Anti-vzv viral antibodies, pharmaceutical compositions thereof, and uses

Abstract

This invention belongs to the field of biomedicine and relates to an anti-VZV virus antibody, its pharmaceutical composition, and its uses. Specifically, the anti-VZV virus antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3, wherein: the amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:7; and the amino acid sequences of LCDR1 are shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. The anti-VZV virus antibody of this invention has good VZV virus neutralizing activity.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to an anti-VZV virus antibody, its pharmaceutical composition, and its uses. Specifically, this invention relates to a monoclonal antibody with VZV virus neutralizing activity. Background Technology

[0002] Varicella-zoster virus (VZV, also known as human herpesvirus 3) is a neurotropic human herpesvirus, a ubiquitous herpesvirus with a double-stranded DNA genome, that can cause both chickenpox and shingles. Primary infection causes chickenpox, characterized by a generalized itchy rash that rapidly develops from spots to papules and vesicles, eventually crusting over. The lesions are scattered and isolated, reflecting viral spread to the skin. VZV then remains latent in cranial ganglia, dorsal root ganglia, intestinal ganglia, and autonomic ganglia. (1) .

[0003] Viral reactivation leads to viral replication, resulting in shingles (herpes zoster), inflammation, and cell death in tissues innervated by the relevant neurons—a process that can cause persistent radicular pain (postherpetic neuralgia). The pathogenesis of postherpetic neuralgia is unclear, making treatment difficult. In addition, other complications of shingles may occur, including myelitis, cranial nerve palsy, meningitis, stroke (vascular complications), retinitis, and gastrointestinal infections such as ulcers, pancreatitis, and hepatitis. The global incidence of shingles ranges from 3 to 5 per 1000 person-years, with an incidence of 5.23 to 10.9 per 1000 person-years in individuals ≥50 years of age. (2) .

[0004] More than 90% of adults are latently infected with VZV, and about one-third of them will develop shingles. In China, approximately 1.5 million people develop shingles each year. Due to the increasing number of elderly people, organ transplant recipients with suppressed immune systems, patients undergoing chemotherapy for cancer or autoimmune diseases, HIV-infected individuals, and patients with chronic diseases, a growing population is showing impaired immunity to VZV and developing shingles. (3) Postherpetic neuralgia is a severe form of pain that can last for years or even longer, and can even lead to suicide, greatly impacting patients' quality of life.

[0005] Currently, there is no specific treatment for shingles. The main medications used are antiviral drugs, such as acyclovir and valacyclovir. While antiviral treatment can alleviate acute pain associated with shingles, it has not been proven to reliably reduce the risk of postherpetic neuralgia (PHN), and is not recommended for treating confirmed PHN. (4)Furthermore, although vaccines against shingles are available, they are ineffective for patients who have already developed the disease. Therefore, finding a rapid and effective treatment is extremely urgent.

[0006] Varicella-zoster virus (VZV) glycoprotein E (gE) is essential for VZV infection. gE is highly conserved in herpes simplex virus and is a key glycoprotein for viral replication. gE interacts with insulin-degrading enzyme (IDE), promoting VZV infection and intercellular viral transmission. (5) Currently available shingles vaccines all target the gE protein.

[0007] For decades, hybridoma technology has been used to isolate mouse monoclonal antibodies. However, the therapeutic applications of these antibodies are limited by the induction and autoreactivity of anti-mouse antibodies. The technique of isolating monoclonal antibodies using phage display libraries has yielded many useful antibodies, but its applicability is limited by the differences in binding properties between antibodies expressed in bacterial and eukaryotic cells. Summary of the Invention

[0008] Through in-depth research and creative work, the inventors isolated memory B cells from the peripheral blood of patients, cultured B cells, collected supernatants for screening using gE protein as a target, and performed B cell lysis for amplification and cloning of immunoglobulin heavy and light chains, thus obtaining an anti-VZV virus antibody. The inventors surprisingly discovered that this antibody can effectively neutralize VZV virus and is a fully human monoclonal antibody with VZV virus neutralizing activity, named 21O16. This provides the following invention:

[0009] One aspect of the present invention relates to an anti-VZV virus antibody or an antigen-binding fragment thereof, said antibody comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising HCDR1 to HCDR3, and said light chain variable region comprising LCDR1 to LCDR3, wherein:

[0010] The amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:7.

[0011] The amino acid sequences of LCDR1 are shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.

[0012] In some embodiments of the present invention, the anti-VZV virus antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:4.

[0013] In some embodiments of the present invention, the anti-VZV virus antibody or its antigen-binding fragment, wherein the heavy chain constant region of the antibody is Ig gamma-1 chain C region (e.g., NCBI ACCESSION: P01857) or Ig ggamma-4 chain C region (e.g., NCBI ACCESSION: P01861.1); and the light chain constant region is Ig kappa chain C region (e.g., NCBI ACCESSION: P01834).

[0014] In some embodiments of the present invention, the anti-VZV virus antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, humanized antibody, chimeric antibody or biantibody.

[0015] In some embodiments of the present invention, the anti-VZV virus antibody or its antigen-binding fragment, wherein the anti-VZV virus antibody binds to the VZV virus gE protein via EC. 50 The values ​​are less than or equal to 10 μg / ml, less than or equal to 5 μg / ml, or less than or equal to 3 μg / ml.

[0016] In some embodiments of the present invention, the anti-VZV virus antibody is a monoclonal antibody, preferably a humanized monoclonal antibody.

[0017] In some embodiments of the present invention, the anti-VZV virus antibody or its antigen-binding fragment is wherein the anti-VZV virus antibody is a fully human antibody, such as a human monoclonal antibody.

[0018] In some embodiments of the present invention, the anti-VZV virus antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain of the anti-VZV virus antibody is as shown in SEQ ID NO:40, and the amino acid sequence of the light chain of the anti-VZV virus antibody is as shown in SEQ ID NO:42.

[0019] Another aspect of the present invention relates to an isolated nucleic acid molecule encoding an anti-VZV virus antibody or an antigen-binding fragment thereof as described in any one of the present invention. Preferably, the sequence of the isolated nucleic acid molecule is shown in SEQ ID NO:41 and SEQ ID NO:43.

[0020] Another aspect of the invention relates to a recombinant vector comprising the isolated nucleic acid molecules of the invention.

[0021] Another aspect of the invention relates to a host cell comprising the isolated nucleic acid molecule of the invention or the recombinant vector of the invention.

[0022] Another aspect of the present invention relates to a conjugate comprising an antibody and a conjugation portion, wherein the antibody is any anti-VZV virus antibody or antigen-binding fragment thereof as described in any one of the present invention, and the conjugation portion is a detectable label; preferably, the conjugation portion is a radioactive isotope, a fluorescent substance, a colored substance, or an enzyme.

[0023] Another aspect of the invention relates to a pharmaceutical composition comprising any anti-VZV virus antibody or antigen-binding fragment thereof as described in any one of the invention, or a conjugate thereof; optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0024] Another aspect of the invention relates to the use of any anti-VZV virus antibody or antigen-binding fragment thereof, or conjugate thereof, as described in any one of the invention, in the preparation of a medicament for the treatment or prevention of varicella or shingles.

[0025] The anti-VZV virus antibody or its antigen-binding fragment or the conjugate of the present invention, as described in any one of the present invention, is used for the treatment or prevention of chickenpox or herpes zoster.

[0026] Another aspect of the invention relates to a method for treating or preventing chickenpox or shingles, comprising the step of administering to a subject in need an effective amount of any of the anti-VZV virus antibodies or antigen-binding fragments thereof or conjugates thereof as described in any one of the invention.

[0027] In some embodiments of the present invention, the method for treating or preventing chickenpox or shingles, wherein...

[0028] The single dose of the anti-VZV virus antibody or its antigen-binding fragment is 0.1-100 mg per kilogram of body weight, preferably 5-50 mg or 5-15 mg per kilogram of body weight;

[0029] Preferably, the medication is administered once every 3 days, every 4 days, every 5 days, every 6 days, every 10 days, every week, every 2 weeks, or every 3 weeks;

[0030] Preferably, the administration method is intravenous infusion or intravenous injection.

[0031] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0032] As used in this article, the term EC 50 The half-maximal concentration (WMC) is the concentration that produces a 50% maximum effect.

[0033] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one "light" (L) chain and one "heavy" (H) chain). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, and the heavy chain also contains "D" regions of 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 regions of antibodies mediate 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. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antibody binding sites. The allocation of amino acids to various regions or domains follows Bethesda Md, Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al. Nature 1989; 342: 878-883, or the definition of the IMGT numbering system, see Ehrenmann F, Kaas Q, Lefranc M P. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF[J]. Nucleic acids research, 2009; 38(suppl_1): D301-D307.

[0034] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different types of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0035] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies can usually be obtained using the hybridoma technique first reported by Kohler et al. G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity[J]. Nature, 1975; 256(5517):495), but it can also be obtained using recombinant DNA technology (see US Patent 4,816,567).

[0036] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (receptor antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human antibody (e.g., mouse, rat, or rabbit) with the expected specificity, affinity, or reactivity. Furthermore, some amino acid residues in the framework region (FR) of the receptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further improve or optimize the antibody's performance. For more detailed information on humanized antibodies, please refer to, for example, Jones et al., Nature 1986; 321:522-525; Reichmann et al., Nature, 1988; 332:323-329; Presta, Curr. Op. Struct. Biol. 1992; 2:593-596; and Clark, Immunol. Today 2000; 21:397-402. In some cases, the antigen-binding fragment of the antibody is a diabetic, where V... H and V LThe domain is expressed on a single polypeptide chain, but the linker is too short to allow pairing between two domains on the same chain, thus forcing the domain to pair with a complementary domain on another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 1993; 90:6444-6448 and Poljak RJ et al., Structure 1994; 2:1121-1123).

[0037] As used herein, the term "single-chain fragment variable (ScFv)" refers to an antibody heavy chain variable region (V) linked by linkers. H ) and antibody light chain variable region (V L The molecules of V. L and V H The domain enables the formation of monovalent molecules by linker pairing with single polypeptide chains (see, for example, Birdet et al., Science 1988; 242:423-426 and Huston et al., Proc. Natl. Acad. Sci. USA 1988; 85:5879-5883). Such scFv molecules can have a general structure: NH2-V L -Connection fragment-V H -COOH or NH2-V H -Connection fragment-V L -COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al, Proc. Natl. Acad. Sci. USA 1993; 90:6444-6448). Other linkers that can be used in this invention are described by Alfthan et al, Protein Eng. 1995; 8:725-731, Choi et al, Eur. J. Immunol. 2001; 31:94-106, Hu et al, Cancer Res. 1996; 56:3055-3061, Kipriyanov et al, J. Mol. Biol. 1999; 293:41-56 and Roovers et al, Cancer Immunology, Immunotherapy, 2001, 50(1):51-59.

[0038] As used herein, the terms "separated" or "isolated" refer to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" entity, it may be due to an alteration of its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide separated from this natural state is called a separated one. The terms "separated" or "isolated" do not exclude the presence of artificial or synthetic substances, nor do they exclude the presence of other impurities that do not affect the substance's activity.

[0039] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0040] As used herein, the term “host cell” refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0041] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to an antigen at a concentration of less than about 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10-8 M, 10 -9 M or 10 -10 M or lower affinity (K) D () binds to the antigen.

[0042] As used in this article, the term "K" D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, antibodies have an equilibrium dissociation constant of less than approximately 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 - 9 M or 10 -10 M or a smaller dissociation equilibrium constant (K) D K binds to antigens (e.g., the gE protein of varicella-zoster virus). K can be determined using methods known to those skilled in the art. D For example, measurements can be taken using a Fortebio molecular interaction analyzer.

[0043] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0044] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0045] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing a disease (e.g., chickenpox or shingles) means an amount sufficient to prevent, stop, or delay the onset of a disease (e.g., chickenpox or shingles); an effective amount for treating a disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient who already has the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0046] Beneficial effects of the invention

[0047] The anti-VZV virus antibody of the present invention can effectively bind to the VZV virus gE protein and has good VZV virus neutralizing activity. Attached Figure Description

[0048] Figure 1 PAGE assay for 21O16 purity. From left to right, lane 1 is for protein markers, lane 2 is for 21O16 reduction electrophoresis, lane 3 is for 21O16 non-reduction electrophoresis, and the rightmost lane shows the molecular weight of each marker band.

[0049] Figure 2A Purity of 21O16 was determined by SEC-HPLC at 214 nm.

[0050] Figure 2B Purity of 21O16 was determined by SEC-HPLC at 280 nm.

[0051] Figure 3 :21O16ELISA binds to gE protein.

[0052] Figure 4A Results of the 21O16 neutralization experiment. The numbers in the upper left corner represent the number of cytopathic spots, indicating the viral load.

[0053] Figure 4B IC obtained from the statistical analysis of the neutralization results of 21O16 50 value. Detailed Implementation

[0054] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0055] Example 1: Isolation, purification, and in vitro expansion culture of memory B cells

[0056] 1. Preparation of peripheral blood PBMCs from patients:

[0057] Peripheral blood PBMCs were extracted from recovered patients using standard methods, counted, and then frozen in liquid nitrogen for later use. Recovered patients were defined as those who had undergone treatment and whose herpes zoster lesions had disappeared 3-4 weeks prior, and who had given informed consent.

[0058] 2. Isolation and purification of memory B cells:

[0059] CD19 Flow sorting + IgA - IgD - IgM - memory B (i.e., CD19) + IgG + The memory B cells (CD19+IgA-IgD-IgM-memory B cells) are selected using a negative selection method (i.e., IgA, IgD, and IgM negative) to avoid the risk of cell death caused by the activation of B cells when directly isolated with IgG antibodies.

[0060] 1) Thaw PBMCs, centrifuge at 335g, 4℃ for 5 min, resuspend in 1 ml PBS-1% (wt / vol) BSA, and transfer 50 μl to each of the six new 15 ml centrifuge tubes for fluorescence compensation (Table 1). Centrifuge the six tubes. Resuspend the original tube (tube 1) in 50 μl of mixed staining solution, and resuspend the remaining five tubes in 50 μl PBS-1% (wt / vol) BSA. As shown in Table 1, four of the tubes (tubes 2-5) were treated with a single antibody:

[0061] PE-Cy7 anti-human CD19 (manufacturer: Biolegend, product number: 302216);

[0062] PE anti-human IgM No. 3 (manufacturer: Biolegend, product number: 314508);

[0063] No. 4 APC anti-human IgA (manufacturer: Jackson Immuno Research, product number 109-135-011);

[0064] FITC anti-human IgD (manufacturer: Biolegend, product number 348206).

[0065] One tube (tube #6) without antibody was used as a blank control.

[0066] 2) Incubate in the dark at 4°C for 30 min. Resuspend the cells in 2 ml of PBS-1% (wt / vol) BSA, centrifuge at 335 g and 4°C for 5 min to remove the supernatant. Resuspend in 500 μl of PBS-1% (wt / vol) BSA and transfer to a sterile flow cytometry tube.

[0067] 3) Use a Beckman MofloXDP flow cytometer to sort CD19+IgA-IgD-IgM-B cells. The cell purity should be above 90%. If it is below 90%, repeat the sorting process. The staining and sorting process should be completed within 3 hours.

[0068] Table 1: Sorting and Staining Scheme

[0069] 1 Mixed staining solution 50 0 2 CD19-PE-Cy7 0.5 50 3 IgM-PE 1 50 4 IgA-APC 2.5 50 5 IgD-FITC 2.5 50 6 - - 50

[0070] 3. In vitro expansion and culture of memory B cells:

[0071] 1) Prepare the cell culture mixture according to Table 2.

[0072] 2) Transfer the cell mixture to a large sterile loading well. Spread 50 μl of the mixture into each of the 308 wells. Add 70 μl of sterile PBS to each well around the outer edge of the 384-well plate, for a total of 76 wells.

[0073] 3) Ensure that the cells in the sample well are frequently and gently mixed to keep the B cells in uniform suspension.

[0074] 4) Transfer the 384 plate to a 5% CO2 incubator and incubate at 37°C.

[0075] 5) After 12 days of incubation, transfer the culture supernatant to a new 384-well plate, seal the plate with aluminum foil, and cap it. Immediately store the supernatant plate at -80°C for subsequent ELISA screening.

[0076] 6) Add 20 μl of lysis buffer* to all wells containing B cells. Seal the plate with aluminum foil and cap it. Immediately store the B cell lysis plate at -80°C for future use in Ig gene amplification and cloning.

[0077] *Lysis buffer: Prepare lysis buffer for 20 plates. Add 2 ml of 1M Tris-HCl (pH 8.0) and 1.7 ml of RNase inhibitor to 132 ml of DEPC-treated H2O. Mix the buffer thoroughly.

[0078] Table 2: Culture System

[0079] IL-2 (10000 U / mL) 3.5ml 100U / ml IL-21 (100 μg / ml) 175μl 50ng / ml CD40L (100μg / ml) 175μl 50ng / ml Memory B cells (number) 28000 4 / well / 50μl <![CDATA[NIH-3T3 cells (3.5×10 6 / ml)]]> <![CDATA[3.5X10 7 ]]> <![CDATA[10 5 / ml(5000 / well)]]> Complete IMDM medium 340ml Total 350ml

[0080] The preparation method of the 3T3 cells used in the feeder layer is as follows:

[0081] ① Add 30 ml of NIH-3T3 cell culture medium (DMEM high-glucose medium containing 10% fetal bovine serum) to a T-175 cell culture flask, and seed 3 × 10⁶ cells. 6 Five NIH-3T3 cell cultures were prepared. When the cells reached 80-90% confluence in the culture flasks, the culture medium was aspirated, and the cells were washed with 10 ml of PBS. 4 ml of trypsin was added, and the cells were incubated at 37°C for 3 minutes to digest them. After detaching the cells from the culture flasks, NIH-3T3 cell culture medium was added to terminate the digestion. The cells were then collected in 50 ml conical tubes.

[0082] ② Centrifuge at 4℃ for 10 minutes with 300g.

[0083] ③ Resuspend the cells in 10 ml of NIH-3T3 cell culture medium. Count the cells.

[0084] ④ Centrifuge at 300g, 4℃ for 10 minutes. Discard the supernatant. Place the cells in an irradiator and irradiate them with 50Gy.

[0085] ⑤ After irradiation at room temperature, resuspend the cells in cell cryopreservation solution, 35 × 10⁻⁶ cells per tube. 6 Cells. Place the cryovials in a programmed cooling box and store them at -80°C overnight. The next day, transfer them to a liquid nitrogen freezer for long-term storage.

[0086] Example 2: ELISA screening of anti-gE protein positive wells

[0087] 1. Dilute commercial gE protein (abcam) with PBS to 500 ng / ml, pre-coat the plate one day in advance, and add 100 μl / well to a 96-well microplate. Coat overnight at 4°C.

[0088] 2. Wash the plate three times with PBS.

[0089] 3. Block the plate with PBS containing 5% skim milk powder at 37°C for 30 minutes.

[0090] 4. Wash the plate three times with PBS.

[0091] 5. Add 80 μl of PBS containing 0.5% Tween-20 (PBST) to each well, followed by 20 μl of cell culture supernatant (prepared in step 5 of Example 1, “3. In Vitro Expansion Culture of Memory B Cells”). The positive control well contains 100 μl of patient serum diluted 1000 times with PBST. Incubate at 37°C for 1.5 h.

[0092] 6. Wash the plate 3 times with PBST.

[0093] 7. Add 100 μl of diluted anti-human-IgG-HRP to each well and incubate at 37°C for 30 min.

[0094] 8. Wash the plate 5 times with PBST.

[0095] 9. Add 100 μl of TMB colorimetric solution per well and incubate at room temperature in the dark for 15 min.

[0096] 10. Add 50 μl of stop solution.

[0097] 11.450nm microplate reader reading.

[0098] Cell lysis buffer (prepared in step 6 of “3 Memory B cell in vitro expansion culture” in Example 1) corresponding to wells with OD values ​​greater than 1 was selected for subsequent Ig gene amplification.

[0099] Example 3: Ig gene amplification and sequence acquisition

[0100] 1. Reverse transcription (RT-PCR):

[0101] Prepare the RT-PCR mixture according to the Qiagen OneStep RT-PCR kit protocol (Table 3).

[0102] Table 3: RT-PCR system

[0103]

[0104] Primers are shown in Table 4.

[0105] Table 4: Primers

[0106]

[0107]

[0108]

[0109] Program: RT 50℃ for 30 minutes, 95℃ for 15 minutes ("Hot Start" to deactivate RT and activate hot Taq), 95℃ for 1 minute, 55℃ for 1 minute, 72℃ for 1 minute, 40 cycles.

[0110] 2. Nested PCR:

[0111] Prepare the PCR mixture according to the Qiagen Hotstart Taq kit instructions (Table 5).

[0112] Table 5: PCR System

[0113]

[0114]

[0115] Primers are shown in Table 4.

[0116] Program: 95℃ for 4 min; 95℃ for 1 min, 57℃ for 1 min, 72℃ for 1.5 min, 40 cycles.

[0117] 3. Run 2 μl per well on a 1% agarose gel (wt / vol). Positive results are determined by visualization of a band at approximately 400 bp.

[0118] 4. Glue recycling:

[0119] Gel recovery was performed according to the kit (OMEGA, cat: D2500-D2) instructions.

[0120] The obtained product was sent to a sequencing company to obtain the DNA sequences of the light and heavy chains.

[0121] This resulted in several antibodies, one of which was 21O16.

[0122] DNA sequence of the heavy chain variable region of antibody 21O16:

[0123] gaggtgcagctggtggagtccgggggaggcttagttcagcctggggggtccctgagactctcctgtgcagcctctggaatcaccttcagtagacactggatgcactgggtccgccaagctccagggaaggggctggtgtgggtctcacgtattaatactgatgggactactacaagc tacgcggactccgtgaagggccgattcaccatctccagagacaacgccaagaacacgctgtatctgcaaatgaacagtctgagagccgaggacacggctgtgtattactgtgtaggcgcatcccactggtatttcgacttctggggccgtggcaccctggtcactgtctcctca(SEQ ID NO:1)

[0124] The amino acid sequence of the heavy chain variable region of antibody 21O16:

[0125] EVQLVESGGGLVQPGGSLRLSCAASGITFSRHWMHWVRQAPGKGLVWVSRINTDGTTTSYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCVGASHWYFDFWGRGTLVTVSS(SEQ ID NO:2)

[0126] DNA sequence of the light chain variable region of antibody 21O16:

[0127] gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattggcagatatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatcaagtttg caaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagtttcactgccccgctcagtttcggcggagggaccaaggtagagatcaaa(SEQ ID NO:3)

[0128] The amino acid sequence of the light chain variable region of antibody 21O16:

[0129] DIQMTQSPSSSLSASVGDRVTITCRASQSIGRYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFTAPLSFGGGTKVEIK(SEQ ID NO:4)

[0130] The heavy chain constant region and light chain constant region of antibody 21O16 are the corresponding sequences of IgG1.

[0131] The full-length amino acid sequence of the antibody 21O16 heavy chain:

[0132] EVQLVESGGGLVQPGGSLRLSCAASGITFSRHWMHWVRQAPGKGLVWVSRINTDGTTTSYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCVGASHWYFDFWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 40)

[0133] Full-length nucleic acid sequence of the heavy chain of antibody 21O16:

[0134]

[0135] The full-length amino acid sequence of the light chain of antibody 21O16:

[0136] DIQMTQSPSSSLSASVGDRVTITCRASQSIGRYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFTAPLSFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:42)

[0137] Antibody 21O16 light chain full-length nucleic acid sequence:

[0138] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGGCAGATATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCAAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTTCACTGCCCCGCTCAGTTTCGGCGGAGGGACCAAGGTAGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT(SEQ ID NO: 43)

[0139] The 6 CDRs of antibody 21O16 obtained by alignment in the IMGT system are as follows:

[0140] Three CDRs of the heavy chain of antibody 21O16:

[0141] HCDR1: GITFSRHW (SEQ ID NO: 5)

[0142] HCDR2: INTDGTTT (SEQ ID NO: 6)

[0143] HCDR3: VGASHWYFDF (SEQ ID NO: 7)

[0144] Three CDRs of the light chain of antibody 21O16:

[0145] LCDR1: QSIGRY (SEQ ID NO:8)

[0146] LCDR2: AAS (SEQ ID NO:9)

[0147] LCDR3: QQSFTAPLS (SEQ ID NO: 10).

[0148] Example 4: Preparation and Identification of Antibodies

[0149] The light and heavy chain gene sequences of the antibody obtained in Example 3 were ligated into the pCDNA3.1(+) eukaryotic expression vector using conventional techniques such as enzyme digestion and ligation, and then sequenced for identification.

[0150] The antibody plasmid was co-transfected into CHO-S cells according to the CHO-S (Gibco, A29129) instructions. When cell viability was >90%, 1.5*10⁻⁶ cells were seeded. 8 25ml ExpiCHO TM Expression culture was performed in 125 ml shake flasks. ExpiFectamine was prepared. TM CHO / plasmid mixture: OptiPRO TM Add 16 μg of light chain plasmid and 8 μg of heavy chain plasmid to 1 ml of SFM and mix well; OptiPRO TM Add 920 μl of SFM and 80 μl of reagent, mix well, and immediately add to the plasmid solution. Mix well again and then add to the cell suspension. Incubate at 37°C, 115 rpm, 8% CO2. The next day, add 150 μl of EptiCHO. TM Enhancer and 6ml EptiCHO TM Feed. After 7 days, the supernatant was collected, purified by affinity column analysis, and then analyzed by SDS-PAGE and SEC-HPLC.

[0151] For antibody 21O16, the results of SDS-PAGE analysis are as follows: Figure 1 As shown, the results of SEC-HPLC analysis are as follows: Figure 2A and Figure 2B As shown.

[0152] The results showed that the purity of the obtained antibody was over 95%, which met the requirements for subsequent experiments.

[0153] Example 5: ELISA detection of gE protein binding capacity

[0154] The binding ability of several antibodies, including 21O16, expressed in Example 4 was tested by ELISA. Antibodies with OD values ​​above 0.5 were selected for subsequent neutralization experiments.

[0155] ELISA detection of antibody binding to gE protein:

[0156] 1. Dilute commercial gE protein (abcam) with PBS to 500 ng / ml, pre-coat the plate one day in advance, and add 100 μl / well to a 96-well microplate. Coat overnight at 4°C.

[0157] 2. Wash the plate three times with PBS.

[0158] 3. PBS containing 5% skim milk powder, sealed in a 37°C plate for 30 minutes.

[0159] 4. Wash the plate three times with PBS.

[0160] 5. Serially dilute the expression sample with PBST to a maximum concentration of 10 μg / ml. Add 100 μl to each well. The positive control well contains 100 μl of patient serum diluted 1000 times with PBST. Incubate at 37°C for 1.5 h.

[0161] 6. Wash the plate 3 times with PBST.

[0162] 7. Add 100 μl of diluted anti-human-IgG-HRP to each well and incubate at 37°C for 30 min.

[0163] 8. Wash the board 5 times.

[0164] 9. Add 100 μl of TMB colorimetric solution per well and incubate at room temperature in the dark for 15 min.

[0165] 10. Add 50 μl of stop solution.

[0166] 11.450nm microplate reader reading.

[0167] Antibody 21O16 showed the best efficacy, as indicated by the ELISA results. Figure 3 As shown.

[0168] The results showed that 21O16 could bind well to gE protein, EC 50 =2.182 (μg / ml).

[0169] Example 6: In vitro neutralization experiment (plaque method)

[0170] The experimental samples were multiple antibodies, including 21O16, expressed in Example 4.

[0171] 1. Pre-fill the 6-well plate with 5x10 mm of water. 5 One ARPE-19 cell (Chinese Academy of Sciences Cell Bank, catalog number GNHu45) was cultured overnight at 37°C in DMEM / F12 (Gibco) containing 10% FBS.

[0172] 2. Dilute the prepared VZV virus (Oka) with PBS to 2000 PFU / ml;

[0173] 3. Dilute the antibody to 200 μg / ml with PBS, and then perform serial dilutions of 2-fold, setting the dilution ratio according to experimental requirements;

[0174] 4. Mix 50 μl of diluted virus, 50 μl of 100 U / ml guinea pig complement (Beijing Bosi Technology Co., Ltd., catalog number BM361Y. Complement enhances antibody neutralization.), and 100 μl of diluted antibody. Incubate at 37°C for 60 min, then add the mixture to 6-well plates containing cells. Add 800 μl of serum-free culture medium to each well (each dilution of sample is replicated in triplicate).

[0175] 5. Change the medium after incubating at 37℃ for 2 hours, and continue culturing for 7-10 days;

[0176] 6. Observe and count the number of lesion spots (Zhongxing red or other cell staining can be performed to facilitate counting);

[0177] 7. The experimental controls were set as ARPE-19 cells that did not undergo infection experiments (referred to as negative wells, with 3 replicates) and ARPE-19 cells that were only infected with VZV virus (referred to as positive wells, with 3 replicates).

[0178] 8. Neutralizing valence IC 50 The determination method is to calculate it using GraphPad Prism.

[0179] Antibody 21O16 showed the best results, as follows: Figure 4A and Figure 4B As shown. Neutralizing valence IC 50 It was 17.27 μg / ml.

[0180] The results showed that the present invention obtained a fully human monoclonal antibody 21O16 that can effectively neutralize VZV virus.

[0181] References:

[0182] 1. Gershon AA, Gershon MD. Pathogenesis and current approaches to control of varicella-zoster virus infections. Clin Microbiol Rev. 2013 Oct; 26 (4): 728-43. doi: 10.1128 / CMR.00052-13. PMID: 24092852; PMCID: PMC3811230.

[0183] 2.Harbecke R,Cohen JI,Oxman MN.Herpes Zoster Vaccines.J InfectDis.2021Sep 30;224(12Suppl 2):S429-S442.doi:10.1093 / infdis / jiab387.PMID:34590136;PMCID:PMC8482024.

[0184] 3.Forbes HJ,Bhaskaran K,Thomas SL,Smeeth L,Clayton T,LanganSM.Quantification of risk factors for herpes zoster:population based case-control study.BMJ.2014May 13;348:g2911.doi:10.1136 / bmj.g2911.PMID:25134101;PMCID:PMC4019782.

[0185] 4.Chen N,Li Q,Yang J,Zhou M,Zhou D,He L.Antiviral treatment forpreventing postherpetic neuralgia.Cochrane Database Syst Rev.2014Feb 6;(2):CD006866.doi:10.1002 / 14651858.CD006866.pub3.PMID:24500927.

[0186] 5.Li Q,Ali MA,Cohen JI.Insulin degrading enzyme is a cellularreceptor mediating varicella-zoster virus infection and cell-to-cellspread.Cell.2006Oct 20;127(2):305-16.doi:10.1016 / j.cell.2006.08.046.PMID:17055432;PMCID:PMC7125743.

[0187] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of this invention. The full scope of this invention is given by the appended claims and any equivalents thereof. SEQUENCE LISTING <110> Xiamen University <120> Anti-VZV virus antibodies, their pharmaceutical compositions and uses <130> IDC220181 <160> 43 <170> PatentIn version 3.5 <210> 1 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of the heavy chain variable region of antibody 21O16 <400> 1 gaggtgcagc tggtggagtc cgggggaggc ttagttcagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggaat caccttcagt agacactgga tgcactgggt ccgccaagct 120 ccagggaagg ggctggtgtg ggtctcacgt attaatactg atgggactac tacaagctac 180 gcggactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacgctgtat 240 ctgcaaatga acagtctgag agccgaggac acggctgtgt attackgtgt aggcgcatcc 300 cactggtatt tcgacttctg gggccgtggc accctggtca ctgtctcctc a 351 <210> 2 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of the heavy chain variable region of antibody 21O16 <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Arg His 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val 35 40 45 Ser Arg Ile Asn Thr Asp Gly Thr Thr Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Gly Ala Ser His Trp Tyr Phe Asp Phe Trp Gly Arg Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 3 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of the light chain variable region of antibody 21O16 <400> 3 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gagcattggc agatatttaa attggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatcaagtt tgcaaagtgg ggtcccatca 180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg caacttacta ctgtcaacag agtttcactg ccccgctcag tttcggcgga 300 gggaccaagg tagagatcaa a 321 <210> 4 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of the light chain variable region of antibody 21O16 <400> 4 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Arg Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Phe Thr Ala Pro Leu 85 90 95 Ser Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 5 Gly Ile Thr Phe Ser Arg His Trp 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <400> 6 Ile Asn Thr Asp Gly Thr Thr Thr 1 5 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 7 Val Gly Ala Ser His Trp Tyr Phe Asp Phe 1 5 10 <210> 8 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 8 Gln Ser Ile Gly Arg Tyr 1 5 <210> 9 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 <400> 9 Ala Ala Ser 1 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 <400> 10 Gln Gln Ser Phe Thr Ala Pro Leu Ser 1 5 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 acaggtgccc actcccaggt gcag 24 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> first <400> 12 aaggtgtcca gtgtgargtg cag 23 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> first <400> 13 cccagatggg tcctgtccca ggtgcag 27 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> first <400> 14 caaggagtct gttccgaggt gcag 24 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> first <400> 15 ggaaggtgtg cacgccgctg gtc 23 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> first <400> 16 atgaggstcc cygctcagct gctgg 25 <210> 17 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 ctcttcctcc tgctactctg gctcccag 28 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 atttctctgt tgctctggat ctctg 25 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 gtttctcgta gtctgctttg ctca 24 <210> 20 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 ggtcctgggc ccagtctgtg ctg 23 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 ggtcctgggc ccagtctgcc ctg 23 <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 gctctgtgac ctcctatgag ctg 23 <210> 23 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 ggtctctctc scagcytgtg ctg 23 <210> 24 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 gttcttgggc caattttatg ctg 23 <210> 25 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 ggtccaattc ycaggctgtg gtg 23 <210> 26 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 gagtggattc tcagactgtg gtg 23 <210> 27 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 caccagtgtg gccttgttgg cttg 24 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 sargtgcagc tcgtggag 18 <210> 29 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 gaggtgcagc tgttggag 18 <210> 30 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 agtagtcctt gaccaggcag cccag 25 <210> 31 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 atgacccagw ctccabycwc cctg 24 <210> 32 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 gtgctgtcct tgctgtcctg ct 22 <210> 33 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 33 ctgctaccgg ttcctgggcc cagtctgtgc tgackcag 38 <210> 34 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 ctgctaccgg ttcctgggcc cagtctgccc tgactcag 38 <210> 35 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 ctgctaccgg ttctgtgacc tcctatgagc tgacwcag 38 <210> 36 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 36 ctgctaccgg ttctctctcs cagcytgtgc tgactca 37 <210> 37 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 37 ctgctaccgg ttcttgggcc aattttatgc tgactcag 38 <210> 38 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 38 ctgctaccgg ttccaattcy cagrctgtgg tgacycag 38 <210> 39 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 39 ctcctcactc gagggyggga acagagtg 28 <210> 40 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Full-length amino acid sequence of the heavy chain of antibody 21O16 <400> 40 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Arg His 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val 35 40 45 Ser Arg Ile Asn Thr Asp Gly Thr Thr Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Gly Ala Ser His Trp Tyr Phe Asp Phe Trp Gly Arg Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 41 <211> 1341 <212> DNA <213> Artificial Sequence <220> <223> Full-length nucleic acid sequence of the heavy chain of antibody 21O16 <400> 41 gaggtgcagc tggtggagtc cgggggaggc ttagttcagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggaat caccttcagt agacactgga tgcactgggt ccgccaagct 120 ccagggaagg ggctggtgtg ggtctcacgt attaatactg atgggactac tacaagctac 180 gcggactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacgctgtat 240 ctgcaaatga acagtctgag agccgaggac acggctgtgt attactgtgt aggcgcatcc 300 cactggtatt tcgacttctg gggccgtggc accctggtca ctgtctcctc agctagcacc 360 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 420 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 480 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 540 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 600 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 660 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 720 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc cgaggtcaca 780 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 840 ggcgtggagg tgcataatgc caacaaag ccgcgggagg agcagtacaa cagcacgtac 900 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggatcaag 960 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1020 gggcagcccc gagaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 1080 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1140 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200 gacggctcct tcttctctca cagcaagctc accgtggaca agagcaggtg gcagcagggg 1260 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1320 ctctccctgt ctccgggtaa a 1341 <210> 42 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Full-length amino acid sequence of the light chain of antibody 21O16 <400> 42 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Arg Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Phe Thr Ala Pro Leu 85 90 95 Ser Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 43 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> Full-length nucleic acid sequence of 21O16 light chain <400> 43 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gagcattggc agatatttaa attggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatcaagtt tgcaaagtgg ggtcccatca 180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg caacttacta ctgtcaacag agtttcactg ccccgctcag tttcggcgga 300 gggaccaagg tagagatcaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg ccaaagtaca gtggaaggtg gataacgcccc tccaatcggg taactcccag 480 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagttcgc ccgtcacaaa gagcttcaac aggggagagt gt 642

Claims

1. An anti-VZV virus antibody or its antigen-binding fragment, wherein the anti-VZV virus antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3, wherein: The amino acid sequence of HCDR1 is shown in SEQ ID NO: 5, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 6, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

7. The amino acid sequences of LCDR1 are shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO:

10.

2. The anti-VZV virus antibody or its antigen-binding fragment according to claim 1, wherein, The amino acid sequence of the heavy chain variable region of the anti-VZV virus antibody or its antigen-binding fragment is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

4.

3. The anti-VZV virus antibody or its antigen-binding fragment according to any one of claims 1 to 2, wherein, The heavy chain constant region of the anti-VZV virus antibody is either the Ig gamma-1 chain C region or the Ig gamma-4 chain C region; the light chain constant region is the Ig kappa chain C region.

4. The anti-VZV virus antibody or its antigen-binding fragment according to any one of claims 1 to 2, wherein, The anti-VZV virus antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv, single-chain antibody or chimeric antibody.

5. The anti-VZV virus antibody or its antigen-binding fragment according to any one of claims 1 to 2, wherein, The anti-VZV virus antibody is a fully human antibody.

6. The anti-VZV virus antibody or its antigen-binding fragment according to any one of claims 1 to 2, wherein, The amino acid sequence of the heavy chain of the anti-VZV virus antibody is shown in SEQ ID NO: 40, and the amino acid sequence of the light chain of the anti-VZV virus antibody is shown in SEQ ID NO:

42.

7. An isolated nucleic acid molecule encoding an anti-VZV virus antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 6.

8. The isolated nucleic acid molecule according to claim 7, wherein, The sequence of the nucleic acid encoding the heavy chain of the anti-VZV virus antibody is shown in SEQ ID NO: 41, and the sequence of the nucleic acid encoding the light chain of the anti-VZV virus antibody is shown in SEQ ID NO:

43.

9. A recombinant vector comprising the isolated nucleic acid molecule as described in claim 7 or 8.

10. A host cell comprising the isolated nucleic acid molecule of claim 7 or 8, or the recombinant vector of claim 9.

11. Conjugates, comprising antibodies and conjugated portions, wherein, The antibody is the anti-VZV virus antibody or its antigen-binding fragment as described in any one of claims 1 to 6, and the conjugated portion is a detectable label.

12. The coupling according to claim 11, wherein, The coupling component is a radioactive isotope, a fluorescent substance, a colored substance, or an enzyme.

13. A pharmaceutical composition comprising an anti-VZV virus antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 6, or a conjugate as described in claim 11 or 12.

14. The pharmaceutical composition of claim 13, further comprising one or more pharmaceutically acceptable excipients.

15. Use of the anti-VZV virus antibody or antigen-binding fragment thereof as described in any one of claims 1 to 6, or the conjugate as described in claim 11 or 12, in the preparation of a medicament for treating varicella or herpes zoster.

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