encoding the capsid protein comprises the nucleotide sequence shown in SEQ ID NO:29, or a sequence having at least 85% identity with SEQ ID NO:

29.

18. An anti-Her2 antibody or an antigen-binding fragment thereof, comprising a light chain variable region and a heavy chain; The light chain variable region comprises the amino acid sequence shown in SEQ ID NO:2, or a sequence having at least 85% identity with SEQ ID NO:2; The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 3 or 5, or a sequence having at least 85% identity with SEQ ID NO: 3 or 5.

19. The anti-Her2 antibody or antigen-binding fragment thereof according to claim 18, wherein The anti-Her2 antibody or antigen-binding fragment thereof does not comprise a light chain constant region.

20. The anti-Her2 antibody or antigen-binding fragment thereof according to claim 18 or 19, wherein The light chain variable region and the heavy chain are connected via a linker.

21. The anti-Her2 antibody or antigen-binding fragment thereof according to any one of claims 18 to 20, wherein: The anti-Her2 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 1 or 4, or a sequence having at least 85% identity with SEQ ID NO: 1 or 4.

22. A pharmaceutical composition comprising the gene expression cassette according to any one of claims 1 to 12, the gene delivery vector according to any one of claims 13 to 17, or the anti-Her2 antibody or antigen-binding fragment thereof according to any one of claims 18 to 21, and, optionally, a pharmaceutically acceptable carrier.

23. Use of the gene expression cassette according to any one of claims 1 to 12, the gene delivery vector according to any one of claims 13 to 17, or the anti-Her2 antibody or antigen-binding fragment thereof according to any one of claims 18 to 21 in the preparation of a medicament for treating breast cancer; Preferably, the breast cancer comprises central nervous system metastasis of breast cancer; Preferably, the breast cancer comprises Her2-positive breast cancer.

24. A method for treating breast cancer, comprising administering to a subject a therapeutically effective amount of the gene expression cassette of any one of claims 1 to 12, the gene delivery vector of any one of claims 13 to 17, or the anti-Her2 antibody or antigen-binding fragment thereof of any one of claims 18 to 21, or the pharmaceutical composition of claim 22; Preferably, the breast cancer comprises central nervous system metastasis of breast cancer; Preferably, the breast cancer comprises Her2-positive breast cancer.

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