Anti-lmp1 antibodies or antigen-binding fragments thereof and uses thereof

Abstract

The application belongs to the field of biological medicine, and particularly relates to an anti-LMP1 antibody and application thereof. The antibody has good affinity and / or biological activity to LMP1, and can be used for detecting LMP1, diagnosing or preventing and treating diseases or disorders related to LMP1.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to anti-LMP1 antibodies or their antigen-binding fragments and their uses. Background Technology

[0002] Epstein-Barr virus (EBV), also known as human herpesvirus 4 (HHV-4), was the first known carcinogenic virus in humans, causing lifelong infection in approximately 95% of the global population. It is estimated that EBV causes up to 357,900 cancer cases annually, accounting for 1.9% of the global cancer burden. EBV infection can lead to a variety of epithelial and lymphoid malignancies, including nasopharyngeal carcinoma, Burkitt lymphoma, Hodgkin's lymphoma, natural killer (NK) / T-cell lymphoma, leiomyosarcoma, and gastric cancer. EBV infection is also associated with autoimmune diseases, including infectious mononucleosis (IM), multiple sclerosis (MS), rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE), as evidenced by the fact that EBNA1 was initially identified as seropositive for EBV antigen in RA patients. Recent studies have found EBV in astrocytes and microglia in the brains of more than 90% (91 / 101) of MS patients, compared to only 24% (5 / 21) in non-MS neuropathological patients. This may represent a new mechanism by which EBV is associated with mild cognitive impairment (MCI) or Alzheimer's disease (AD), supporting the hypothesis that EBV infection promotes cognitive dysfunction.

[0003] The EBV genome consists of linear double-stranded DNA, approximately 172 kilobase pairs (kb) in length. The B95–8 strain of EBV, derived from infectious mononucleosis, was the first herpesvirus to have its complete genome cloned and sequenced. Recently, the complete sequences of two more EBV strains have been determined: the GD1 strain isolated from a Chinese nasopharyngeal carcinoma patient and the AG876 strain isolated from a Burkitt lymphoma patient in Ghana. The virus encodes nearly 100 viral genes, but not all of the proteins encoded by these genes have been identified or characterized. Among the factors encoded by EBV involved in the regulation of malignant processes, latent membrane protein 1 (LMP1) plays a central role and is considered the virus's major oncogenic protein. LMP1 is expressed in most EBV-associated lymphoproliferative disorders and malignancies and plays a crucial role in the early development of EBV-mediated pathogenesis and disease phenotypes.

[0004] LMP1 is expressed in Epstein-Barr virus (EBV) latency phases II and III and exhibits high expression in various EBV-related tumors, such as infectious mononucleosis, PTLD, X-linked proliferative disorders, Hodgkin's disease, peripheral T-cell lymphoma, and undifferentiated nasopharyngeal carcinoma. The oncogenic potential of LMP1 is supported by multiple pieces of evidence. In in vitro experiments, LMP1 can transform rat fibroblasts into carcinoid cells, which then form tumors after implantation into nude mice. Its transforming ability was further validated in in vivo experiments: in a mouse model lacking T lymphocytes, conditional activation of LMP1 at the pre-B / early B cell stage can induce rapid lethal lymphoproliferation. Viral particles lacking functional LMP1 or carrying disrupted LMP1-TRAF6 complex structures lose their ability to transform into B cells, further highlighting the central role of LMP1 in B cell transformation.

[0005] Furthermore, studies have shown that EBV infection is associated with various autoimmune diseases such as SLE, Sjögren's syndrome (SS), MS, and RA. LMP1, an EBV-encoded protein expressed on the cell membrane surface of EBV-infected cells, participates in the regulation of various autoimmune disease-related cytokines such as B lymphocyte activating factor (BAFF), the B lymphocyte surface marker CD23, and interferon (IFN), and can mimic CD4. + T cell surface CD40 ligands promote B lymphocyte activation and differentiation, and their numerous regulatory effects on cells contribute to the development of various autoimmune diseases. Tripterygium wilfordii, a commonly used drug for rheumatic diseases, has been found to reduce the survival rate of EBV-positive B lymphocytes. Its mechanism of action may involve inhibiting the ED-L1 promoter of LMP1, thereby downregulating the expression and transcription levels of LMP1 in EBV-positive B lymphocytes.

[0006] In conclusion, targeting LMP1 may bring new breakthroughs in the treatment of LMP1-related diseases or conditions. Summary of the Invention

[0007] In order to solve one of the above-mentioned technical problems in the prior art, the present invention provides an anti-LMP1 antibody or its antigen-binding fragment and its use.

[0008] The first aspect of the present invention provides an anti-LMP1 antibody or an antigen-binding fragment thereof, the anti-LMP1 antibody or the antigen-binding fragment thereof comprising: a1) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the amino acid sequence shown in SEQ ID NO: 3, CDR-H2 with the amino acid sequence shown in SEQ ID NO: 4, and CDR-H3 with the amino acid sequence shown in SEQ ID NO: 5; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the amino acid sequence shown in SEQ ID NO: 6, CDR-L2 with the amino acid sequence shown in SEQ ID NO: 7, and CDR-L3 with the amino acid sequence shown in SEQ ID NO: 8; CDR is defined according to the IMGT numbering system; a2) Heavy chain variable regions (VH) including the following three CDRs: CDR-H1 with the amino acid sequence shown in SEQ ID NO: 9, CDR-H2 with the amino acid sequence shown in SEQ ID NO: 10, and CDR-H3 with the amino acid sequence shown in SEQ ID NO: 11; and / or, light chain variable regions (VL) including the following three CDRs: CDR-L1 with the amino acid sequence shown in SEQ ID NO: 12, CDR-L2 with the amino acid sequence shown in SEQ ID NO: 13, and CDR-L3 with the amino acid sequence shown in SEQ ID NO: 14; CDR is defined according to the Kabat numbering system; a3) Heavy chain variable regions (VH) including the following three CDRs: CDR-H1 with the amino acid sequence shown in SEQ ID NO: 15, CDR-H2 with the amino acid sequence shown in SEQ ID NO: 16, and CDR-H3 with the amino acid sequence shown in SEQ ID NO: 17; and / or Light chain variable regions (VL) including the following three CDRs: CDR-L1 with the amino acid sequence shown in SEQ ID NO: 18, CDR-L2 with the amino acid sequence shown in SEQ ID NO: 19, and CDR-L3 with the amino acid sequence shown in SEQ ID NO: 20; CDR is defined according to the AbM numbering system; a4) Heavy chain variable regions (VH) including the following three CDRs: CDR-H1 with the amino acid sequence shown in SEQ ID NO: 21, CDR-H2 with the amino acid sequence shown in SEQ ID NO: 22, and CDR-H3 with the amino acid sequence shown in SEQ ID NO: 23; and / or, light chain variable regions (VL) including the following three CDRs: CDR-L1 with the amino acid sequence shown in SEQ ID NO: 24, CDR-L2 with the amino acid sequence shown in SEQ ID NO: 25, and CDR-L3 with the amino acid sequence shown in SEQ ID NO: 26; Wherein, CDR is defined according to the Chothia numbering system; or a5) Heavy chain variable regions (VH) including the following three CDRs: CDR-H1 with the amino acid sequence shown in SEQ ID NO: 27, CDR-H2 with the amino acid sequence shown in SEQ ID NO: 28, and CDR-H3 with the amino acid sequence shown in SEQ ID NO: 29; and / or, light chain variable regions (VL) including the following three CDRs: CDR-L1 with the amino acid sequence shown in SEQ ID NO: 30, CDR-L2 with the amino acid sequence shown in SEQ ID NO: 31, and CDR-L3 with the amino acid sequence shown in SEQ ID NO: 32; CDR is defined according to the Contact numbering system.

[0009] In some embodiments, the heavy chain variable region of the anti-LMP1 antibody or its antigen-binding fragment further includes the framework region of the heavy chain variable region.

[0010] In some embodiments, the framework region of the heavy chain variable region includes the framework region of the heavy chain variable region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, or geese; further includes the framework region of the heavy chain variable region or a mutant thereof derived from immunoglobulins of mice, rats, lemurs, macaques, chimpanzees, or humans; and even further includes the framework region of the heavy chain variable region or a mutant thereof derived from human immunoglobulins.

[0011] In some embodiments, the light chain variable region of the anti-LMP1 antibody or its antigen-binding fragment further includes a framework region of the light chain variable region.

[0012] In some embodiments, the framework region of the light chain variable region includes the framework region of the light chain variable region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, or geese; further includes the framework region of the light chain variable region or a mutant thereof derived from immunoglobulins of mice, rats, lemurs, macaques, chimpanzees, or humans; and even further includes the framework region of the light chain variable region or a mutant thereof derived from human immunoglobulins.

[0013] In some embodiments, the anti-LMP1 antibody or its antigen-binding fragment comprises: The heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and / or the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0014] In some embodiments, the anti-LMP1 antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region.

[0015] In some embodiments, the heavy chain constant region includes at least a portion of the heavy chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; further includes at least a portion of the heavy chain constant region or a mutant thereof derived from immunoglobulins of mice, rats, lemurs, macaques, chimpanzees, or humans; and even further includes the heavy chain constant region or a mutant thereof derived from human immunoglobulins.

[0016] In some embodiments, the light chain constant region includes at least a portion of the light chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; further includes at least a portion of the light chain constant region or a mutant thereof derived from immunoglobulins of mice, rats, lemurs, macaques, chimpanzees, or humans; and even further includes the light chain constant region or a mutant thereof derived from human immunoglobulins.

[0017] In some embodiments, the heavy chain constant region includes the heavy chain constant region or a mutant thereof derived from IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM immunoglobulin.

[0018] In some embodiments, the light chain constant region includes light chain constant regions derived from κ and λ immunoglobulins.

[0019] In some embodiments, the heavy chain constant region includes the amino acid sequence shown in SEQ ID NO: 33, or an amino acid sequence having at least 80% sequence identity with it, or having up to 20 conserved amino acid substitutions compared to it.

[0020] In some embodiments, the light chain constant region includes the amino acid sequence shown in SEQ ID NO: 34, or an amino acid sequence having at least 80% sequence identity with it, or having up to 20 conserved substitutions with it.

[0021] In some embodiments, the anti-LMP1 antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0022] In some embodiments, the antigen-binding fragment of the LMP1 antibody is a Fab fragment, a Fab' fragment, a Fab'-SH fragment, an F(ab')2 fragment, an Fv fragment, a single-chain Fv (scFv), a dsFv, or an Fd fragment.

[0023] In some embodiments, the anti-LMP1 antibody or its antigen-binding fragment comprises: The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 35, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and / or, the light chain comprises the amino acid sequence shown in SEQ ID NO: 36, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0024] A second aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an anti-LMP1 antibody or an antigen-binding fragment thereof of the first aspect.

[0025] Those skilled in the art will understand that nucleotides in nucleic acid molecules can be substituted based on codon degeneracy. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon-optimized.

[0026] A third aspect of the present invention provides a carrier comprising the nucleic acid molecule of the second aspect.

[0027] In some embodiments, the vector may be an expression vector. In some embodiments, the expression vector may include eukaryotic expression vectors and / or prokaryotic expression vectors. In some embodiments, the eukaryotic expression vector includes, but is not limited to, yeast expression vectors, mammalian expression vectors, and insect expression vectors. For example, the expression vector may include, but is not limited to, plasmids, retroviral vectors, lentiviral vectors, bacteriophage vectors, adenovirus vectors, adeno-associated vectors, or herpes simplex vectors.

[0028] In some embodiments, the carrier may be selected from nanoparticles, liposomes, exogenous bodies, microbubbles, or gene guns.

[0029] A fourth aspect of the present invention provides a cell comprising an anti-LMP1 antibody or an antigen-binding fragment thereof as described in the first aspect, a nucleic acid molecule as described in the second aspect, or a carrier as described in the third aspect.

[0030] In some embodiments, the cells do not involve reproductive material.

[0031] In some embodiments, the cell can be a host cell conventionally used in the art, as long as the expression vector stably expresses the carried nucleic acid molecule as the LMP1 antibody or its antigen-binding fragment described above. In some embodiments, the host cell can be a prokaryotic cell and / or a eukaryotic cell. The prokaryotic cell may include, for example, *Escherichia coli*, and the eukaryotic cell may include, for example, CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, HeLa cells, Vero cells, Expi293 cells, hybridoma cells, yeast cells, and insect cells.

[0032] The fifth aspect of the present invention provides a method for preparing an anti-LMP1 antibody or an antigen-binding fragment thereof of the first aspect, the method comprising: culturing cells of the fourth aspect, and collecting the anti-LMP1 antibody or an antigen-binding fragment thereof from the cultured cell culture.

[0033] A sixth aspect of the present invention provides a conjugate comprising the anti-LMP1 antibody of the first aspect or an antigen-binding fragment thereof; and a conjugation portion.

[0034] In some implementations, the coupling portion may include, but is not limited to, a detectable marker or a therapeutic agent.

[0035] In some embodiments, the detectable marker can be any substance detectable by means of fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, chemical, etc. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridine esters, magnetic beads, calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) microbeads, and biotin for binding avidin (e.g., streptavidin) modified with the above markers. In some embodiments, such markers are suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable marker is selected from radioactive isotopes, fluorescent substances, luminescent substances, colored substances, or enzymes. In some embodiments, the detectable marker described above can be linked to the LMP1 antibody or its antigen-binding fragment of the present invention via linkers of different lengths to reduce potential steric hindrance.

[0036] In some embodiments, the detectable marker may include, but is not limited to, enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (e.g., chemiluminescent substances), colored substances, biotin, etc.

[0037] In some embodiments, the therapeutic agent may include, but is not limited to, other medications for treating diseases or conditions associated with LMP1.

[0038] In some embodiments, the therapeutic agent may include, but is not limited to, cytotoxins.

[0039] In some embodiments, the cytotoxin is monomethyl auristatin E (MMAE).

[0040] In some embodiments, the coupling has a structure as shown in the following formula: ; Wherein, mAb is the anti-LMP1 antibody of the first aspect or its antigen-binding fragment, and n is an integer (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) or a decimal between 1 and 8.

[0041] In some embodiments, the coupling portion is selected from substances that can improve the biological properties of the antibody (e.g., increase serum half-life), such as chemical groups, such as polyethylene glycol (PEG), methyl, ethyl, or glycosyl groups.

[0042] The seventh aspect of the present invention provides a pharmaceutical composition comprising: an anti-LMP1 antibody or an antigen-binding fragment thereof (as described in the first aspect), a nucleic acid molecule (as described in the second aspect), a carrier (as described in the third aspect), a cell (as described in the fourth aspect), or a conjugate (as described in the sixth aspect); and a pharmaceutically acceptable carrier.

[0043] In some embodiments, the pharmaceutical composition may also include other pharmaceutically active agents.

[0044] In some embodiments, the other pharmaceutically active agent may be a biologically active drug, such as a drug capable of treating diseases or conditions associated with LMP1. In some embodiments, the other pharmaceutically active agent may include, but is not limited to, other drugs for treating diseases or conditions associated with LMP1. In some embodiments, the other pharmaceutically active agent is a drug with antitumor activity, or with activity in preventing or treating autoimmune diseases or cognitive impairment-related diseases.

[0045] In some embodiments, the antibody or its antigen-binding fragment is provided as a separate component or as a mixed component with the other pharmaceutically active agent.

[0046] In some embodiments, the pharmaceutical composition can be administered via, for example, parenteral, subcutaneous, sublingual, rectal, nasal, intravenous, intramuscular, oral, ocular, or topical routes.

[0047] In some embodiments, the pharmaceutical composition is in the form of, for example, an aqueous solution, suspension, powder, tablet, capsule, granule, powder, pill, disintegrant, syrup, spray, gel, emulsion, injection, elixir, lozenge, suppository, etc.

[0048] The eighth aspect of the present invention provides a diagnostic or therapeutic kit comprising: an anti-LMP1 antibody or an antigen-binding fragment thereof (as described in the first aspect), a nucleic acid molecule (as described in the second aspect), a carrier (as described in the third aspect), a cell (as described in the fourth aspect), a conjugate (as described in the sixth aspect), or a pharmaceutical composition (as described in the seventh aspect); and optionally, a specification and / or a delivery device.

[0049] In some embodiments, the kit can be used to diagnose diseases or conditions associated with LMP1.

[0050] In some implementations, the LMP1-related disease or condition includes EBV infection or a disease or condition caused by EBV infection.

[0051] In some implementations, the disease or condition caused by EBV infection is a tumor, an autoimmune disease, or a cognitive impairment-related disease.

[0052] The ninth aspect of the present invention provides the use of at least one of the following: the anti-LMP1 antibody of the first aspect or its antigen-binding fragment; the nucleic acid molecule of the second aspect; the carrier of the third aspect; the cell of the fourth aspect; the conjugate of the sixth aspect; or the pharmaceutical composition of the seventh aspect: (1)-(5) (1) Diagnose diseases or conditions related to LMP1; (2) Prevention or treatment of diseases or conditions related to LMP1 (3) Inhibits cell proliferation; (4) Detect the presence or level of LMP1 in the sample; (5) Prepare a product, which is used in at least one of (1)-(4).

[0053] In some implementations, the LMP1-related disease or condition includes EBV infection or a disease or condition caused by EBV infection.

[0054] In some implementations, the disease or condition caused by EBV infection is a tumor, an autoimmune disease, or a cognitive impairment-related disease.

[0055] In some embodiments, the LMP1 antibody or its antigen-binding fragment, nucleic acid molecule, carrier, cell, conjugate, or pharmaceutical composition is administered in combination with other pharmaceutically active agents, such as simultaneously, separately, or sequentially.

[0056] In some embodiments, the other pharmaceutically active agent is a biologically active drug, such as a drug that can treat diseases or conditions associated with LMP1.

[0057] In some embodiments, the other pharmaceutically active agent is a drug having antitumor activity or activity in preventing or treating autoimmune diseases or cognitive impairment-related diseases.

[0058] In some embodiments, inhibiting cell proliferation includes contacting the cells with the LMP1 antibody or its antigen-binding fragment of the present invention, the nucleic acid molecule, the carrier, the cells, the conjugate, or the pharmaceutical composition.

[0059] In some embodiments, the cells are cells expressing LMP1.

[0060] In some embodiments, the cells may be in vitro. In some embodiments, the cells may be in vivo in a subject who suffers from a disease or condition associated with LMP1 (e.g., a tumor or autoimmune disease).

[0061] The tenth aspect of the present invention provides a method for preventing or treating diseases or conditions related to LMP1, the method comprising administering to a subject in need an effective amount of an anti-LMP1 antibody of the first aspect or an antigen-binding fragment thereof, a nucleic acid molecule of the second aspect, a carrier of the third aspect, a cell of the fourth aspect, a conjugate of the sixth aspect, or a pharmaceutical composition of the seventh aspect.

[0062] In some embodiments, the method may further include administering a second therapy to the subject, the second therapy being selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

[0063] In some embodiments, the second therapy may be applied simultaneously, separately, or sequentially with the LMP1 antibody or its antigen-binding fragment of the present invention, the nucleic acid molecule, the carrier, the cell, the conjugate, or the pharmaceutical composition thereof.

[0064] In some implementations, the LMP1-related disease or condition includes EBV infection or a disease or condition caused by EBV infection.

[0065] In some implementations, the disease or condition caused by EBV infection is a tumor, an autoimmune disease, or a cognitive impairment-related disease.

[0066] The eleventh aspect of the present invention provides a method for detecting the presence or level of LMP1 in a sample, the method comprising the steps of: contacting the sample with an antibody or antigen-binding fragment thereof of the first aspect to allow the antibody or antigen-binding fragment thereof to form a complex with LMP1, and detecting the formation of the complex.

[0067] In some implementations, the method may include the steps of: detecting the presence or level of LMP1 in a sample from a subject and comparing it with a reference value (e.g., from a healthy subject); wherein an increase in the expression level compared to the reference value indicates that the subject has an LMP1-related disease or condition.

[0068] In some implementations, the LMP1-related disease or condition includes EBV infection or a disease or condition caused by EBV infection.

[0069] In some implementations, the disease or condition caused by EBV infection is a tumor, an autoimmune disease, or a cognitive impairment-related disease. Attached Figure Description

[0070] Figure 1 The chimeric antibody against EB virus LMP1 was shown to bind to the LMP1-overexpressing HEK293 cell line.

[0071] Figure 2 The binding activity of the chimeric antibody against EB virus LMP1 to the CNE2z cell line endogenously expressing LMP1 was demonstrated.

[0072] Figure 3 The antibody-drug conjugate (ADC) against EB virus LMP1 was demonstrated to kill HEK293-LMP1 cells.

[0073] Figure 4 The antibody-drug conjugate (ADC) against EB virus LMP1 was demonstrated to kill CNE2z cells. Detailed Implementation

[0074] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. Such structures and techniques have also been described in many publications.

[0075] definition Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0076] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0077] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0078] The term "LMP1" used in this article, also known as LMP-1 or CD340, is a membrane protein encoded by Epstein-Barrvirus (EBV) and belongs to the tumor necrosis factor receptor superfamily. It is 386 amino acids in length, and its structure includes a short N-terminal intracellular domain of 26 amino acids, a six-transmembrane domain of 162 amino acids, and a C-terminal intracellular domain of 200 amino acids. As a constitutive activating receptor, its transmembrane region possesses the inherent ability to form homomeric oligomers within the membrane, mimicking ligand-induced receptor cross-linking, thereby initiating signal transduction through its signal transduction domains. Cryo-electron microscopy resolved the structure of the LMP1 transmembrane domain and the LMP1-TRAF3 complex, revealing that LMP1 forms homodimers as its basic assembly units, unlike the trimer structures of CD40 or TNFR1. These dimers aggregate to form higher-order oligomeric filamentary structures with signal transduction activity. Within these filamentous structures, three LMP1 dimers recruit two TRAF3 trimers, efficiently occupying all TRAF3 trimer binding sites without steric hindrance. The C-terminus of LMP1 contains three activation regions (CTAR1-3) for recruiting tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) or TNFR-associated death domain (TRADD) proteins. These proteins, in turn, activate multiple signaling cascades, including the nuclear factor κB (NF-κB), c-Jun N-terminal kinase (JNK) / activator protein 1 (AP1), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and Janus kinase (JAK) pathways. LMP1 is expressed in EBV-infected B lymphocytes, and its mechanism of action involves the activation of multiple signaling pathways. LMP1 expression helps the virus evade host immune surveillance, promoting persistent viral infection and tumor cell survival.

[0079] As used herein, the term "antibody" refers to a globulin produced by plasma cells, which are produced by the proliferation and differentiation of B lymphocytes in response to antigen stimulation. Antibodies specifically bind to the corresponding antigens and mediate immune effects. They are mainly found in serum and body fluids and are important immune molecules mediating humoral immunity. Antibodies can encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific, trispecific, or tetraspecific antibodies), single-chain molecules, and antigen-binding fragments. The chemical basis of antibodies is immunoglobulin (Ig). It should be understood that the antibodies described herein can be used to prepare chimeric antigen receptors (CARs), for example, by integrating the antigen-binding domain of the antibody into a CAR construct, or to prepare immune cells (such as T cells or NK cells) expressing the CAR, as well as for therapeutic methods or pharmaceutical compositions for related diseases.

[0080] As used herein, the term "monoclonal antibody" refers to antibodies derived from a substantially homogeneous group of antibodies, meaning that, apart from possible trace amounts of variant antibodies (e.g., containing naturally occurring mutations or generated during the production of the monoclonal antibody formulation, typically present in small quantities), the individual antibodies within the group are identical and / or bind to the same epitopes. Unlike polyclonal antibody formulations, which typically comprise different antibodies targeting different antigenic determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen.

[0081] As used herein, the term "multispecific antibody" is used in its broadest sense to encompass antibodies exhibiting multi-epitope specificity. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit exhibits multi-epitope specificity; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full-length antibodies, antibody fragments, bispecific antibodies, and trispecific antibodies, antibody fragments covalently or non-covalently linked, etc.

[0082] The terms “full-length antibody” and “intact antibody” as used herein are used interchangeably to refer to antibodies that are structurally similar to natural antibodies. “Natural antibody” refers to a naturally occurring immunoglobulin molecule. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) (also called a variable heavy chain domain or heavy chain variable domain) and three constant domains (CH1, CH2, and CH3) (also called heavy chain constant regions, CH). From the N-terminus to the C-terminus, each light chain has a variable region (VL) (also called a variable light chain domain or light chain variable domain) and a light chain constant domain (CL) (also called light chain constant region). The heavy chain of an antibody can be one of five types: α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), and can be further subdivided into subtypes such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chain of an antibody, based on the amino acid sequence of its constant domain, can be one of two types: the κ (kappa) light chain and the λ (lambda) light chain.

[0083] Within the light and heavy chains, variable and constant regions are linked by a "J" region containing approximately 12 or more amino acid residues, and the heavy chain also contains a "D" region containing approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0084] The term "Fd fragment" as used herein refers to an antibody fragment consisting of VH and CH1 domains. The term "dAb fragment" as used herein refers to an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544 546 (1989)). The term "Fab fragment" as used herein refers to an antibody fragment consisting of VL, VH, CL, and CH1 domains. The term "F(ab')2 fragment" as used herein refers to an antibody fragment containing two Fab fragments linked by disulfide bridges on the hinge region. The term "Fab' fragment" as used herein refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light and heavy chain Fd fragment (composed of VH and CH1 domains). The term "Fab'-SH" as used herein refers to a Fab fragment containing free thiol groups.

[0085] As used in this article, the term "Fv fragment" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. The Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0086] As used herein, the term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker. In some cases, a disulfide bond may also exist between the VH and VL domains of the scFv.

[0087] As used herein, the term "variable region" or "variable domain" refers to the domain of the antibody heavy or light chain involved in the binding of the antigen-binding molecule to the antigen. The variable regions (VH and VL) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR1-4) and three hypervariable regions (HVR1-3), arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL domain is sufficient to confer antigen-binding specificity. The three HVRs within the VH and VL together constitute the antigen-binding site of Ig, which can bind complementary to the corresponding antigenic epitope; therefore, the HVRs are also called complementarity-determining regions (CDRs), denoted as CDR1, CDR2, and CDR3, respectively. The VH or VL chain of the antibody may further contain all or part of the constant regions of the heavy or light chain.

[0088] The CDR of the antibody or antigen-binding fragment of the present invention can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment of the present invention is preferably determined by the IMGT, Kabat, Contact, Chothia, or AbM numbering system.

[0089] As used in this article, the term "variable" refers to the fact that certain segments of the variable region are generally different in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed throughout the variable region, but is concentrated in three segments called hypervariable regions (HVRs) within the variable regions of the light and heavy chains. The relatively highly conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, mostly in a β-sheet configuration, linked by three HVRs that form loops and, in some cases, form part of a β-sheet structure. The HVRs in each chain are tightly held together by the FRs and, together with the HVRs of other chains, contribute to the formation of the antibody's antigen-binding site. Constant regions do not directly participate in antibody-antigen binding but have other effector functions, such as participating in antibody-dependent cytotoxicity.

[0090] Antibody "classes" refer to the types of constant structural domains or constant regions possessed by the antibody's heavy chain. Based on differences in heavy chain structure and antigenicity, they can be classified into five classes: μ chain, γ chain, α chain, δ chain, and ε chain. Immunoglobulins composed of different heavy and light chains are respectively called IgA, IgD, IgE, IgG, and IgM. Even within the same class of Ig, the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain differ, thus further subdividing the same class of Ig into different subclasses. For example, human IgG can be divided into IgG1–IgG4; IgA can be divided into IgA1 and IgA2. Based on differences in light chain structure and antigenicity, immunoglobulin (Ig) light chains are divided into κ (kappa) chains and λ (lambda) chains, thus classifying Ig into two types: κ type and λ type.

[0091] "Humanized antibodies" comprise amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, humanized antibodies comprise at least one, typically two, variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to the HVRs of the non-human antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Humanized antibodies may optionally comprise at least a portion of the antibody constant region derived from a human antibody. Antibodies in a "humanized form," such as non-human antibodies, refer to antibodies that have undergone humanization.

[0092] As used herein, the terms “polynucleotide,” “nucleic acid,” or “nucleotide sequence” refer to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA), virus-derived RNA, or plasmid DNA (pDNA). Polynucleotides may contain conventional phosphodiester bonds or unconventional bonds (such as amide bonds, as found in peptide nucleic acids (PNAs)). The term “nucleic acid molecule” refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[0093] An "antibody fragment" or "antigen-binding fragment" contains a portion of a complete antibody that retains the antibody's antigen-binding activity. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv; bisomatic antibodies, trisomatic antibodies, tetrasomatic antibodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments and single-domain antibodies (single-domain antibodies).

[0094] As used herein, the terms "antigen-binding domain" or "antigen-binding site" refer to the portion of an antigen-binding molecule that specifically binds to an antigenic determinant. More specifically, the term "antigen-binding domain" refers to a portion of an antibody containing a region that specifically binds to and is complementary to a portion or all of the antigen. In cases where the antigen molecule is large, the antigen-binding molecule may bind only a specific portion of the antigen, called an epitope. The antigen-binding domain may be provided by, for example, one or more variable domains (also called variable regions). Preferably, the antigen-binding domain comprises a variable region (VL) of the antibody light chain and a variable region (VH) of the antibody heavy chain. In one aspect, the antigen-binding domain is capable of binding its antigen and blocking or partially blocking the function of said antigen.

[0095] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule (e.g., a continuous amino acid sequence or a conformation composed of different regions of non-continuous amino acids) to which an antigen-binding moiety binds, thereby forming an antigen-binding moiety-antigen complex. Antigenic determinants can be present, for example, on the surface of tumor cells, on the surface of microbially infected cells, on the surface of other diseased cells, on the surface of immune cells, in serum, and / or in the extracellular matrix (ECM). Unless otherwise stated, proteins used as antigens in this invention can be any naturally occurring form of protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). Antigens can also be human proteins, or antigens can be "full-length," unprocessed proteins, and any form of protein produced by intracellular processing, or naturally occurring protein variants, such as splice variants or allelic variants.

[0096] The specific “binding strength” or “affinity” of an antibody or its antigen-binding fragment to an antigen refers to the strength of the non-covalent interaction between a single binding site and its binding ligand (e.g., antigen), and can be distinguished from unwanted or non-specific binding. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) techniques and conventional binding assays.

[0097] As used herein, the term "isolated" nucleic acid molecule or polynucleotide refers to a nucleic acid molecule, DNA, or RNA, that has been separated from its natural environment. In this invention, the recombinant polynucleotide encoding an antibody or its antigen-binding fragment contained in the vector is also isolated. Other examples of isolated polynucleotides include recombinant polynucleotides in heterologous host cells or polynucleotides purified in solution. Isolated polynucleotides include polynucleotide molecules typically found in cells containing the polynucleotide molecule, but which are located extrachromosomally or at chromosomal locations different from their natural chromosomal locations. Isolated RNA molecules include in vivo or in vitro RNA transcripts of this invention, in positive and negative strand forms, and in double strand forms. The isolated polynucleotides or nucleic acids of this invention further include synthetically generated molecules of this type. Additionally, the polynucleotide or nucleic acid may be or may include regulatory elements such as promoters, ribosome binding sites, or transcription terminators.

[0098] As used herein, the terms "vector" or "expression vector" and "expression construct" are used interchangeably to refer to a DNA molecule to which a specific gene, operatively linked, is introduced into a target cell and directed for expression. The vector includes a vector as a self-replicating nucleic acid structure and a vector incorporated into the genome of the host cell into which it has been introduced. The expression vector of the present invention comprises an expression cassette. The expression vector can be transcribed into a large amount of stable mRNA. Once the expression vector is in the target cell, a ribonucleic acid molecule or protein encoded by the gene is generated by cellular transcription and / or translation mechanisms. In one embodiment, the expression vector of the present invention comprises an expression cassette containing a polynucleotide sequence encoding an antibody of the present invention or an antigen-binding fragment thereof. The term "expression cassette" of the present invention refers to a recombinant or synthetically generated polynucleotide having a series of nucleic acid elements that allow a specific nucleic acid to be transcribed in a target cell. The recombinant expression cassette can be introduced into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, in addition to other sequences, the recombinant expression cassette portion of the expression vector includes the nucleic acid sequence to be transcribed and a promoter. In some embodiments, the expression cassette of the present invention comprises a polynucleotide sequence encoding an antibody of the present invention or an antigen-binding fragment thereof.

[0099] As used herein, the terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” including primary transformed cells and their derived progeny. The nucleic acids of the progeny may not be completely identical to those of the parent cells and may contain mutations. Host cells are any type of cell that can be used to generate the bispecific antigen-binding molecule of this invention. Host cells include cultured cells, such as cultured mammalian cells, such as CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, as well as cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0100] As used herein, the term "antibody-drug conjugate" or "ADC" refers to a binding protein (such as an antibody or its antigen-binding fragment) chemically linked to one or more chemical drugs. In a preferred embodiment, an ADC comprises a binding protein, a drug, and a connector linking the binding protein to the drug.

[0101] An "effective amount" of a drug is the amount necessary to produce physiological changes in the cells or tissues to which it is administered. An "effective amount" includes the amount sufficient to improve or prevent the symptoms or signs of a medically diagnosed disease. An effective amount also means the amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of any side effects. An effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0102] The "therapeutic effective amount" of a drug (such as a pharmaceutical composition) refers to the amount necessary to effectively achieve the desired therapeutic or preventive effect in terms of dosage, dosing intervals, and time. For example, a therapeutically effective amount of a drug eliminates, mitigates / reduces, delays, minimizes, or prevents the adverse effects of a disease.

[0103] As used herein, the term “individual” or “subject” refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). Specifically, an individual or subject is a human being.

[0104] As used herein, the term "pharmaceutical composition" refers to a mixture containing one or more antibodies or antigen-binding fragments of the present invention, along with other chemical components, such as physiological / pharmaceutical-grade carriers or excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0105] As used herein, the term "pharmaceuticalally acceptable excipient" refers to a component in a pharmaceutical composition that, apart from the active ingredient, is non-toxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, and / or preservatives.

[0106] As used herein, the term "treatment" refers to the administration of an oral or topical therapeutic agent, such as a composition comprising any antibody of the present invention or an antigen-binding fragment thereof, or a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof, to a patient having one or more LMP1-related diseases or symptoms, the therapeutic agent having a therapeutic effect on these diseases or symptoms. Typically, the therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more diseases or symptoms, to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable extent.

[0107] As used herein, the term "prevention" refers to delaying, suppressing, or preventing the onset of LMP1-related disease in mammals where the initiation of cancer or tumor development has not been confirmed, but a susceptibility to cancer has been identified, for example, through genetic screening or other methods. The term also includes treating mammals with precancerous lesions to halt the progression of the precancerous lesions to malignancy or to induce their regression.

[0108] In this article, "diseases or conditions associated with LMP1" refers to diseases or conditions caused by EBV infection, including tumors, autoimmune diseases, or cognitive impairment-related diseases. Specifically, tumors include malignant tumors of the lymphatic system (including B-cell lymphomas such as Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and diffuse large B-cell lymphoma (DLBCL); and T / NK-cell lymphomas such as extranodal NK / T-cell lymphoma (ENKTL) and aggressive NK-cell leukemia (ANKL); also including post-transplant lymphoproliferative disorders (PTLD), epithelial carcinomas (including nasopharyngeal carcinoma (NPC) and EBV-associated gastric cancer (EBVaGC)) and other related malignant tumors (including leiomyosarcoma (LMS) in immunocompromised children; and lymphomas occurring in the nasopharynx, stomach, salivary glands, etc.). Leukocyte carcinoma (LEC); autoimmune diseases including infectious mononucleosis (IM), systemic lupus erythematosus (SLE), Sjögren's syndrome (SS), rheumatoid arthritis (RA), systemic sclerosis (SSc), multiple sclerosis (MS), inflammatory bowel disease (IBD), autoimmune hepatitis, primary biliary cirrhosis (PBC), type 1 diabetes (T1D), Hashimoto's thyroiditis, Graves' disease, celiac disease, juvenile idiopathic arthritis (JIA), antiphospholipid syndrome (APS), myasthenia gravis, or autoimmune hemolytic anemia; cognitive impairment-related diseases including mild cognitive impairment (MCI) or Alzheimer's disease (AD).

[0109] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are counted, but those from the reference sequence are not.

[0110] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the NCBI BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0111] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0112] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0113] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are substantially based on those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, and F.M. Ausubel et al., Molecular Biology: A Concise Laboratory Manual, 3rd Edition, John Wiley & Sons, Inc. Those skilled in the art will appreciate that the examples are described by way of illustration and are not intended to limit the scope of the invention.

[0114] Example 1: Animal immunization and preparation of mouse hybridoma antibodies against EB virus LMP1 Mice were immunized with EBV LMP1 mRNA LNP and EBV LMP1-overexpressing HEK293 cells. SP2 / 0 cells were fused, and positive clones were screened by flow cytometry to obtain hybridomas. The specific methods are as follows: Animal immunization: EBV LMP1 mRNA LNP (nearshore protein) and HEK293 cells expressing EBV LMP1 (HEK293-LMP1, obtained by packaging lentivirus into the PLVX-ef1a-IRES-puro vector containing the encoding sequence of EBV LMP1 protein (UniProtKB / Swiss-Prot:P03230), transfected into HEK293 cells) were used as immunogens to immunize Balb / c and SJL female mice (Shanghai Silex Laboratory Animal Co., Ltd., SJL / J) intraperitoneally (2-5 × 10⁻⁶). 6 Cell / mouse). LNP immunization was administered at 25 μg per mouse, repeated three times at 2-week intervals; cell immunization was administered three times at 2-week intervals, with HEK293-LMP1 cells (2-5 × 10⁻⁵) injected intraperitoneally 10 days after the last immunization. 6 The mice were given a shock immunization (cells / mouse), and the spleens of the mice were harvested 3 days later for cell fusion.

[0115] Cell fusion: Mouse spleen cells and SP2 / 0 cells (ATCC No. CRL-1581) were electrofused at a ratio of 2:1 (BTX electrofusion instrument: ECM2001+). The cells were cultured in 96-well plates with HAT (Sigma, catalog number: H0262) medium. Antibody screening of hybridoma cell supernatant was performed after 10 days.

[0116] Screening for LMP1-specific positive clones: HEK293 cells expressing LMP1 (HEK293-LMP1) were screened at a ratio of 5 × 10⁻⁶ cells / year. 7Cells were seeded at a density of 100 μL / well in a 96-well U-shaped plate. 100 μL of hybridoma cell culture supernatant was added and incubated at 4°C for 1 hour. Cells were washed twice by centrifugation at 300 g with FACS buffer (PBS containing 1% FBS, Adamas, #C8020). Cells were then incubated at 4°C for 1 hour with Alexa488-labeled anti-mouse IgG antibody (INVITROGEN, #A21202). Cells were washed three times by centrifugation at 300 g with FACS buffer. Mean fluorescence intensity (MFI) was read using a flow cytometry system (BECKMAN COULTER cytoFLEX). Positive clones were screened by comparison with the negative control (HEK293 cells that do not express LMP1). The mean fluorescence intensity (MFI) of clone 4B5 binding to LMP1 (HEK293-LMP1) in Table 1 was higher than that of the blank control cells (HEK293), indicating that clone 4B5 specifically binds to EBV LMP1.

[0117] Table 1: Binding of supernatant from anti-EBV LMP1 positive clonal hybridomas (mean fluorescence intensity, MFI)

[0118] Example 2: Preparation and identification of murine monoclonal antibody against EB virus LMP1 The positive clone 4B5 in Table 1 was sequenced, and the light and heavy chain variable regions obtained from the sequencing (the amino acid sequences of the variable regions are shown in Table 2) were constructed onto the human constant regions (the amino acid sequences of the light and heavy chain constant regions are shown in Table 3) to construct the chimeric antibody M004 (the amino acid sequences of the chimeric antibody light and heavy chains are shown in Table 4). The sequence was verified by sequencing and used for in vitro functional identification.

[0119] Table 2: CDR and variable region sequences of chimeric antibodies against EBV LMP1

[0120]

[0121] Table 3: Constant Region Sequence

[0122] Table 4: Chimeric Antibodies Against EBV LMP1

[0123] Preparation of chimeric antibodies against EB virus LMP1 The coding sequences of the light and heavy chains of the chimeric antibody M004 were inserted into the Abvector vector to obtain light and heavy chain expression plasmids. Expression was performed in CHO cells, and purification was carried out using a Protein A column. Antibody expression was outsourced to Beijing Qingke Biotechnology Co., Ltd., and the specific methods are as follows: CHO cells expressing chimeric antibodies: The CHO cell density was diluted to 1.5 × 10⁻⁶ cells one day before transfection. 6 Cells / mL were cultured at 37°C and 8% CO2 in a shaker at 120 rpm. On the second day, viable cell density and survival rate were measured; the cell transfection density should be 3 × 10⁻⁶ cells / mL. 6 Cells / mL, cell viability >95%. Preparation of the PEI / plasmid complex: Mix PEI (1 mg / mL, Polysciences, catalog number: 24765-1) by inverting. Dilute the plasmid with serum-free CHO basal medium to a total plasmid volume of 1 μg / mL. The volume of the diluted medium should be 1 / 20 of the transfection volume. Gently mix, with a light to heavy chain ratio of 1:1.5. Dilute the PEI reagent with medium to a volume 1 / 20 of the transfection volume. Gently invert and incubate for 5 minutes at room temperature. Add the diluted PEI reagent to the diluted plasmid and gently invert. Incubate the PEI / plasmid complex at room temperature for 15 minutes, then slowly add the solution dropwise to a transfer flask, gently rotating the flask during addition. After transfection, incubate the flask at 37°C and 8% CO2 in a shaker at 120 rpm. Incubate for 7-10 days and harvest the supernatant.

[0124] Protein A column purification of antibodies: Prepare a gravity chromatography column. Open the column cap and place the gasket at the bottom of the column, pressing it firmly. Prepare the packing material, Protein A (Cytiva, catalog number: 17549801). Calculate the required packing suspension volume precisely based on the target packing volume and the packing suspension ratio: Required packing suspension volume = Target packing volume / Packing suspension ratio. Vortex the packing material thoroughly to ensure complete suspension. Add the packing suspension to the bottom of the gravity chromatography column. Add at least 10 CV of equilibration buffer (PBS) to the column. After equilibration, check the outlet pH. If the target pH is not reached, continue adding equilibration buffer until the target pH is reached. Slowly add a certain volume of sample to the column. Add at least 10 CV of eluent to the column. Slowly add 5 CV of elution buffer (10-50 mM NaAc, pH 3.0-pH 3.5) to the column and incubate for 3-5 minutes. Collect the eluent. Repeat the elution step as needed.

[0125] Neutralization: Adjust the pH of the elution product to the target pH using neutralization buffer (1M Tris). Determine protein concentration using Nanodrop. Replace the buffer used to store the antibody with PBS via ultrafiltration.

[0126] Example 3: Functional identification of chimeric antibodies against EB virus LMP1 1. Binding activity of anti-EBV LMP1 chimeric antibody to HEK293 cell line overexpressing EBV LMP1 The concentration of HEK293 cells (Shanghai Hongcheng) overexpressing EBV LMP1 was adjusted to 1×10⁻⁶ using FACS buffer (PBS solution containing 1% FBS). 6 Cells / mL were collected and placed at 100 μL / well in a 96-well U-bottom plate. After centrifugation, the supernatant was discarded. The chimeric antibody M004 against EBV LMP1 and the negative control antibody (Baiying Biotechnology) were diluted to the initial working concentration using FACS buffer (PBS solution containing 1% FBS), followed by serial dilutions. Cells were resuspended in the serially diluted antibody at 100 μL / well, mixed thoroughly by pipetting, and incubated at 4°C for 1 hour. After incubation, cells were centrifuged and washed three times with FACS buffer. The AlexaFlour-647-labeled anti-human secondary antibody (Invitrogen, A-21445) was diluted 1:1000 with FACS buffer (PBS solution containing 1% FBS). 100 μL of the secondary antibody dilution was added to each well, the cell clumps were resuspended, mixed thoroughly by pipetting, and incubated at 4°C for approximately 45 minutes. After incubation, cells were centrifuged, washed three times with FACS buffer, and then resuspended in FACS buffer at 100 μL / well. Mean fluorescence intensity (MFI) was read using a flow cytometer (BDCelesta). The experimental data were analyzed using Graphpad Prism 8.0 software, with the logarithm of antibody concentration on the x-axis and the corresponding MFI value on the y-axis. A four-parameter regression model was used to fit the antibody dose-response curve and calculate the EC50. 50 The result is as follows Figure 1 As shown in Table 5.

[0127] Table 5: Binding activity of anti-EBV LMP1 chimeric antibody against HEK293 LMP1 cell line

[0128] 2. Binding activity of cell lines endogenously expressing EBV LMP1. The concentration of CNE2z cells (Nanjing Kebai, catalog number: CBP60653) endogenously expressing EB virus LMP1 was adjusted to 1×10 using FACS buffer (PBS solution containing 1% FBS). 6 Cells / mL were collected and placed at 100 μL / well in a 96-well U-bottom plate. After centrifugation, the supernatant was discarded. The chimeric antibody M004 against EBV LMP1 and the negative control antibody (Baiying Biotechnology) were diluted to the initial working concentration using FACS buffer (PBS solution containing 1% FBS), followed by serial dilutions with FACS buffer. Cells were resuspended in the serially diluted antibody at 100 μL / well, mixed by pipetting, and incubated at 4°C for 1 hour. After incubation, cells were centrifuged and washed three times with FACS buffer. The AlexaFlour-647-labeled anti-human secondary antibody (Invitrogen, catalog number: A-21445) was diluted 1:1000 with FACS buffer (PBS solution containing 1% FBS). 100 μL of the secondary antibody dilution was added to each well, the cell clumps were resuspended, mixed by pipetting, and incubated at 4°C for approximately 45 minutes. Cells were centrifuged after incubation. After incubation, cells were centrifuged, washed three times with FACS buffer, and then resuspended in FACS buffer at 100 μL / well. Mean fluorescence intensity (MFI) was read using a flow cytometer (BDCelesta). The experimental data were analyzed using GraphpadPrism 8.0 software, with the logarithm of antibody concentration on the x-axis and the corresponding MFI value on the y-axis. A four-parameter regression model was used to fit the antibody dose-response curve and calculate the EC50. 50 The result is as follows Figure 2 As shown in Table 6.

[0129] Table 6: Cell line binding activity of chimeric antibodies against EBV LMP1

[0130] The results showed that the anti-EBV LMP1 chimeric antibody M004 had strong binding activity to the CNE2z cell line, which endogenously expresses EBV LMP1.

[0131] Example 4: Identification of the cytotoxic activity of anti-EBV LMP1 antibody-drug conjugate (ADC) 1. Preparation of ADC by conjugating anti-EBV LMP1 chimeric antibody with MMAE Add antibody M004 and PBS buffer to bring the antibody concentration to approximately 5 mg / mL. Add 2-3 equivalents of TCEP (Thermo, 20490) for reduction, and incubate at 37°C with gentle shaking for 1-2 hours. Then adjust the mixer temperature to approximately 25°C to cool the reaction solution. Add 6-8 equivalents of DMSO-dissolved VcMMAE (MCE, HY-15575), and incubate at 25°C for 1-2 hours. Finally, transfer the reaction solution to a 30 K ultrafiltration tube and replace the medium with 10 mM, pH 6.0 histidine hydrochloride solution for 10 DV. 3. Concentrate to a suitable volume, centrifuge to obtain the purified ADC product, and use a BCA protein concentration assay kit (Beyotime, #P0009) to detect the concentration. Use hydrophobic interaction chromatography (HIC) to detect the DAR value, as shown in Table 7, to obtain the antibody-MMAE conjugate.

[0132] The structure of the antibody-MMAE conjugate is as follows: ; Where mAb is antibody M004, and n is approximately 4.8.

[0133] Table 7: Concentration and DAR value of anti-EBV LMP1 antibody-drug conjugate.

[0134] Using the same method, a negative control ADC (i.e., hIgG1-MMAE) was prepared based on a negative control antibody (Baiying Bio).

[0135] 2. The killing effect of antibody-drug conjugates against EBV LMP1 on HEK293-LMP1 and CNE2z The inhibitory effect of the anti-LMP1 antibody-drug conjugate (ADC) on cell proliferation was evaluated by incubating LMP1-positive human nasopharyngeal carcinoma cells CNE2z and HEK293 cells overexpressing EBV LMP1 (HEK293-LMP1, obtained by packaging lentivirus with the PLVX-ef1a-IRES-puro vector containing the EBV LMP1 protein coding sequence and transfecting HEK293 cells) for 7 days, respectively, using a chemiluminescent cell viability assay (CTG method).

[0136] Cells in the logarithmic growth phase were collected and placed in 96-well cell culture plates at a density of 500 cells / well, with 100 μL of cell culture solution per well. The cell culture plates were incubated at 37°C and 5% CO2 for 6 hours. The ADC drug M004-MMAE and the negative control ADC (hIgG1-MMAE) were serially diluted 5-fold with complete DMEM (corning, #10-013-CVRC) to obtain 9 concentration gradients (starting with the highest concentration of 600 nM). 20 μL of each drug was added to the cell culture plate, with the complete DMEM serving as a blank control. The plates were incubated at 37°C and 5% CO2 for 7 days. After incubation, the cell culture plates were removed and allowed to equilibrate to room temperature. 60 μL of CTG detection reagent (VKEY-BIO, Cat#: A20100-50 mL) was added to each well, vortexed, and incubated in the dark for 10 minutes before the signal value was read using a microplate reader. Using GraphPad Prism software, a nonlinear regression model was used to plot an S-shaped dose-response curve and calculate the IC50. 50 Cell viability is calculated as follows: RLU of test drug / RLU of solvent (blank control) × 100%.

[0137] according to Figures 3-4 The results in Table 8 indicate that the antibody-drug conjugate M004-MMAE has significant inhibitory activity on the proliferation of LMP1-positive human nasopharyngeal carcinoma cells CNE2z and LMP1-overexpressing HEK293 cells.

[0138] Table 8: In vitro killing activity of antibody-drug conjugates against EBV LMP1 against HEK293-LMP1 and CNE2z (human nasopharyngeal carcinoma cells).

[0139] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An anti-LMP1 antibody or an antigen-binding fragment thereof, said anti-LMP1 antibody or antigen-binding fragment comprising: a1) includes the heavy chain variable region (VH) of the following three CDRs: CDR-H1 with the amino acid sequence shown in SEQ ID NO: 3, CDR-H2 with the amino acid sequence shown in SEQ ID NO: 4, and CDR-H3 with the amino acid sequence shown in SEQ ID NO: 5; and a light chain variable region (VL) of the following three CDRs: CDR-L1 with the amino acid sequence shown in SEQ ID NO: 6, CDR-L2 with the amino acid sequence shown in SEQ ID NO: 7, and CDR-L3 with the amino acid sequence shown in SEQ ID NO: 8; CDR is defined according to the IMGT numbering system; a2) includes heavy chain variable regions (VH) comprising the following three CDRs: CDR-H1 with amino acid sequence as shown in SEQ ID NO: 9, CDR-H2 with amino acid sequence as shown in SEQ ID NO: 10, and CDR-H3 with amino acid sequence as shown in SEQ ID NO: 11; and light chain variable regions (VL) comprising the following three CDRs: CDR-L1 with amino acid sequence as shown in SEQ ID NO: 12, CDR-L2 with amino acid sequence as shown in SEQ ID NO: 13, and CDR-L3 with amino acid sequence as shown in SEQ ID NO: 14; CDR is defined according to the Kabat numbering system; a3) includes heavy chain variable regions (VH) comprising the following three CDRs: CDR-H1 with amino acid sequence as shown in SEQ ID NO: 15, CDR-H2 with amino acid sequence as shown in SEQ ID NO: 16, and CDR-H3 with amino acid sequence as shown in SEQ ID NO: 17; and light chain variable regions (VL) comprising the following three CDRs: CDR-L1 with amino acid sequence as shown in SEQ ID NO: 18, CDR-L2 with amino acid sequence as shown in SEQ ID NO: 19, and CDR-L3 with amino acid sequence as shown in SEQ ID NO: 20; CDR is defined according to the AbM numbering system; a4) includes heavy chain variable regions (VH) comprising the following three CDRs: CDR-H1 with amino acid sequence as shown in SEQ ID NO: 21, CDR-H2 with amino acid sequence as shown in SEQ ID NO: 22, and CDR-H3 with amino acid sequence as shown in SEQ ID NO: 23; and light chain variable regions (VL) comprising the following three CDRs: CDR-L1 with amino acid sequence as shown in SEQ ID NO: 24, CDR-L2 with amino acid sequence as shown in SEQ ID NO: 25, and CDR-L3 with amino acid sequence as shown in SEQ ID NO: 26; Wherein, CDR is defined according to the Chothia numbering system; or a5) includes the heavy chain variable region (VH) of the following three CDRs: CDR-H1 with the amino acid sequence shown in SEQ ID NO: 27, CDR-H2 with the amino acid sequence shown in SEQ ID NO: 28, and CDR-H3 with the amino acid sequence shown in SEQ ID NO: 29; and, includes the light chain variable region (VL) of the following three CDRs: CDR-L1 with the amino acid sequence shown in SEQ ID NO: 30, CDR-L2 with the amino acid sequence shown in SEQ ID NO: 31, and CDR-L3 with the amino acid sequence shown in SEQ ID NO: 32; CDR is defined according to the Contact numbering system.

2. The anti-LMP1 antibody or its antigen-binding fragment according to claim 1, characterized in that, The anti-LMP1 antibody or its antigen-binding fragment includes: The heavy chain variable region (VH) includes the amino acid sequence shown in SEQ ID NO: 1; and / or, the light chain variable region (VL) includes the amino acid sequence shown in SEQ ID NO:

2.

3. The anti-LMP1 antibody or its antigen-binding fragment according to claim 1, characterized in that, The anti-LMP1 antibody or its antigen-binding fragment includes: The heavy chain variable region (VH) comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; and / or, the light chain variable region (VL) comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

2.

4. The anti-LMP1 antibody or its antigen-binding fragment according to claim 1, characterized in that, The anti-LMP1 antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region.

5. The anti-LMP1 antibody or its antigen-binding fragment according to claim 1, characterized in that, The anti-LMP1 antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, or a humanized antibody.

6. The anti-LMP1 antibody or its antigen-binding fragment according to claim 1, characterized in that, The antigen-binding fragment of the LMP1 antibody is a Fab fragment, a Fab' fragment, a Fab'-SH fragment, an F(ab')2 fragment, an Fv fragment, a single-chain Fv (scFv), or a dsFv.

7. An isolated nucleic acid molecule, said nucleic acid molecule encoding the anti-LMP1 antibody or its antigen-binding fragment as described in any one of claims 1 to 6.

8. A vector comprising the nucleic acid molecule of claim 7.

9. A cell comprising the anti-LMP1 antibody or its antigen-binding fragment as described in any one of claims 1 to 6, the nucleic acid molecule as described in claim 7, or the vector as described in claim 8.

10. A method for preparing the anti-LMP1 antibody or its antigen-binding fragment according to any one of claims 1 to 6, the method comprising: The anti-LMP1 antibody or its antigen-binding fragment is obtained by culturing the cells of claim 9 and collecting the cultured cell culture.

11. A conjugate comprising the anti-LMP1 antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 6; and a conjugation portion; said conjugation portion being a detectable marker or therapeutic agent.

12. A pharmaceutical composition comprising: The anti-LMP1 antibody or its antigen-binding fragment as described in any one of claims 1 to 6, the nucleic acid molecule as described in claim 7, the vector as described in claim 8, the cell as described in claim 9, or the conjugate as described in claim 11; and a pharmaceutically acceptable vector.

13. Diagnostic or therapeutic reagent kits, comprising: The anti-LMP1 antibody or its antigen-binding fragment according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the vector according to claim 8, the cell according to claim 9, the conjugate according to claim 11, or the pharmaceutical composition according to claim 12; and optionally, the instruction manual and / or the delivery device.

14. Use of the anti-LMP1 antibody or its antigen-binding fragment according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the carrier according to claim 8, the cell according to claim 9, the conjugate according to claim 11, or the pharmaceutical composition according to claim 12 in at least one of (1)-(2): (1) To prepare products for the diagnosis of diseases or conditions related to LMP1; (2) Prepare a product for detecting the presence or level of LMP1 in a sample; The disease or condition associated with LMP1 is either nasopharyngeal carcinoma or Hodgkin's lymphoma.

15. Use of the conjugate in the preparation of products for the prevention or treatment of diseases or conditions associated with LMP1; The conjugate comprises the anti-LMP1 antibody or its antigen-binding fragment as described in any one of claims 1 to 6; and a conjugation portion; wherein the conjugation portion is a therapeutic agent; and the therapeutic agent is a cytotoxic agent. The disease or condition associated with LMP1 is either nasopharyngeal carcinoma or Hodgkin's lymphoma.

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