Anti-varicella zoster virus antibody

Abstract

The present invention relates to neutralizing monoclonal antibodies or antigen-binding fragments thereof that are specific to and bind with high affinity to varicella-zoster virus, and to preparative methods for producing such antibodies. The antibodies of the present invention have high efficiency in neutralizing varicella-zoster virus infection. The present invention also relates to the epitopes to which the antibodies bind, and the use of the antibodies in the diagnosis, prevention, and treatment of infected individuals.

Description

Technical Field

[0001] The varicella-zoster virus (VZV), an enveloped virus, belongs to the human herpes simplex virus. VZV has high species specificity, and its natural infection occurs only in humans and gorillas. Various glycoproteins such as gE, gB, gH, gI, gC, and gL exist on the envelope of VZV. VZV glycoproteins not only participate in the entry of the virus into cells but also may be transferred from infected cells to uninfected cells and initiate humoral and cellular immune responses to the virus.

Background Art

[0002] VZV enters the body through the conjunctiva and respiratory mucosa during the initial infection, causing primary infection with varicella, a highly contagious disease common in children. In healthy children, varicella is a self-limiting disease with a duration of about 4 to 5 days. However, in immunodeficient children and non-immune newborns, varicella infection may cause severe complications such as viral pneumonia and encephalitis and may lead to fatal outcomes. A small number of adults are infected with varicella, show severe symptoms, often develop pneumonia (20% - 30%), and have a high mortality rate. In the case of pregnant women, varicella may cause fetal malformations, miscarriage, or stillbirth in addition to a severe condition. Tests have shown that the incidence of varicella during pregnancy is 0.1‰ - 0.7‰. When the initial infection occurs within 6 months of pregnancy, the intrauterine infection rate is about 25%, and the incidence of congenital varicella syndrome is about 12% of the infected fetuses.

[0003] VZV may also potentially lie latent within ganglia. Factors such as aging, immune weakening or immunosuppression due to disease or drugs will reactivate the latent virus, causing recurrent infection of herpes zoster distributed along the nerves, i.e., shingles (zoster). VZV recurrent infection may cause skin lesions and abnormal disseminated infection of damage to organs such as the lungs, brain, liver, kidneys, heart and eyes. VZV often is accompanied by postherpetic neuralgia and, in severe cases, may even cause death. The pain of postherpetic neuralgia is severe and may persist for several years, greatly affecting the quality of life of patients. The incidence of herpes zoster and postherpetic neuralgia increases with age, causing a greater social burden in an aging society.

[0004] Currently, the most important preventive measure for VZV virus infection is vaccination. For the two clinical symptoms caused by primary infection and recurrent infection of the VZV virus, varicella vaccine and herpes zoster vaccine are currently available for prevention. Other treatment methods mainly involve the use of antivirals such as acyclovir, famciclovir, valacyclovir, acyclovir, vidarabine, and the like. Therefore, there is currently no effective and specific treatment.

[0005] Currently, antibody therapy based on virus-neutralizing antibodies has been applied to the treatment of many diseases. For VZV virus infection, the US FDA has approved varicella-zoster immune globulin (VZIG) produced by Cangene of Canada for use in high-risk populations of VZV infection, mainly pregnant women and newborns. However, VZV immune globulin is a blood product with batch-to-batch variation using its own scarce sources, with a blank period and a risk of pathogen transmission. In addition, this type of product is associated with safety issues such as the transmission of blood-borne pathogens such as HIV, hepatitis B virus, hepatitis C virus, etc., so it has drawbacks for application. Currently, drugs related to VZV immune globulin are not commercially available in China and most countries.

[0006] So far, existing vaccines and systemic antiviral drugs can prevent and control VZV infection, but they cannot eliminate various complications of VZV infection. Therefore, there is still a need to find effective neutralizing antibodies against VZV that can effectively detect VZV and block host VZV infection and inter-host transmission. In addition, improved methods for the treatment of VZV infection, particularly methods suitable for emergency intervention against VZV infection, are still needed. The anti-VZV antibodies and their compositions provided in this application meet the above requirements.

Summary of the Invention

Problems to be Solved by the Invention

[0007] The present invention provides a new neutralizing anti-VZV antibody capable of binding to VZV with high affinity, a composition, a kit, an anti-VZV antibody, and methods for using and preparing the antibody.

Means for Solving the Problems

[0008] In one aspect, the present invention provides an isolated anti-VZV antibody and antigenic fragments thereof. In a specific embodiment, the anti-VZV antibody and antigenic fragments thereof comprise one, two or three CDRs (preferably three CDRs) selected from the VH region sequences of any of the antibodies shown in Table I. In other embodiments, the antibodies of the present invention comprise one, two or three CDRs (preferably three CDRs) selected from the VL region sequences of any of the antibodies shown in Table I. In some embodiments, the antibodies of the present invention comprise the six CDR region sequences of any of the antibodies shown in Table I. In a preferred embodiment, the CDR sequences of the antibody are the CDR sequences shown in Table II.

[0009] In some embodiments, the anti-VZV antibody or antigen-binding fragment thereof of the present invention comprises: A) heavy chain complementarity determining regions (CDRs): (i) a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 1, 7, and 13, or a sequence comprising 1 to 5 amino acid substitutions (such as conservative substitutions), deletions, or insertions with respect to SEQ ID NO: 1, 7, or 13; (ii) a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 2, 8, and 14, or a sequence comprising 1 to 5 amino acid substitutions (such as conservative substitutions), deletions, or insertions with respect to SEQ ID NO: 2, 8, or 14; (iii) a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 3, 9, and 15, or a sequence comprising 1 to 5 amino acid substitutions (such as conservative substitutions), deletions, or insertions with respect to SEQ ID NO: 3, 9, or 15; and B) light chain complementarity determining regions (CDRs): (i) a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 4, 10, and 16, or a sequence comprising 1 to 5 amino acid substitutions (such as conservative substitutions), deletions, or insertions with respect to SEQ ID NO: 4, 10, or 16; (ii) a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 5, 11, and 17, or a sequence comprising 1 to 5 amino acid substitutions (such as conservative substitutions), deletions, or insertions with respect to SEQ ID NO: 5, 11, or 17; (iii) a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 6, 12, and 18, or a sequence comprising 1 to 5 amino acid substitutions (such as conservative substitutions), deletions, or insertions with respect to SEQ ID NO: 6, 12, or 18, and an anti-VZV antibody comprising a modified CDR still has the ability to bind to VZV.

[0010] In some embodiments, the anti-VZV antibody or antigen-binding fragment thereof of the present invention comprises: A) heavy chain complementarity-determining regions (CDRs): (i) CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, (ii) CDR2 consisting of the amino acid sequence of SEQ ID NO: 2, (iii) CDR3 consisting of the amino acid sequence of SEQ ID NO: 3; and B) light chain complementarity-determining regions (CDRs): (i) CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, (ii) CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, (iii) CDR3 consisting of the amino acid sequence of SEQ ID NO: 6.

[0011] In some embodiments, the anti-VZV antibody or antigen-binding fragment thereof of the present invention comprises: A) heavy chain complementarity-determining regions (CDRs): (i) CDR1 consisting of the amino acid sequence of SEQ ID NO: 7, (ii) CDR2 consisting of the amino acid sequence of SEQ ID NO: 8, (iii) CDR3 consisting of the amino acid sequence of SEQ ID NO: 9; and B) light chain complementarity-determining regions (CDRs): (i) CDR1 consisting of the amino acid sequence of SEQ ID NO: 10, (ii) CDR2 consisting of the amino acid sequence of SEQ ID NO: 11, (iii) CDR3 consisting of the amino acid sequence of SEQ ID NO: 12.

[0012] In some embodiments, the anti-VZV antibody or antigen-binding fragment thereof of the present invention comprises: A) heavy chain complementarity-determining regions (CDRs): (i) CDR1 consisting of the amino acid sequence of SEQ ID NO: 13, (ii) CDR2 consisting of the amino acid sequence of SEQ ID NO: 14, (iii) CDR3 consisting of the amino acid sequence of SEQ ID NO: 15; and B) light chain complementarity-determining regions (CDRs): (i) CDR1 consisting of the amino acid sequence of SEQ ID NO: 16, (ii) CDR2 consisting of the amino acid sequence of SEQ ID NO: 17, (iii) CDR3 consisting of the amino acid sequence of SEQ ID NO: 18.

[0013] In some embodiments, the anti-VZV antibody or antigen-binding fragment thereof of the invention comprises, or consists of, a heavy chain variable region VH comprising an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or higher identity to the amino acid sequence selected from SEQ ID NO: 19, 21, 23 or 25, and the anti-VZV antibody comprising the VH has the ability to bind to VZV. In some embodiments, the anti-VZV antibody or antigen-binding fragment thereof of the invention comprises the six CDRs of any of the antibodies shown in Table II and comprises a heavy chain variable region VH having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence selected from SEQ ID NO: 19, 21, 23 or 25, and the anti-VZV antibody comprising the VH has the ability to bind to VZV. In some embodiments, the heavy chain variable region VH of the anti-VZV antibody comprises an amino acid sequence having one or more substitutions (such as conservative substitutions), insertions or deletions with respect to the amino acid sequence selected from SEQ ID NO: 19, 21, 23 or 25, and the anti-VZV antibody comprising the VH has the ability to bind to VZV.

[0014] In some embodiments, the anti-VZV antibody or antigen-binding fragment thereof of the present invention comprises, or consists of, a light chain variable region (VL) having an amino acid sequence with at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or higher identity, to an amino acid sequence selected from SEQ ID NO: 20, 22, 24 or 26, and the anti-VZV antibody comprising the VL has the ability to bind to VZV. In some embodiments, the anti-VZV antibody or antigen-binding fragment thereof of the present invention comprises the six CDRs of any of the antibodies shown in Table II and has a light chain variable region (VL) with at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 20, 22, 24 or 26, and the anti-VZV antibody comprising the VL has the ability to bind to VZV. In some embodiments, the light chain variable region (VL) of the anti-VZV antibody comprises an amino acid sequence having one or more substitutions (such as conservative substitutions), insertions or deletions with respect to an amino acid sequence selected from SEQ ID NO: 20, 22, 24 or 26, and the anti-VZV antibody comprising the VL has the ability to bind to VZV.

[0015] In some embodiments, the anti-VZV antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL). 1) The heavy chain variable region VH comprises, or consists of, an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or higher identity, to an amino acid sequence selected from SEQ ID NO: 19, 21, 23 or 25, and the light chain variable region VL comprises, or consists of, an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or higher identity, to an amino acid sequence selected from SEQ ID NO: 20, 22, 24 or 26, or

[0016] 2) The heavy chain variable region VH comprises the six CDRs of any of the antibodies shown in Table II and has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 19, 21, 23 or 25, the light chain variable region VL comprises the six CDRs of any of the antibodies shown in Table II and has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 20, 22, 24 or 26, and the anti-VZV antibody comprising the VH and VL has the ability to bind to VZV, or

[0017] 3) The heavy chain variable region VH comprises, or consists of, an amino acid sequence having one or more substitutions (such as conservative substitutions), insertions or deletions with respect to the amino acid sequence selected from SEQ ID NO: 19, 21, 23 or 25, and the light chain variable region VL comprises, or consists of, an amino acid sequence having one or more substitutions (such as conservative substitutions), insertions or deletions with respect to the amino acid sequence selected from SEQ ID NO: 20, 22, 24 or 26, and the anti-VZV antibody comprising the VH and VL has the ability to bind to VZV.

[0018] In a preferred embodiment, the present invention provides an anti-VZV antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region VH comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 19, and the light chain variable region VL comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 20.

[0019] In a preferred embodiment, the present invention provides an anti-VZV antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region VH comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 21, and the light chain variable region VL comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 22.

[0020] In a preferred embodiment, the present invention provides an anti-VZV antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region VH comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 23, and the light chain variable region VL comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 24.

[0021] In a preferred embodiment, the present invention provides an anti-VZV antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region VH comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 25, and the light chain variable region VL comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 26.

[0022] In some embodiments, the anti-VZV antibody or antigen-binding fragment thereof further comprises a heavy chain and / or light chain constant region sequence derived from a human antibody germline consensus sequence.

[0023] In some embodiments, the antibodies of the invention also include antibodies that compete with any of the previously described antibodies for binding to VZV, and antibodies that bind to the same VZV epitope as any of the previously described antibodies.

[0024] In some embodiments, at least a portion of the framework region of the anti-VZV antibody is a human consensus framework sequence. In one embodiment, the anti-VZV antibody of the invention also includes an antibody fragment thereof, preferably an antibody fragment selected from Fab, Fab’-SH, Fv, scFv, and (Fab’) 2 fragments.

[0025] In some embodiments, the anti-VZV antibody of the invention is a neutralizing antibody for use in neutralizing VZV.

[0026] In one aspect, the invention provides a nucleic acid encoding any of the foregoing anti-VZV antibodies or fragments thereof. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from mammalian cells or other cells suitable for preparing an antibody or antigen-binding fragment thereof. In another embodiment, the host cell is prokaryotic.

[0027] In one embodiment, the invention provides a method for preparing an anti-VZV antibody or antigen-binding fragment thereof, the method comprising culturing a host cell under conditions suitable for expressing a nucleic acid encoding the antibody or antigen-binding fragment thereof, and optionally isolating the antibody or antigen-binding fragment thereof. In certain embodiments, the method further comprises recovering the anti-VZV antibody or antigen-binding fragment thereof from the host cell.

[0028] In one embodiment, the present invention provides an anti-VZV antibody or an antigen-binding fragment thereof prepared by the method of the present invention.

[0029] In some embodiments, the present invention provides a composition comprising any anti-VZV antibody or an antigen-binding fragment thereof described herein, preferably the composition is a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutical carrier. In one embodiment, the anti-VZV antibody or an antigen-binding fragment thereof contained in the composition is coupled to a coupling moiety. In some embodiments, the present invention provides that the anti-VZV antibody and its antigen-binding fragment contained in the composition are coupled to a coupling moiety capable of extending the half-life of the antibody or its antigen-binding fragment.

[0030] In another aspect, provided herein is a composition comprising any anti-VZV antibody or a fragment thereof. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the composition is a pharmaceutical composition.

[0031] In one aspect, the present invention relates to a method for neutralizing VZV contained in a subject or a sample, the method comprising: (a) contacting the subject or the sample with any anti-VZV antibody or a fragment thereof described herein, and (b) detecting a complex formed by the anti-VZV antibody or a fragment thereof and VZV. In a preferred embodiment, the anti-VZV antibody and its antigen-binding fragment of the present invention further comprise a detectable label. The present invention also relates to the use of any anti-VZV antibody or a fragment thereof in the preparation of a composition or a drug or a kit for neutralizing VZV in a subject.

[0032] In another aspect, the present invention relates to a method for preventing or treating VZV infection, or one or more diseases or symptoms associated with VZV infection (e.g., chickenpox, herpes zoster) in a subject, the method comprising administering to the subject an effective amount of any anti-VZV antibody or fragment thereof described herein, or administering a pharmaceutical composition of the present invention. In one embodiment, the subject is a neonate, premature infant, pregnant woman, and an immunocompromised subject who has received immunosuppressive agents, cytotoxic agents, radiotherapy, or the like due to organ transplantation surgery, blood cancer, malignancy, nephrotic syndrome, and the like.

[0033] The present invention also relates to the use of any anti-VZV antibody or fragment thereof described herein in the preparation of a medicament for the treatment or prevention of VZV infection, or one or more diseases or symptoms associated with VZV infection. In some embodiments, the condition associated with VZV is chickenpox or herpes zoster.

[0034] In another aspect, the present invention relates to a method for enhancing, augmenting or stimulating an immune response or function in a subject, the method comprising administering to the subject an effective amount of any anti-VZV antibody or fragment thereof described herein, thereby enhancing, augmenting or stimulating an immune response or function in the subject.

[0035] In another aspect, any anti-VZV antibody or fragment thereof described herein for use as a medicament.

[0036] In another aspect, the present invention provides a method for diagnosing whether a subject is infected with VZV, comprising detecting the presence and / or level of VZV in a sample from the subject using the anti-VZV antibody and antigen-binding fragment thereof of the present invention. In a preferred embodiment, the anti-VZV antibody and antigen-binding fragment thereof of the present invention further comprise a detectable label.

[0037] In another aspect, the present invention provides a kit comprising an antibody or composition of the present invention, such as a diagnostic kit, detection kit, and treatment kit.

[0038] The present invention also encompasses any combination of any of the embodiments described herein. Any embodiment described herein or any combination thereof is applicable to any anti-VZV antibody or fragment thereof, method, and use of the invention described herein.

Advantages of the Invention

[0039] The present invention provides a fully human antibody and an antigen-binding fragment thereof that can specifically recognize VZV / bind to VZV. The full human antibody and its antigen-binding fragment are neutralizing antibodies having a neutralizing effect and capable of inhibiting VZV infection. The antibody and its antigen-binding fragment have good affinity, strong specificity, non-responsiveness to heterologous sera, and no risk of transmitting other infectious diseases, and can be used in a subject for preventing and treating VZV infections such as chickenpox and herpes zoster, or diseases or symptoms related to such infections.

Brief Description of the Drawings

[0040]

Figure 1

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DETAILED DESCRIPTION OF THE INVENTION

[0041] 1.1 Definitions Before the present invention is described in detail below, it should be understood that the specific methodologies, protocols, and reagents described herein may vary and that the present invention is not limited thereto. The terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention, which is to be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0042] For the purpose of interpreting this specification, the following definitions are used, and terms used in the singular may include the plural and, where appropriate, vice versa. It is understood that the terms used herein are for the purpose of describing particular embodiments and are not intended to be limiting.

[0043] The term "about" when used in connection with a numerical value means a value within a range that is 5% lower than the lower limit and 5% higher than the upper limit from the specified numerical value.

[0044] The term "and / or" is understood to mean either or both of the available alternatives.

[0045] As used herein, the term "comprising" or "including" means including the recited element, integer or step without excluding any other element, integer or step. When the term "comprising" or "including" is used herein, unless otherwise specified, it also includes situations consisting of the recited element, integer or step. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to include an antibody variable region consisting of the specific sequence.

[0046] The term "antibody" is used herein in the broadest sense and includes, without limitation, various antibody structures such as monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity. Intact antibodies generally contain at least two full-length heavy chains and two full-length light chains, but in some cases, they may contain fewer chains. For example, antibodies that occur naturally in camels may contain only heavy chains.

[0047] As used herein, "monoclonal antibody" or "mAb" refers to a single antibody or a cloned antibody, e.g., derived from eukaryotes, prokaryotes, phage clones, i.e., each of the antibodies constituting the population is identical and / or binds to the same epitope, except for possible variant antibodies that are present usually in very small amounts (e.g., naturally occurring mutations(s), or variants containing mutations(s) generated during the production of monoclonal antibody products). The modifier "monoclonal" refers to the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring any particular method for generating the antibody. Monoclonal antibodies can be generated, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic technologies such as CDR grafting, or combinations of such or other techniques known in the art.

[0048] One of ordinary skill in the art will understand that a "complete antibody" (which may be used interchangeably herein with "whole antibody") comprises at least two heavy chains (H) and two light chains (L). Each heavy chain is composed, from the N-terminus to the C-terminus, of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further separated into complementarity determining regions (CDRs) and intervening framework regions (FRs). Each of VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0049] "Natural antibody" refers to naturally occurring immunoglobulin molecules having various structures. "Natural sequence of the Fc domain" contains the same amino acid sequence as the amino acid sequence of the Fc domain found in nature. Examples of the natural sequence of the human Fc domain include the natural sequence of the human IgG1 Fc domain (non-A and A allotypes); the natural sequence of the human IgG2 Fc domain; the natural sequence of the human IgG3 Fc domain; and the natural sequence of the human IgG4 Fc domain; as well as their naturally occurring variants.

[0050] "Human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or an antibody from a non-human source that utilizes a human antibody library or other human antibody coding sequence. Such a definition for a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residue(s).

[0051] The term "neutralizing antibody" refers to an antibody or antibody fragment capable of binding to a pathogen and eliminating or significantly reducing the virulence of the pathogen (e.g., the ability to infect cells). Such neutralizing antibodies typically play a role in killing cells and preventing the pathogen from invading cells.

[0052] In some embodiments, the invention includes fragments of anti-VZV antibodies. Examples of antibody fragments include Fv, Fab, Fab’, Fab’-SH, F(ab’) 2 , diabodies, linear antibodies, single-chain antibody molecules (such as scFv); and multispecific antibodies formed by antibody fragments, but are not limited thereto. Digestion of an antibody with papain yields two identical antigen-binding fragments, called “Fab” fragments, each having a single antigen-binding site, and the remaining “Fc” fragment, the name of which immediately reflects the ability to crystallize. Pepsin treatment yields an F(ab’) 2 fragment that has two antigen-binding sites and is still capable of cross-linking antigens.

[0053] A “complementary determining region” or “CDR region” or “CDR” or “hypervariable region” is an amino acid sequence within the variable region of an antibody that is primarily responsible for binding to an epitope. Heavy and light chain CDRs are typically designated CDR1, CDR2, and CDR3 and are numbered in order from the N-terminus.

[0054] Various schemes for determining the CDR sequences of a given VH or VL amino acid sequence are well known in the art: Kabat Complementary Determining Regions (CDRs) are determined based on sequence variability and are most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), Chothia refers to the positions of structural loops (Chothia et al., (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:877-883), AbM HVRs are a compromise between Kabat HVRs and Chothia structural loops and are used by Oxford Molecular's AbM Antibody Modeling Software. "Contact" HVRs are based on the analysis of available complex crystal structures. Depending on the different schemes for determining CDRs, the respective residues of these HVR / CDRs are as follows.

Table 1

[0055] In one embodiment, the antibody CDRs of the present invention are CDR sequences at the following Kabat residue positions according to the Kabat numbering system: Positions 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3) within VL, and positions 27-35 (CDR1), 50-65 (CDR2), and 93-102 (CDR3) within VH.

[0056] The CDRs can also be determined based on the same Kabat numbering positions as a reference CDR sequence (e.g., any of the exemplary CDRs of the present invention).

[0057] The term "variant" related to an antibody, as used herein, refers to an antibody having amino acid modification(s) (s) in a target antibody region(s) (e.g., heavy chain variable region or light chain variable region or heavy chain CDR region or light chain CDR region) that has been subjected to at least one amino acid substitution, deletion, and / or insertion, such as 1 to 30, or 1 to 20, or 1 to 10, etc., one or two or three or four or five, etc., and the variant basically retains the biological properties of the antibody molecule before modification. In one aspect, the present invention encompasses variants of any of the antibodies described herein. In one embodiment, a variant of an antibody retains at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen-binding ability) of the antibody before modification. It can be understood that the heavy chain variable region or light chain variable region or each CDR region of the antibody can be individually or collectively converted. In some embodiments, the amino acid modification(s) that occur in one or more or all three heavy chain CDRs are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or less. Preferably, the amino acid modification(s) are amino acid substitution(s), preferably conservative substitution(s).

[0058] The term "conservative substitution" refers to the substitution of an amino acid by another amino acid belonging to the same classification, for example, an acidic amino acid is substituted by another acidic amino acid, a basic amino acid is substituted by another basic amino acid, or a neutral amino acid is substituted by another neutral amino acid. Exemplary substitutions are shown in the following table.

Table 2

[0059] In some embodiments, the antibody variant has at least 80%, 90%, 95%, 99%, or higher amino acid sequence identity to the parental antibody within the target region of the antibody.

[0060] As used herein, the term "vector" refers to a nucleic acid molecule capable of increasing another nucleic acid to which it is ligated. This term encompasses vectors as self-replicating nucleic acid structures and vectors incorporated into the genome of an introduced host cell. Certain vectors are capable of directing the expression of operably linked nucleic acids. Such vectors are referred to herein as "expression vectors."

[0061] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which foreign nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells" that include primary transformed cells and progeny derived therefrom regardless of the number of passages. Progeny may not have exactly the same nucleic acid content as the parental cell and may contain mutations. Mutant progeny having the same function or biological activity as that originally secreted or selected in the transformed cell are included herein.

[0062] Suitable host cells for cloning or expressing nucleic acids encoding antibodies or vectors include prokaryotic or eukaryotic cells as described herein. The antibody can be produced, for example, in bacteria, particularly when glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523, and also see Charlton, Methods in Molecular Biology, Volume 248 (B. K. C. Lo, ed., Humana Press, Totowa, NJ, 2003), Pages 245-254, which describes the expression of antibody fragments in E. coli. After expression, the antibody can be isolated from the bacterial paste in soluble fragments and further purified.

[0063] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing an antibody or antigen-binding fragment thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeasts, including fungi and yeasts in which the glycosylation pathway has been humanized and which result in the production of antibodies having a partial or complete human glycosylation pattern, are suitable cloning or expression hosts for vectors encoding antibodies. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006). Host cells suitable for expressing glycosylated antibodies also are derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells also can be used as hosts. For example, mammalian cell lines modified to be suitable for growth in suspension can be used. Other examples of useful mammalian host cell lines include the simian kidney CV1 line transformed with SV40 (COS-7); human fetal kidney lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)) and the like. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Volume 248 (B. K. C. Lo, ed., Humana Press, Totowa, NJ), pages 255-268 (2003).

[0064] An "isolated" antibody is one that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to a purity of greater than 95% or 99% as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing electrophoresis (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B848:79-87 (2007).

[0065] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from the components of its natural environment. Isolated nucleic acids include nucleic acid molecules that are normally contained in cells that contain the nucleic acid molecule, but are present extrachromosomally or at a chromosomal locus different from the natural chromosomal locus.

[0066] The "percent amino acid sequence identity (%)" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignments for the purpose of determining percent amino acid sequence identity can be achieved using various methods in the art, such as publicly available computer software, such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared.

[0067] When percent sequence identities are referred to in this application, these percentages are calculated relative to the full length of the longer sequence, unless specifically indicated otherwise. Calculations relative to the full length of the longer sequence apply to both nucleic acid sequences and polypeptide sequences.

[0068] "Affinity" or "binding affinity" refers to the specific binding affinity that reflects the interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity between molecule X and its partner Y can usually be represented by the equilibrium dissociation constant (K D ). The equilibrium dissociation constant is the ratio of the dissociation rate constant and the association rate constant (k dis and k on respectively). Affinity can be measured by common methods known in the art, including methods known in the prior art and the methods described herein.

[0069] "Immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, including but not limited to a carrier.

[0070] The term "pharmaceutical composition" refers to a formulation that contains no additional components that would be toxic and unacceptable to the subject being administered, in a form that effectively allows the biological activity of the active ingredient contained therein.

[0071] In another aspect, the present invention provides a pharmaceutical composition comprising one or more monoclonal antibodies that bind to VZV or an immunologically active fragment thereof. It should be understood that the anti-VZV antibodies or pharmaceutical compositions provided by the present invention can be incorporated into appropriate carriers, excipients, and other reagents in the formulation for combination administration, thereby providing improved uptake, delivery, tolerance, and the like.

[0072] The term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant (e.g., complete or incomplete Freund's adjuvant), excipient, or vehicle that is administered together with a therapeutic agent.

[0073] Pharmaceutically acceptable carriers suitable for use in the present invention may be conventional compositions and formulations suitable for drug delivery of the disclosed antibodies, such as excipients for pharmaceutical preparations described in “Handbook of Pharmaceutical Excipients”, 7th edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago; and “Remington’s Pharmaceutical Sciences”, E.W. Martin, Mack Publishing Co, Easton, PA, 21st edition, 2012.

[0074] In some embodiments, the nature of the carrier depends on the specific mode of administration used. For example, parenteral preparations usually contain an injection solution as the carrier. Injection solutions include water, physiological saline, emulsions in oily or aqueous media, and other pharmaceutical and physiologically acceptable liquids, and may contain agents such as suspensions, preservatives, excipients, stabilizers, surfactants, chelating agents, and / or binders. In some embodiments, pharmaceutically acceptable carriers also include low molecular weight polypeptides, proteins (e.g., serum albumin and gelatin), amino acids (e.g., glycine, glutamine, asparagine, glutamate, aspartate, methionine, arginine, and lysine), saccharides and carbohydrates (e.g., polysaccharides and monosaccharides), and sugar alcohols (e.g., mannitol and sorbitol). When preparing an aqueous solution for injection, physiological saline, and isotonic solutions containing glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride can be used, and if necessary, can be used in combination with suitable solubilizers such as alcohol (e.g., ethanol), polyols (e.g., propylene glycol and PEG), and nonionic surfactants (e.g., polysorbate 80, polysorbate 20, poloxamer 188, and HCO-50). In solid compositions (e.g., powders, pills, tablets, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grade mannitol, lactose, starch, or magnesium stearate. Solid compositions can also be formulated as injection solutions in a liquid medium immediately before administration (e.g., the lyophilized composition Herceptin (trademark)).

[0075] The pharmaceutical composition of the present invention can be administered by various routes including, but not limited to, oral, intravenous, intramuscular, intracranial, transdermal, topical, intranasal, and other modes of administration.

[0076] The term "effective amount" refers to an amount or dosage sufficient to achieve, or at least partially achieve, the predicted effect after administration in a single or multiple doses, and a "therapeutically effective amount" refers to an amount that produces a desired effect in a subject being treated, such as improvement (e.g., one or more improvements) and / or delay in the progression of the subject's symptom(s), including but not limited to the same. An amount effective for prophylaxis refers to an amount sufficient to prevent, block, or delay the occurrence of a disease. Determining the effective amount is well within the ability of one of ordinary skill in the art. For example, the therapeutically effective amount depends, among other things, on the specific disease involved, the degree or severity of the disease, the response of the individual patient, the specific antibody administered, the mode of administration, the bioavailability profile of the preparation administered, the dosing regimen selected, and the use of any combination therapies.

[0077] As used herein, "treatment" refers to slowing, interfering with, blocking, reducing, stopping, decreasing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

[0078] The term "varicella" refers to an acute infectious disease caused by a primary infection with varicella-zoster virus (VZV), characterized primarily by fever, the appearance of numerous red spots and papules, herpes, and blisters over the entire body.

[0079] The term "herpes zoster" refers to a condition caused by the reactivation of latent VZV virus present in ganglia, in which the latent VZV virus migrates and proliferates in the skin along the nerve axons where the nerves are distributed.

[0080] The term "subject" or "individual" is a primate (e.g., a human and non-human primates such as monkeys). In certain embodiments, the individual or subject is a human.

[0081] 1.2 Sequences of exemplary anti-VZV antibodies of the present invention [Table 3] [Table 4]

Example

[0082] The present invention is further illustrated by the following examples. However, it should be understood that the examples are described more as illustrations rather than limiting methods, and various improvements can be made by those skilled in the art.

[0083] Unless clearly indicated to the contrary, the present invention will be carried out by conventional methods in the art of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology and cell biology. Descriptions of these methods can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition, 2001); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, revised July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I&II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Perbal, A Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998) Current Protocols in Immunology Q.E. Coligan, A.M.It can be found in journals such as Annual Review of Immunology; and Advances in Immunology, eds. Kruisbeek, D.H., Margulies, E.M., Shevach, and W. Strober, 1991).

[0084] Example 1 Selection of Plasma Cells Volunteers were vaccinated with varicella vaccine (Merck, Zostavax) according to the manufacturer's protocol, and blood samples were collected on the 7th day after vaccination. Plasma cells and PBMC cells were separated by density gradient centrifugation respectively. The specific method was referred to in the specification of Chinese Patent Application Publication No. 107760690B of the obtained patent. The VZV gH / gL protein complex (CAMBRIDGEBIO, Cat No: 01-11-0045) was selected as the antigen for ELISA to detect serum antibody titers, and the samples with the highest antibody titers and significant fold changes (50-fold dilution, OD value > 2.0) were selected for flow sorting. Single plasma cells were sorted through flow cytometry with the gate of CD3 / CD14 / CD16 / cd235a-CD19+ CD20+ / -CD38hi CD27hi, and a specific population of plasma cells was separated, and the gene sequences of fully human monoclonal antibodies against VZV were isolated. The specific method was referred to in the specification of Chinese Patent Application Publication No. 107760690B of the obtained invention patent.

[0085] Example 2 Isolation of Variable Region Genes of Target Antibodies First, cDNA (first strand) was synthesized from the plasmablasts obtained in Example 1 by reverse transcription with primers for the constant region (refer to the primer information disclosed in the specification of Chinese Patent Application Publication No. 107760690B) and Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA). The antibody genes were then isolated according to the following PCR procedure: In the first round of PCR, 5 μl of the reverse transcription reaction product, 5 units of Taq enzyme, 0.2 mM dNTP, and 0.5 μM of primers for each of the antibody subtype heavy chain and light chain quantification regions (sequences) were contained in a 50 μl system. The reaction conditions were: pre-denaturation at 95°C for 5 minutes, followed by 35 cycles of PCR with each cycle being 95°C × 30 seconds, 55°C × 60 seconds, 72°C × 90 seconds, and elongation at 72°C for 7 minutes. In the second round of PCR, 2.5 μl of the PCR product from the first round, 5 units of Taq Plus enzyme, 0.2 mM dNTP, and 0.5 μM of primers for each of the antibody subtype heavy chain and light chain variable regions were contained in a 50 μl system. The reaction conditions were: pre-denaturation at 95°C for 5 minutes, followed by 35 cycles of PCR with each cycle being 95°C × 30 seconds, 58°C × 60 seconds, 72°C × 90 seconds, and elongation at 72°C for 7 minutes. The obtained PCR products were identified by 1.2% agarose gel electrophoresis.

[0086] The PCR products of the antibody genes identified as positive and with the heavy chain and light chain capable of being paired were purified with a Qiagen PCR product purification kit, sequenced from the front and back respectively, and analyzed with the IMGT online server (http: / / imgt.cines.fr / ).

[0087] Example 3 Construction of Recombinant Antibodies for Expression The obtained PCR product of the antibody variable region gene was ligated into the pcDNA3.3 vector containing the human IgG1 constant region by using the TA cloning method to construct an expression vector for a fully human neutralizing antibody against varicella-zoster virus. Subsequently, the expression vector was transformed into DH5α competent bacteria for vector amplification, and the recombinant plasmid was extracted. HEK293 cells were co-transfected with the obtained recombinant plasmid and the transfection reagent PolyFect and cultured in an incubator at 37 °C and 8% CO 2 2. The expression vectors for the paired heavy and light chain genes were expressed intracellularly. After 96 hours of culture, the supernatant was collected. Cell debris was discarded by centrifugation, and the supernatant was purified by protein A affinity chromatography. The purified antibody was tested by SDS-PAGE. As shown in Figure 1, the results showed that the non-reduced bands of antibodies TRN1024, TRN1025, and TRN1026 were between 135 and 180 KD. Heavy and light chain bands were clearly observed after reduction, that is, the target antibody against varicella-zoster virus, namely the recombinant fully human neutralizing antibody, was observed.

[0088] Example 4 Detection of the binding activity of the recombinant antibody In this experiment, the recombinant antibody obtained from the previous example was tested by ELISA assay to determine the binding activity.

[0089] A 96-well ELISA plate was coated overnight at 4°C with 100 ng / well of the VZV gH / gL protein complex, and then blocked with a blocking solution for 2 hours at room temperature. Then, 100 μL of the HEK293 cell culture supernatant containing the recombinant antibody of the present invention and a negative control (the anti-rabies virus antibody TRN006, an antibody unrelated to VZV) was added to the 96-well plate and incubated at 37°C for 1 hour. 100 μL of goat anti-IgG-Fab-HRP diluted 1:10,000 with the blocking solution was added to each well and incubated at 37°C for 1 hour. After the reaction was completed, the OD value was detected and the results were calculated. And it was revealed that three fully human VZV monoclonal antibodies TRN1024, TRN1025, and TRN1026 capable of binding to the VZV gH / gL protein had a low EC50 of 0.002 μg / mL (shown in Figure 2), indicating that the antibodies obtained in the present invention were capable of specifically binding to the VZV gH / gL protein.

[0090] Example 5 Detection of the Affinity of the Recombinant Antibody The antibody of the present invention was measured by surface plasmon resonance technology (instrument, BIACORE 3000) for the determination of the KD value. The capture molecule was coupled to the CM5 chip to activate the dextran surface of the chip, and the coupling amount was determined according to the injection time. The prepared antibody was used as a ligand, and the calculated signal value was used to determine the injection concentration and contact time of the monoclonal antibody. The VZV gH / gL protein complex (CAMBRIDGEBIO, Cat No: 01-11-0045) used as an analyte was diluted with HBS-EP buffer, and then analytes at increasing concentrations were flowed through the chip, thereby each resulting in a signal curve. Each concentration was regarded as one cycle. After one cycle was completed, the chip was regenerated with 10 mmol / L glycine hydrochloride to restore the original state where no antigen was bound. BiaCore X-100 System software was used for the analysis. The specific procedure was as follows:

[0091] Anti-human IgG (Fc) was coupled to each of the two channels of the CM5 chip by utilizing amino coupling. For the captured TRN1024, TRN1025, and TRN1026, the concentration was 1 μg / mL and the binding time was 60 seconds. As shown in Figure 3, the minimum and maximum concentrations of the bound VZV gH / gL protein complex were 1.56 μg / mL and 200 μg / mL, respectively, the binding time was 90 seconds, and the dissociation time was 600 seconds. The regeneration solution was 3M MgCl 2 and the regeneration time was 30 seconds.

[0092] The association rate (ka), dissociation rate (kd), and equilibrium dissociation constant were calculated by fitting the association and dissociation sensorgrams simultaneously (Figure 3). The results are shown in the following table. All three antibodies, TRN1024, TRN1025, and TRN1026, had higher affinity, that is, the antibodies of the present invention can efficiently bind to the antigen.

Table 5

[0093] Example 6 Identification of in vitro neutralizing activity of recombinant antibodies Human fetal lung fibroblasts (MRC-5) were used to determine the neutralizing activity of the antibodies. According to the conventional method, the cells were detected by enzyme immunoassay after being infected with the VZV virus. The number of positive cells with signals was the number of infected cells, which could be regarded as the unit of the VZV virus used in the infection experiment. The titer of the VZV virus was determined by the method of gradient dilution spot count. For use in the neutralization experiment the next day, the infected MRC-5 cells as described above were digested by the conventional method and then seeded into a 96-well plate for conventional culture. On the day of the experiment, each of the antibodies TRN1024, TRN1025, and TRN1026 was serially diluted 2-fold with PBS solution starting from 100 μg / mL, for a total of 10 serial dilutions, and each of the antibody serial dilutions in PBS solution was added to a new 96-well plate at a concentration of 50 μL / well. Then, 50 μL of 100 CCID50 (50% cell culture infective dose) of the VZV Oka standard strain was added to each well, and neutralization was carried out at 37 °C for 1 hour. Then, the neutralization solution was added to the 96-well plate containing the MRC-5 cells cultured the previous day. 100 μL of virus medium was supplemented to each well, and the pathological state of the cells was observed daily.

[0094] The results showed that the antibodies of the present invention could clearly neutralize the activity of the VZV Oka standard strain and inhibit apoptosis of MRC-5 cells, while the control without addition of the antibodies could not neutralize the virus activity. After calculation, the neutralizing titers of the three antibodies TRN1024, TRN1025, and TRN1026 reached 1.56 μg / mL, indicating that the three antibodies could specifically recognize the cells infected with the VZV virus and play a role in neutralizing the virus and inhibiting virus transmission to the whole cells.

[0095] Example 7 Detection of anti-nuclear antibody resistance of recombinant antibodies Hep-2 cells are human laryngeal cancer epithelial cells. The international standard method for the detection of antinuclear antibodies is the indirect immunofluorescence method using Hep-2 cells as a substrate because of its rich antigen regions (about 100 - 150 types), strong antigen specificity, and high antigen content. In this example, the staining response of the antibody to Hep2 cells was detected by an immunofluorescence method that determines whether the antibody has an autoimmune response. An antinuclear antibody (ANA) detection kit (for 200 people) was used for the detection, and observations were made under a fluorescence microscope. The results are shown in Figure 4. The experimental groups and the negative control group of antibodies TRN1024, TRN1025, and TRN1026 (100 μg / ml) all showed no fluorescence (GFP) specific to Hep-2 cells, that is, the antibody did not bind to the antigen of Hep-2 cells, but obvious GFP fluorescence was observed in the positive control group. This indicates that the antibody of the present invention does not have an autoimmune response to Hep-2 cells.

[0096] Example 8 In Vitro Test for Neutralizing Viruses Derived from Clinical Samples Fluids were collected from the herpes of patients with varicella or herpes zoster virus, and the virus was isolated from the clinical samples (the isolation method is referred to in Liu J, Wang M, Gan L et al. Genotyping of Clinical Varicella-Zoster Virus Isolates Collected in China[J]. Journal of Clinical Microbiology, 2009, 47(5):1418-1423. / Liu J J, Wang M L, Gan L et al.[Seroepidemiology of varicella-zoster virus infection measured by the fluorescent antibody to membrane antigen test][J]. Zhonghua liu xing bing xue za zhi = Zhonghua liuxingbingxue zazhi, 2009, 30(4):371), and 10 VZV clinical isolates were isolated. These clinical isolates of VZV were subjected to virus culture to prepare cell-free virus (CFV). The in vitro neutralization ability of three antibodies, TRN1024, TRN1025, and TRN1026, was determined by the conventional plaque reduction neutralization test (gold standard), VariZIG (Cangene, immunoglobulin) was used as a positive control, and an irrelevant antibody TRN006 was used as a negative control. The results showed that all three antibodies, TRN1024, TRN1025, and TRN1026, could specifically recognize and neutralize these 10 innVZV clinical isolates, suggesting clinical significance.

[0097] Example 9 In Vivo Prevention Test In this example, the in vivo prophylactic functions of three antibody strains, TRN1024, TRN1025, and TRN1026, against VZV Oka strain virus infection were investigated. Four-week-old guinea pigs weighing 300 - 350 g were selected and assigned to a blank group, a control group (VariZIG), and an experimental group (TRN1026, 5 mg / kg). First, equal amounts of physiological saline, VariZIG, and TRN1026 were administered, and 1 day later, each animal was intravenously injected with 50 μL of PBMC infected with the VZV Oka virus strain (available from ATCC). The viral load in the blood was determined on days 1, 3, and 7 by blood sampling (the viral gene copy number of Wisel was detected). The results of the viral gene copy number showed that the experimental group and the control group exhibited significantly lower viral loads compared to the blank group, indicating that the low-dose antibody could protect guinea pigs against VZV virus challenge, that is, the recombinant anti-VZV antibody of the present invention has in vivo activity and a protective effect. 6

[0098] Example 10 Analysis of the identity of the variable region sequences of recombinant antibodies The applicant has discovered that, for the antibody sequences discovered in the present invention, one or more amino acid sequence modifications such as substitutions (such as conservative substitutions), insertions, or deletions introduced into the framework regions of the heavy chain variable region and / or the light chain variable region do not substantially affect the binding ability of the variable region to the antigen. In the antibody TRN1026 of the present application, the heavy chain variable region sequence (VH) is as shown in SEQ ID NO:23, and the light chain variable region sequence (VL) is as shown in SEQ ID NO:24. As an example, antibody derivatives generated by incorporating one or more amino acid sequence modifications such as substitutions (such as conservative substitutions), insertions, or deletions in the framework region of the variable region of antibody TRN1026 still retain the binding activity of the anti-VZV antibody. For example, an antibody variant derived from antibody TRN1026 having VH as shown in SEQ ID NO:25 and VL as shown in SEQ ID NO:26 was expressed, the culture supernatant was collected therefrom, and the binding activity was determined by ELISA. The results showed that the antibody variant derived from antibody TRN1026 still retained the binding activity to the VZV gH / gL protein complex and had a low EC50 of 0.05 μg / mL. It can be recognized that one or more amino acid sequence modifications such as substitutions (such as conservative substitutions), insertions, or deletions in the framework region of the antibody variable region do not affect the ability and function of the recombinant antibody.

Claims

**Claim 1** An isolated anti - varicella - zoster virus (VZV) monoclonal antibody and antigen - binding fragment thereof, comprising (i) three complementarity - determining regions CDR of the heavy - chain variable region contained in SEQ ID NO: 19, and three complementarity - determining regions CDR of the light - chain variable region contained in SEQ ID NO: 20, or (ii) three complementarity - determining regions CDR of the heavy - chain variable region contained in SEQ ID NO: 21, and three complementarity - determining regions CDR of the light - chain variable region contained in SEQ ID NO: 22, or (iii) three complementarity - determining regions CDR of the heavy - chain variable region contained in SEQ ID NO: 23, and three complementarity - determining regions CDR of the light - chain variable region contained in SEQ ID NO: 24, or (iv) three complementarity - determining regions CDR of the heavy - chain variable region contained in SEQ ID NO: 25, and three complementarity - determining regions CDR of the light - chain variable region contained in SEQ ID NO: 26, An isolated anti - varicella - zoster virus (VZV) monoclonal antibody and antigen - binding fragment thereof containing the same. **Claim 2** An isolated anti - VZV monoclonal antibody or antigen - binding fragment thereof containing a heavy - chain variable region and a light - chain variable region, comprising (i) the heavy - chain variable region contains CDR1 comprising the amino - acid sequence contained in SEQ ID NO: 7, CDR2 comprising the amino - acid sequence contained in SEQ ID NO: 8, and CDR3 comprising the amino - acid sequence contained in SEQ ID NO: 9, and the light - chain variable region contains CDR1 comprising the amino - acid sequence contained in SEQ ID NO: 10, CDR2 comprising the amino - acid sequence contained in SEQ ID NO: 11, and CDR3 comprising the amino - acid sequence contained in SEQ ID NO: 12; or (ii) the heavy - chain variable region contains CDR1 comprising the amino - acid sequence contained in SEQ ID NO: 1, CDR2 comprising the amino - acid sequence contained in SEQ ID NO: 2, and CDR3 comprising the amino - acid sequence contained in SEQ ID NO: 3, and the light - chain variable region contains CDR1 comprising the amino - acid sequence contained in SEQ ID NO: 4, CDR2 comprising the amino - acid sequence contained in SEQ ID NO: 5, and CDR3 comprising the amino - acid sequence contained in SEQ ID NO: 6; or (iii) the heavy chain variable region comprises CDR1 comprising the amino acid sequence included in SEQ ID NO: 13, CDR2 comprising the amino acid sequence included in SEQ ID NO: 14, and CDR3 comprising the amino acid sequence included in SEQ ID NO: 15; and the light chain variable region comprises CDR1 comprising the amino acid sequence included in SEQ ID NO: 16, CDR2 comprising the amino acid sequence included in SEQ ID NO: 17, and CDR3 comprising the amino acid sequence included in SEQ ID NO: 18; An isolated anti-VZV monoclonal antibody or an antigen-binding fragment thereof.

3. Comprising a heavy chain variable region and a light chain variable region, (i) the heavy chain variable region comprises the amino acid sequence included in SEQ ID NO: 21, and the light chain variable region comprises the amino acid sequence included in SEQ ID NO: 22; or (ii) the heavy chain variable region comprises the amino acid sequence included in SEQ ID NO: 19, and the light chain variable region comprises the amino acid sequence included in SEQ ID NO: 20; or (iii) the heavy chain variable region comprises the amino acid sequence included in SEQ ID NO: 23, and the light chain variable region comprises the amino acid sequence included in SEQ ID NO: 24; or (iv) the heavy chain variable region comprises the amino acid sequence included in SEQ ID NO: 25, and the light chain variable region comprises the amino acid sequence included in SEQ ID NO: 26, The isolated anti-VZV monoclonal antibody or an antigen-binding fragment thereof according to claim 1 or 2.

4. The isolated anti-VZV monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody is a human monoclonal antibody.

5. The antigen-binding fragment is selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab') 2 The isolated anti-VZV monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is selected from the group consisting of fragments.

6. Comprising heavy chain and light chain constant region sequences derived from a human antibody germline consensus sequence, wherein at least a part of the heavy chain and light chain constant region sequences are the human heavy chain and light chain consensus constant region sequences, the isolated anti-VZV monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5.

7. An isolated nucleic acid encoding the isolated anti-VZV monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6.

8. A vector comprising the nucleic acid according to claim 7.

9. A host cell comprising the nucleic acid according to claim 7 or the vector according to claim 8.

10. A method for preparing an anti-VZV monoclonal antibody or an antigen-binding fragment thereof, which comprises culturing the host cell according to claim 9 under conditions suitable for expressing the nucleic acid encoding the anti-VZV or its antigen-binding fragment according to any one of claims 1 to 6.

11. An anti-VZV monoclonal antibody or an antigen-binding fragment thereof prepared by the method according to claim 10.

12. A pharmaceutical composition comprising the anti-VZV monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6 and 11.

13. The anti-VZV monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6 and 11 for the preparation of a medicament or agent for the detection, treatment, prevention and / or alleviation of varicella-zoster virus infection or varicella-zoster virus disease.

14. The pharmaceutical composition according to claim 12 for treating a human individual infected with varicella-zoster virus.

15. The pharmaceutical composition according to claim 12 for enhancing the resistance of a human individual to varicella-zoster virus infection.

16. The pharmaceutical composition according to claim 14 or 15, wherein the individual is immunocompromised.

17. The pharmaceutical composition according to claim 14 or 15, wherein the individual is a neonate, a premature infant, a woman in labor, and an immunocompromised subject receiving immunosuppressants, cytotoxic agents or radiotherapy other than organ transplantation surgery, hematological malignancies, malignancies, nephrotic syndrome and the like.

18. The pharmaceutical composition according to claim 12 for neutralizing varicella-zoster virus in a subject.

19. An in vitro method for neutralizing varicella-zoster virus in a sample, which comprises adding an appropriate amount of the anti-VZV monoclonal antibody or its antigen-binding site according to any one of claims 1 to 6 and 11 to the sample.

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