Treatment for shingles

Abstract

Antibodies targeting VZV glycoprotein E (gE) bind recombinant gE with high affinity, bind to gE on the surface of VZV-infected cells, interact with immune system effectors (e.g., Fc receptors), mediate antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), and prevent VZV from infecting cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS Not applicable.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH Not applicable.

[0003] The present invention relates generally to the fields of medicine, virology, immunology, and antibodies (Abs). [Background technology]

[0004] Varicella-zoster virus (VZV) is a human alphaherpesvirus that infects over 90% of people worldwide. VZV causes chickenpox (varicella), primarily in children and young adults. In chickenpox, the virus targets the skin and peripheral nerves, causing a rash characterized by pustular vesicles that rupture and form scabs before healing. After the clinical symptoms of chickenpox subside, VZV remains latent in the trigeminal nerve and dorsal root ganglion of infected individuals. Later in life, in about one in three infected people, VZV reactivates and causes shingles (herpes zoster or "HZ"), a disease that causes burning or shooting pain, tingling or itching, chills, fever, headache, upset stomach, and a rash or blisters on one side of the body, usually around the face and lower back.

[0005] Most cases of shingles last 3–5 weeks, but approximately 9–19% of all shingles patients progress to postherpetic neuralgia (PHN). The incidence of PHN increases with age; the risk of PHN is approximately 2% in patients under 50 years of age, approximately 20% in patients over 50 years of age, and approximately 35% in patients over 80 years of age. Pain from PHN can significantly impact quality of life. A global research initiative involving 130 studies in 26 countries showed a temporary increase in the incidence of shingles in seven countries over the past few decades, with more than 30% of patients with PNH experiencing pain for more than one year. The incidence of shingles is up to 15 times higher in HIV-infected patients than in uninfected individuals, and 25% of patients with Hodgkin lymphoma develop shingles.

[0006] Currently, there is no cure for shingles. An ideal treatment for shingles would address three main objectives: (1) treating the acute viral infection, (2) treating the acute pain associated with shingles, and (3) preventing postherpetic neuralgia. Currently, antiviral drugs such as acyclovir, valacyclovir, and famciclovir are prescribed along with painkillers, which may reduce the duration and severity of the disease in some patients, but these three objectives are not achieved. Summary of the Invention

[0007] Described herein is the development of novel antibody (Ab)-based therapies for varicella zoster. These therapies utilize antibodies targeting VZV glycoprotein E (gE), the most abundant and immunogenic glycoprotein on the surface of VZV. The anti-gE antibodies described herein bind recombinant gE with high affinity, bind to gE on the surface of VZV-infected cells, interact with immune system effectors (e.g., Fc receptors), and mediate antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) of cells expressing gE on their surface, preventing VZV cell infection.

[0008] Prior to the studies described herein, it was unclear whether anti-gE antibodies could mediate ADCC, ADCP, and prevent VZV cell infection, because gE itself neutralizes the function of antiviral IgG. In alphaherpesviruses such as VZV, gE heterodimerizes with glycoprotein I (gI) to form a functional membrane Fc receptor. Experiments with a related alphaherpesvirus, herpes simplex virus (HSV), suggested that gE / gI binds to anti-HSV IgG in a manner that allows the Fc domain of anti-HSV IgG to bind gE, thereby preventing antibody- and complement-mediated neutralization on the surface of virions and virus-infected cells.

[0009] In some embodiments, an anti-gE antibody can be combined with other agents to treat shingles. By way of example, an anti-gE antibody can be administered along with an agent that inhibits or reduces IL-1alpha activity (e.g., an anti-IL-1alpha antibody) to a subject with a VZV infection (e.g., shingles), where the anti-gE antibody treats the infection and the agent that inhibits or reduces IL-1alpha activity reduces neuropathic pain behavior and aberrant neural activity associated with shingles.

[0010] Thus, described herein are pharmaceutical compositions comprising purified antibodies comprising antigen-binding variable regions that exhibit very high binding affinity for VZV glycoprotein E (gE). Such antibodies may be monoclonal antibodies ("mAbs") and may have a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1 (or its CDRs) and a light chain comprising the amino acid sequence of SEQ ID NO:2 (or its CDRs). In some embodiments, the antibody comprises a heavy chain variable region amino acid sequence having CDRs of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, and a light chain variable region amino acid sequence having CDRs of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. The antibody may be an IgG, such as an IgG1.

[0011] Also described herein is a set of isolated nucleic acids comprising a first nucleic acid encoding a heavy chain of a mAb that specifically binds to gE and a second nucleic acid encoding a light chain of a mAb that specifically binds to gE, wherein the first nucleic acid can encode the amino acid sequence of SEQ ID NO: 1 (or its CDRs), and the second nucleic acid can encode the amino acid sequence of SEQ ID NO: 2 (or its CDRs).

[0012] In another aspect, described herein is an expression vector comprising both a nucleic acid encoding the amino acid sequence of SEQ ID NO: 1 (or a CDR thereof) and a nucleic acid encoding the amino acid sequence of SEQ ID NO: 2 (or a CDR thereof). Also described herein is a set of expression vectors comprising a first expression vector encoding the amino acid sequence of SEQ ID NO: 1 (or a CDR thereof) and a second expression vector encoding the amino acid sequence of SEQ ID NO: 2 (or a CDR thereof).

[0013] Further described herein is an isolated host cell (e.g., a mammalian cell such as a CHO cell) comprising a nucleic acid encoding the amino acid sequence of SEQ ID NO:1 (or a CDR thereof) and a nucleic acid encoding the amino acid sequence of SEQ ID NO:2 (or a CDR thereof).

[0014] Also described herein is the use of the above-described antibody for the treatment of shingles. A method for treating shingles in a mammal comprises administering to the mammalian subject a therapeutically effective amount of a purified antibody comprising an antigen-binding variable region that exhibits very high binding affinity for gE. Such a method may also comprise administering to the mammalian subject an agent that inhibits the function of IL-1 alpha, such as an anti-IL-1 alpha antibody, a protein that binds to the extracellular portion of the interleukin-1 receptor component (IL-1R1), or a protein that binds to the IL-1 receptor accessory protein (IL-1RAcP).

[0015] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Commonly understood definitions of biological terms can be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994.

[0016] As used herein, the word "a" or "an" before a noun refers to one or more of that particular noun. For example, the phrase "an antibody" refers to "one or more antibodies."

[0017] The term "antibody" or "Ab" refers to any immunoglobulin (e.g., a human, rodent, cartilaginous fish, or camelid antibody) or conjugate thereof that specifically binds to an antigen (e.g., gE). A wide variety of antibodies are known to those of skill in the art. Non-limiting examples of antibodies include monoclonal antibodies (including, e.g., full-length antibodies), antigen-binding fragments of antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies (e.g., single-domain antibodies, camelid antibodies, and cartilaginous fish antibodies), chimeric (e.g., humanized) antibodies, and fully human antibodies (including those that can be found in or derived from humans (i.e., truly human antibodies)). The term antibody also includes antibody conjugates (e.g., antibodies conjugated to a stabilizing protein, a label, or a therapeutic agent (e.g., any of the therapeutic agents described herein or known in the art)).

[0018] The term "antigen-binding fragment" refers to any portion of a full-length antibody that contains at least one variable domain (e.g., a mammalian (e.g., human, mouse, rat, rabbit, or goat) heavy or light chain immunoglobulin variable domain, a camelid variable antigen-binding domain (VHH), or a cartilaginous fish immunoglobulin novel antigen receptor (Ig-NAR) domain) capable of specifically binding to an antigen. For example, an antigen-binding fragment described herein can include at least a portion of an antibody Fc region sufficient to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) in a mammal (e.g., a human) and / or linked to a therapeutic agent (e.g., any of the therapeutic agents described herein or known in the art). As another example, an antigen-binding fragment described herein can include at least a portion of an antibody Fc region that does not mediate ADCC and / or CDC in a mammal (e.g., a human). Non-limiting examples of antibody fragments include Fab, Fab', F(ab'), Fv fragments, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments. Additional antibody fragments comprising at least one camelid VHH domain or at least one cartilaginous fish Ig-NAR domain include minibodies, microbodies, sub-nanobodies, nanobodies, and any of the other forms of antibodies described in U.S. Patent Application Publication No. 2010 / 0092470.

[0019] The term "human antibody" refers to an antibody encoded by nucleic acid present in the human genome (e.g., a rearranged human immunoglobulin heavy or light chain locus). In some embodiments, a human antibody is produced in mammalian (e.g., human) cell culture (e.g., a Chinese hamster ovary cell line). In some embodiments, a human antibody is produced in non-human cells (e.g., a mouse or hamster cell line). In some embodiments, a human antibody is produced in bacterial cells or yeast cells.

[0020] The term "single-chain antibody" refers to a single polypeptide comprising at least one variable binding domain capable of specifically binding to an antigen. Non-limiting examples of single-chain antibodies are described herein and are known in the art (see, e.g., the antibodies described in U.S. Patent Application Publication No. 2010 / 0092470).

[0021] An antibody or antigen-binding fragment thereof "specifically binds" or "binds specifically" to a particular antigen, e.g., gE (via an epitope described herein for a full-length antibody including the incorporation of a light chain variable region and a heavy chain variable region) if it binds to the antigen but recognizes and binds to a lesser extent (e.g., does not recognize and bind) other molecules in a sample. In some embodiments, an antibody or antigen-binding fragment thereof is measured at 1×10 -10 M or less (e.g., 1×10 -11 Less than M or 1 x 10 -12 Selectively bind to the epitope with an affinity (KD) (e.g., as determined by surface plasmon resonance) of less than M. The ability of an antibody or antigen-binding fragment to specifically bind to a protein epitope may be determined using any of the methods known in the art or described herein.

[0022] The term "complementarity-determining region" or "CDR" refers to a region within an Ig (heavy or light chain Ig) that forms part of the antigen-binding site in an antibody or antigen-binding fragment thereof. As is known in the art, a heavy chain Ig contains three CDRs (CDR1, CDR2, and CDR3, respectively), and a light chain Ig contains three CDRs (CDR1, CDR2, and CDR3, respectively). In any antibody or antigen-binding fragment thereof, the three CDRs of the heavy chain Ig and the three CDRs of the light chain Ig together form the antigen-binding site in the antibody or antigen-binding fragment thereof. The Kabat database is one system used in the art for numbering the CDR sequences present in a light or heavy chain Ig.

[0023] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All applications and publications mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Additionally, the specific embodiments discussed below are merely illustrative and not intended to be limiting. [Brief explanation of the drawings]

[0024] [Figure 1] Figure 1 shows two graphs demonstrating that XB22-14 specifically binds to gE on the plasma membrane of VZV-infected ARPE19 cells. Varicella-zoster virus-infected ARPE19 cells (A) and uninfected ARPE19 cells (B) were fixed with 10% neutral-buffered formalin, washed with D-PBS, and then surface-stained with antibodies. For primary staining, cells were stained with 10 μg / mL biotinylated VZ27 (blue peak) or 10 μg / mL biotinylated IgG1κ isotype control antibody (orange peak) for 20 minutes at 4°C. Cells were washed twice with FACS buffer. For secondary staining, cells with or without primary antibody staining (green peak) were incubated with streptavidin-APC (Biolegend, 0.2 mg / mL) diluted 1:200 in FACS buffer. At the end of the 20 min incubation, cells were washed twice with FACS buffer and analyzed on a BD Accuri flow cytometer. [Figure 2] Figure 2 shows a series of representative Octet BLI sensorgrams and 2:1 heterogeneous fitting curves of XB22-14 antibody binding to various receptors. The top two panels show representative Octet BLI sensorgrams and 2:1 heterogeneous fitting curves of XB22-14 antibody binding to the FcRn receptor at pH 6.0 and 7.2. The bottom six panels show representative Octet BLI sensorgrams and 2:1 heterogeneous fitting curves of XB22-14 antibody binding to the CD64, CD32a, CD32b, CD16a, CD16b, and CD89 receptors. [Figure 3]FIG. 3 is a graph showing that XB22-14 can mediate antibody-dependent cellular cytotoxicity (ADCC) of cells expressing gE on their surface. [Figure 4] FIG. 4 is a graph showing that XB22-14 can mediate antibody-dependent cellular phagocytosis (ADCP) of cells expressing gE on their surface. [Figure 5] 5A-5D are a series of photomicrographs and graphs showing that XB22-14 can prevent VZV infection of MeWo cells. [Figure 6] 6A-6B are a series of photomicrographs and graphs showing that XB22-14 can prevent VZV infection of ARPE-19 cells. DETAILED DESCRIPTION OF THE INVENTION

[0025] Described herein are compositions and methods for treating shingles with antibodies comprising an antigen-binding variable region that exhibits very high binding affinity for gE. The preferred embodiments described below exemplify the application of these compositions and methods. Nevertheless, from the description of these embodiments, other aspects of the invention can be made and / or practiced based on the description provided below.

[0026] General Methodology Methods using conventional immunological and molecular biology techniques are described herein. Immunological methods (e.g., assays for detecting and localizing antigen-antibody complexes, immunoprecipitation, immunoblotting, etc.) are generally known in the art and are described in methodological treatises such as *Current Protocols in Immunology*, Coligan et al., ed., John Wiley & Sons, New York. Molecular biology techniques are described in detail in *Molecular Cloning: A Laboratory Manual*, 2nd ed., vols. 1-3, Sambrook et al., ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; and *Current Protocols in Molecular Biology*, Ausubel et al., ed., Greene Publishing and Wiley-Interscience, New York. Antibody methods are described in *Handbook of Therapeutic Abs*, Dubel, S., ed., Wiley-VCH, 2007. Cell culture techniques are generally known in the art and are described in detail in methodological treatises such as "Culture of Animal Cells: A Manual of Basic Technique, 4th edition, by R Ian Freshney, Wiley-Liss, Hoboken, NJ, 2000; and "General Techniques of Cell Culture," by Maureen A Harrison and Ian F Rae, Cambridge University Press, Cambridge, UK, 1994. Protein purification methods are discussed in "Guide to Protein Purification: Methods in Enzymology," Vol. 182, Deutscher MP, ed., Academic Press, San Diego, Calif., 1990.

[0027] Treatment and prevention of VZV infection The compositions and methods described herein are useful for treating or preventing VZV infection. Antibodies such as XB22-14 that bind with high affinity to gE (e.g., on the cell membrane of VZV-infected cells) can neutralize gE on the cell surface and mediate antibody-dependent cellular cytotoxicity (ADCC) of cells expressing gE on their surface and antibody-dependent cellular phagocytosis (ADCP) of cells expressing gE on their surface (e.g., when FcγRIIa is present on the effector cell surface). Furthermore, such antibodies can also bind to VZV virions and mediate ADCP to kill circulating virus. Such antibodies can also bind to gE on the virus, neutralizing the virus and preventing cell membrane fusion and invasion of skin cells. Therefore, the anti-gE antibodies described herein can be administered to a mammalian subject in an amount effective to prevent or treat VZV infection, such as shingles.

[0028] The mammalian subject may be any animal suffering from or at risk of developing VZV infection or VZV reactivation, including humans. Human subjects may be male, female, adult, child, elderly (over 65 years of age), or those with other diseases, particularly those with weakened immune systems. Particularly preferred subjects include those with chickenpox, those with latent VZV infection, those with shingles, those suffering from postherpetic neuralgia, those suffering from ophthalmic shingles, those infected with HIV, and those diagnosed with Hodgkin's lymphoma. When the anti-gE antibody is a truly human antibody (e.g., an antibody having all V regions naturally expressed in a human subject), such as XB22-14, the subject is preferably one who has developed a human anti-human antibody response following previous administration of a therapeutic antibody.

[0029] Antibodies and other agents targeting gE The compositions, methods, and uses described herein may utilize (i) mAbs comprising an antigen-binding variable region that exhibits very high binding affinity to gE. An anti-gE mAb may, for example, comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 (or its CDRs) and a light chain comprising the amino acid sequence of SEQ ID NO: 2 (or its CDRs). The light chain variable region and heavy chain variable region described herein (which together form Fab) can be conjugated to Fc or a portion thereof using conventional molecular biology techniques to fuse the desired Fc portion to the Fab or antigen-binding fragment. In this manner, full-length immunoglobulins such as human IgG1 (e.g., IgG1a or IgG1b), IgG2 (e.g., IgG2a or IgG2b), IgG3 (e.g., IgG3a or IgG3b), IgG4 (e.g., IgG4a or IgG4b), IgD, IgA (e.g., IgA1 and IgA2), IgE, or IgM (e.g., dimers, pentamers, and hexamers) (and different allotypes of the above) incorporating the light chain variable regions and heavy chain variable regions described herein can be produced. Generally, IgG is preferred, with IgG1 and IgG3 being more preferred, as they are believed to be more effective in neutralizing VZV.

[0030] Any suitable type of antibody that specifically binds to gE and reduces or prevents the development of characteristics of VZV infection in a subject can be used in the methods described herein. For example, the anti-gE antibody can be a mAb, a polyclonal antibody, a mixture of mAbs, or an antibody fragment or engineered antibody-like molecule, such as an scFv. The K of the antibody is preferably at least 1 x 10 9 M -1 or more (e.g., 9×10 10 M -1 , 8×10 10 M -1 , 7×10 10 M -1 , 6×10 10 M -1 , 5×10 10 M -1 , 4×10 10 M -1 , 3×10 10 M-1 , 2 × 10 10 M -1 , or 1×10 10 M -1 In a preferred embodiment, the antibody is a fully human mAb comprising (i) an antigen-binding variable region that exhibits very high binding affinity for gE (e.g., at least nanomolar or picomolar), and (ii) a constant region. The human antibody is preferably IgG1, but may be of a different isotype, such as IgM, IgA, or IgE, or a subclass, such as IgG2, IgG3, or IgG4. One particularly useful mAb is XB22-14. Other useful mAbs include those that comprise at least one (preferably all) of the CDRs of XB22-14, those that neutralize gE, and those that compete with XB22-14 for binding to gE (e.g., in a competitive ligand-receptor interaction assay).

[0031] The currently preferred method for producing mAbs is to first isolate such B lymphocytes from a subject and then immortalize them so that they can replicate continuously in culture. Subjects lacking large numbers of naturally occurring B lymphocytes expressing Igs specific for gE may be immunized with one or more gE antigens to expand the number of such B lymphocytes. Human mAbs are prepared by immortalizing human antibody-secreting cells (e.g., human plasma cells). See, e.g., U.S. Patent No. 4,634,664.

[0032] In an exemplary method, one or more (e.g., 5, 10, 25, 50, 100, 1000, or more) human subjects are screened for the presence of such gE-specific antibodies in their blood. Subjects expressing the desired antibodies can then be used as B-lymphocyte donors. In one possible method, peripheral blood is collected from a human donor whose B-lymphocytes express gE-specific antibodies. Such B-lymphocytes are then isolated from the blood sample, for example, by cell sorting (e.g., fluorescence-activated cell sorting (FACS) or magnetic bead cell sorting), and B-lymphocytes expressing gE-specific Igs are selected. These cells can then be immortalized by viral transformation (e.g., using EBV) or by fusion with another immortalized cell, such as a human myeloma, by known techniques. B lymphocytes expressing gE-specific Ig within this population can then be isolated by limiting dilution (e.g., cells positive for gE-specific Ig in the wells of a microtiter plate are selected and subcultured, and the process is repeated until the desired clonal line can be isolated). See, for example, Goding, MAbs: Principles and Practice, pp. 59-103, Academic Press, 1986. Clonal cell lines expressing Ig with at least nanomolar or picomolar binding affinity for gE are preferred. mAbs secreted by these clonal cell lines can be purified from culture medium or body fluids (e.g., ascites) by conventional Ig purification procedures such as salt precipitation, size exclusion, ion exchange separation, and affinity chromatography.

[0033] Although immortalized B lymphocytes can be used in in vitro culture to produce mAbs directly, in certain cases it may be desirable to produce mAbs using heterologous expression systems. See, e.g., the methods described in U.S. Patent Application No. 11 / 754,899. For example, genes encoding mAbs specific for gE can be cloned and introduced into expression vectors (e.g., plasmid-based expression vectors) for expression in heterologous host cells (e.g., CHO cells, COS cells, myeloma cells, and E. coli cells). Because Ig contains a heavy (H) and a light (L) chain in an H2L2 configuration, the genes encoding each can be isolated separately and expressed in different vectors.

[0034] Although generally less preferred due to a higher likelihood of the subject generating an anti-antibody response, chimeric mAbs (e.g., "humanized" mAbs), which are antigen-binding molecules having different portions derived from different animal species (e.g., a variable region of a mouse Ig fused to a constant region of a human Ig), may also be used in the methods described herein. Such chimeric antibodies can be prepared by methods known in the art. See, e.g., Morrison et al., Proc. Nat'l. Acad. Sci. USA, 81:6851, 1984; Neuberger et al., Nature, 312:604, 1984; Takeda et al., Nature, 314:452, 1984. Similarly, antibodies can be humanized by methods known in the art. For example, mAbs with the desired binding specificity can be humanized by various suppliers or as described in U.S. Pat. Nos. 5,693,762, 5,530,101, or 5,585,089.

[0035] The mAbs described herein may be affinity matured to enhance or alter their binding specificity by known methods, such as VH and VL domain shuffling (Marks et al. Bio / Technology 10:779-783, 1992), random mutagenesis of hypervariable region (HVR) and / or framework residues (Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813, 1994; Schier et al. Gene 169:147-155, 1995; Yelton et al. J. Immunol. 155:1994-2004, 1995; Jackson et al., J. Immunol. 154(7):3310-9, 1995; and Hawkins et al., J. Mol. Biol. 226:889-896, 1992). Amino acid sequence variants of an antibody can be generated by introducing appropriate changes into the nucleotide sequence encoding the antibody. Furthermore, modifications of the nucleic acid sequence encoding the mAb (e.g., without altering the amino acid sequence of the mAb) can be altered (e.g., intron removal and / or codon optimization for a given expression system) to enhance production of the mAb in a particular expression system. The mAbs described herein can also be modified by conjugation with another protein (e.g., another mAb) or a non-protein molecule. For example, the mAb can be conjugated to a water-soluble polymer such as polyethylene glycol or carbon nanotubes (see, e.g., Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605, 2005). See U.S. Patent Application No. 11 / 754,899.

[0036] Amino acid mutations may be introduced into the constant regions of these IgG subclasses. Examples of amino acid mutations that can be introduced include those that enhance binding to FcK receptors (e.g., those described in Proc. Natl. Acad. Sci. USA 103(11):4005-4010, 2006; MAbs 1(6):572-579, 2009; US2010 / 0196362; US2013 / 0108623; US2014 / 0171623; US2014 / 0093496; and US2014 / 0093959), or those that enhance or reduce binding to FcRn (e.g., those described in J. Biol. Chem. 276(9):6591-6604, 2001; Int Immunol. 18(12):1759-1769, 2006; and J. Biol. Chem. 281(33):23514-23524, 2006).

[0037] Two types of H chains can be heterogeneously linked to generate bispecific antibodies. Knobs-into-holes technology (e.g., as described in J. Immunol. Methods 248(1-2):7-15, 2001 and J. Biol. Chem. 285(27):20850-20859, 2010), electrostatic repulsion technology (e.g., as described in WO 06 / 106905), SEEDbody technology (e.g., as described in Protein Eng. Des. Sel. 23(4):195-202, 2010), and the like can be used to heterogeneously link two types of H chains via the CH3 domain. Any of the antibodies described herein may have modified or deleted glycosylation. Examples of antibodies with modified glycosylation include glycosylated antibodies (e.g., as described in WO 99 / 54342), antibodies with defucosylated glycosylation (e.g., as described in WO 00 / 61739, WO 02 / 31140, WO 06 / 067847, and WO 06 / 067913), and antibodies with glycosylation containing a bisecting GlcNAc (e.g., as described in WO 02 / 79255). Methods for producing IgG antibodies lacking glycosylation include introducing a mutation into asparagine at EU numbering position 297 of the heavy chain (J. Clin. Pharmacol. 50(5):494-506, 2010) and producing IgG using Escherichia coli (J. Immunol. Methods 263(1-2):133-147, 2002 and J. Biol. Chem. 285(27):20850-20859, 2010). Furthermore, heterogeneity associated with deletion of the C-terminal lysine of IgG and heterogeneity associated with mispairing of disulfide bonds in the hinge region of IgG2 can be reduced by introducing amino acid deletions / substitutions (for example, as described in WO 09 / 041613). Any of the antibodies or antigen-binding fragments described herein contain at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acids (e.g., added, inserted, or substituted amino acids not within a CDR) that are not present in the corresponding human antibody.Any of the antibodies or antigen-binding fragments described herein may also have at least one amino acid deleted (e.g., compared to the corresponding human antibody), such as a deletion from the N-terminus or C-terminus of the light or heavy chain, or a deletion of an amino acid from a constant domain (e.g., the Fc domain).

[0038] Preferably, to ensure that high-titer gE-specific mAbs can be administered to subjects with minimal side effects, the mAb compositions of the present invention are at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.9 percent pure by weight (excluding any excipients). The mAb compositions of the present invention may contain only a single type of mAb (i.e., generated from a single clonal B lymphocyte line). In addition to anti-gE mAbs, the Ab compositions of the present invention may also contain other mAbs that specifically bind to antigens other than gE (e.g., human IL-1 alpha).

[0039] The mAb may be conjugated to another molecule, such as a cytotoxin or a detectable label, to modify or enhance its function. To more effectively kill cells expressing gE, the gE-specific mAb may be conjugated to one or more cytotoxins. The cytotoxin used in the present invention can be any cytotoxic agent (e.g., a molecule capable of killing cells after contacting them) that can be conjugated to a human gE-specific mAb. Examples of cytotoxins include radionuclides (e.g., 35 S, 14 C. 32 P, 125 I, 131 I, 90 Y, 89 Zr, 201 Tl, 186 Re, 188 Re, 57 Cu, 213 Bi, and 211Examples of cytotoxins include, but are not limited to, antimetabolites (e.g., 5-fluorouricil (5-FU), methotrexate (MTX), fludarabine, etc.), microtubule inhibitors (e.g., vincristine, vinblastine, colchicine, taxanes (paclitaxel and docetaxel, etc.)), alkylating agents (e.g., cyclophosphamide, melphalan, bischloroethylnitrosourea (BCNU), etc.), platinum agents (e.g., cisplatin (also called cDDP), carboplatin, oxaliplatin, JM- 216, CI-973, etc.), anthracyclines (e.g., doxorubicin, daunorubicin, etc.), antibiotics (e.g., mitomycin C), topoisomerase inhibitors (e.g., etoposide, tenoposide, and camptothecin), or other cytotoxic agents such as ricin, diphtheria toxin (DT), Pseudomonas exotoxin (PE) A, PE40, abrin, saporin, pokeweed virus protein, ethidium bromide, glucocorticoids, anthrax toxin, etc. See, e.g., U.S. Patent No. 5,932,188.

[0040] The gE-specific mAb can also be conjugated to a detectable label. Detectable labels useful in the present invention include biotin or streptavidin, magnetic beads, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, green fluorescent protein, etc.), radioactive labels (e.g., 3 H, 125 I, 35 S, 14 C. 32 P, 111 In, 97 Ru, 67 Ga, 68 Ga, or 72Examples of suitable labels include radioactive materials such as radioactive dyes (e.g., As), radiopaque materials such as metals for radioimaging, paramagnetic agents for magnetic resonance imaging, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Means for detecting such labels are well known to those skilled in the art. Thus, for example, radioactive labels may be detected using photographic film or scintillation counters. Fluorescent markers may also be used and can be detected using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, while colorimetric labels are detected by simply visualizing the colorimetric label.

[0041] The present invention also encompasses nucleic acid molecules encoding mAbs specific for gE. The same nucleic acid molecule may encode both the heavy and light chains of a gE-specific mAb, or a set of two different nucleic acid molecules, one encoding the heavy chain and the other encoding the light chain, may be used. Any other suitable nucleic acid encoding the amino acid sequence of the mAbs described herein may also be used.

[0042] For the production of mAbs, nucleic acid molecules encoding the heavy and light chains may be incorporated into an expression vector in an orientation such that such nucleic acid molecules are operably linked to expression control sequences, such as transcriptional and translational control sequences. Examples of expression vectors include plasmid-derived vectors and vectors derived from viruses, such as adenoviruses, adeno-associated viruses, and retroviruses. The nucleic acid molecules encoding the light and heavy chains may be incorporated into a single vector or different vectors. The vectors of the invention may also include regulatory sequences, such as promoters and / or enhancers (see U.S. Pat. Nos. 5,168,062, 4,510,245, and 4,968,615), selectable markers, or sequences encoding affinity tags (to facilitate purification) or detectable labels.

[0043] To produce mAbs, the vectors of the invention can be introduced into suitable host cells, e.g., prokaryotic cells such as bacteria, or preferably eukaryotic cells such as mammalian, plant, or yeast host cells. Exemplary methods for introducing heterologous polynucleotides into host cells include the use of viral vectors, electroporation, encapsulation of polynucleotides in liposomes, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, Agrobacterium-mediated transformation, biolistic transformation, and direct microinjection of DNA into the nucleus. Mammalian cell lines are currently preferred for expression of mAbs from vectors. Examples of mammalian host cells include Chinese hamster ovary (CHO) cells (e.g., the DG44 CHO cell line or the CHO-K1 cell line), HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), NSO cells, SP2 cells, HEK-293T cells, 293 Freestyle cells, and NIH-3T3 cells. mAbs may also be expressed in transgenic animals or plants. See, e.g., U.S. Patent Nos. 5,827,690, 5,756,687, 5,750,172, 5,741,957, 6,046,037, and 5,959,177.

[0044] Pharmaceutical Compositions and Methods Anti-gE antibody compositions (and other agents that specifically target gE) may be administered to animals or humans in a pharmaceutically acceptable carrier (e.g., sterile saline), selected based on the mode and route of administration and standard pharmaceutical practice. Lists of pharmaceutically acceptable carriers and pharmaceutical formulations can be found in Remington's Pharmaceutical Sciences and USP / NF, standard texts in the field. Other substances may be added to the composition, and other steps may be taken, to stabilize and / or preserve the composition and / or to facilitate administration to a subject.

[0045] For example, the antibody composition may be lyophilized (see Draber et al., J. Immunol. Methods. 181:37, 1995 and PCT / US90 / 01383), dissolved in a solution containing sodium and chloride ions, dissolved in a solution containing one or more stabilizers such as albumin, glucose, maltose, sucrose, sorbitol, polyethylene glycol, and glycine, filtered (e.g., using a 0.45 micron and / or 0.2 micron filter), contacted with beta-propiolactone, and / or dissolved in a solution containing a disinfectant (e.g., a surfactant, an organic solvent, and a mixture of a surfactant and an organic solvent).

[0046] The antibody compositions may be administered to animals or humans by any suitable technique. Typically, such administration is parenteral (e.g., intravenous, subcutaneous, intramuscular, or intraperitoneal introduction). The compositions may also be administered directly to the target site (e.g., skin), for example, by topical application. Other delivery methods, such as liposomal delivery or diffusion from a device impregnated with the composition, are known in the art. The compositions may be administered by a single bolus, multiple injections, or continuous infusion (e.g., by intravenous or peritoneal dialysis).

[0047] A therapeutically effective amount is an amount capable of producing a medically desirable result in the treated animal or human. An effective amount of an anti-gE antibody composition is an amount that shows clinical efficacy in a patient as measured by the amelioration or prevention of one or more symptoms of VZV infection. As is well known in the medical field, the dosage administered per animal or human depends on many factors, including the subject's size, body surface area, age, the specific composition administered, sex, time and route of administration, general health, and other drugs administered concomitantly. A preferred dosage range is about 3-20 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) mg / kg body weight. In other cases, a single dose may be effective to eliminate symptoms / prevent the onset of VZV infection. In other cases, the dose may be given repeatedly, for example, every other week, every week, every two weeks, every three weeks, every half month, once every three weeks, monthly, every two months, or as needed (in the event of a recurrence of symptoms of VZV infection or to prevent the recurrence of VZV symptoms that have resolved (e.g., an episode of shingles after chickenpox)).

[0048] The antibodies and antigen-binding fragments described herein can be formulated as pharmaceutical compositions comprising the antibodies and antigen-binding fragments and at least one pharmaceutically acceptable carrier (e.g., a non-natural pharmaceutically acceptable carrier). Non-limiting examples of pharmaceutically acceptable carriers include sterile water, saline, stabilizers, excipients, antioxidants (e.g., ascorbic acid), buffers (e.g., phosphate, citrate, histidine, and other organic acids), preservatives, surfactants (e.g., PEG and Tween), chelating agents (e.g., EDTA or EGTA), and binders. Additional examples of pharmaceutically acceptable carriers include low molecular weight polypeptides, proteins (e.g., serum albumin and gelatin), amino acids (e.g., glycine, glutamine, asparagine, glutamic acid, aspartic acid, methionine, arginine, and lysine), sugars and carbohydrates (e.g., polysaccharides and monosaccharides), and sugar alcohols (e.g., mannitol and sorbitol). When preparing an injectable aqueous solution, isotonic solutions containing saline and other auxiliary agents such as glucose, D-sorbitol, D-mannose, D-mannitol, and sodium chloride may be used, and if necessary, may be used in combination with suitable solubilizing agents such as alcohol (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol and PEG), and nonionic surfactants (e.g., polysorbate 80, polysorbate 20, poloxamer 188, and HCO-50). By incorporating hyaluronidase into the formulation, larger volumes of liquid can be administered subcutaneously (see, for example, Expert. Opin. Drug. Deliv. 4(4):427-440, 2007).

[0049] The antibodies and antigen-binding fragments provided herein may be encapsulated, for example, in microcapsules (e.g., made from hydroxymethylcellulose, gelatin, and poly(methyl methacrylate)) or incorporated as components of colloid drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) (see, e.g., "Remington's Pharmaceutical Science 16th edition," Oslo Ed. (1980)). Methods for preparing pharmaceutical compositions as controlled-release medicaments are also well known, and such methods may be applied to the antibodies and antigen-binding fragments of the present invention (see, e.g., Langer et al., J. Biomed. Mater. Res. 15:267-277, 1981; Langer, Chemtech. 12:98-105, 1982; U.S. Pat. No. 3,773,919; European Patent Application Publication No. EP 58,481; Sidman et al., Biopolymers 22:547-556, 1983; and EP 133,988). The pharmaceutical compositions provided herein can be formulated for intravenous, intraarterial, intradermal, subcutaneous, intramuscular, intraperitoneal, or oral administration.

[0050] Combination with IL-1-targeting agents Postherpetic neuralgia (PHN), also known as a neuropathic pain syndrome, is characterized by severe pain. The pain is caused by peripheral and central nerve damage resulting from the immune and inflammatory responses associated with VZV reactivation. Pain can persist for months to years after the shingles rash subsides. The frequency and severity of PHN increase with age, occurring in 20% of people aged 60-65 with acute shingles and more than 30% of people over 80 years of age. The pain associated with PHN is caused by peripheral and central neuronal damage, which may be a by-product of the inflammatory response associated with varicella-zoster virus reactivation. Patients with postherpetic neuralgia have been reported to experience reduced quality of life and impaired activities of daily living. PHN is associated with a range of sensory abnormalities, including allodynia (painful responses to stimuli that do not normally cause pain, such as the touch of a feather), hyperalgesia (increased sensitivity to and extreme responses to pain), and dysesthesias (abnormal sensations such as painful burning, tingling, and throbbing sensations).

[0051] Medical conditions associated with pain and hyperalgesia in peripheral and central compartments are accompanied by increased IL-1 expression. Animal studies have shown that mice with genetic disorders of IL-1 signaling exhibit reduced neuropathic pain behavior. Therefore, along with anti-gE antibodies, agents that target and inhibit IL-1 function can be administered to VZV-infected subjects (e.g., subjects with shingles). Agents that target and inhibit IL-1 function include agents that specifically bind to IL-1a or IL-1b. Examples of such agents include anti-IL-1a antibodies, anti-IL-1b antibodies, proteins that bind to the extracellular portion of the interleukin-1 receptor component (IL-1R1), or proteins that bind to the IL-1 receptor accessory protein (IL-1RAcP). [Example]

[0052] Example 1—A newly discovered human monoclonal antibody of the IgG1κ subclass that binds to gE with high affinity has been designated XB22-14 (also referred to herein as VZ27). The molecular weight of intact XB22-14 was approximately 149.6 KDa as determined by mass spectrometry. The amino acid sequences of the light and heavy chain variable regions of the human monoclonal antibody designated XB22-14 are as follows, with the CDRs shown in bold and underlined (determined by IMGT / DomainGapAlign; Ehrenmann, F., Lefranc, M.-P. Cold Spring Harb Protoc., 2011(6):737-749(2011). DOI:10.1101 / pdb.prot5636. PMID:21632775): Heavy chain (IgG1) QVQLVQSGAEVKKPGASVKVSCKVS GYSLIELS MHWVRQAPGKGLEWMGG YDPGVRQT VYARKFRGRLTMTEDTSADTAYMELSSLRSDDTAVYYC ATLFFLSGTYYVDPRWFDP WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [SEQ ID NO: 1] Light chain (kappa) EIVMTQSPATLSVSPGERATLSCRAS QSVSSN LAWYQQKPGQAPRLLIY GAS TRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNWPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 2]

[0053] Thus, the XB22-14 heavy chain has a CDR1 with the amino acid sequence GYSLIELS (SEQ ID NO: 3), a CDR2 with the amino acid sequence YDPGVRQT (SEQ ID NO: 4), and a CDR3 with the amino acid sequence ATLFFLSGTYYVDPRWFDP (SEQ ID NO: 5). Similarly, the XB22-14 light chain has a CDR1 with the amino acid sequence QSVSSN (SEQ ID NO: 6), a CDR2 with the amino acid sequence GAS (SEQ ID NO: 7), and a CDR3 with the amino acid sequence QQYNNWPPLT (SEQ ID NO: 8).

[0054] Example 2: XB22-14 binds to recombinant glycoprotein E with high affinity. The binding of XB22-14 to the recombinant extracellular domain of gE was tested using an Octet RED-based binding kinetics assay. Briefly, 10 μg / mL gE was immobilized on a Ni-NTA biosensor and subsequently bound to 0.03125–1 μg / mL XB22-14 (VZ27) for 600 seconds and dissociated for 1200 seconds. The KD was 1.0 × 10 -12 M was found to be less than M, indicating that XB22-14 (VZ27) binds gE with picomolar binding affinity.

[0055] The Shingrix® vaccine is routinely administered to people over the age of 50 and is known to induce a strong humoral immune response. Therefore, we used an Octet RED-based BLI assay to evaluate whether XB22-14 binds to gE present in the Shingrix® vaccine. Because the gE component of the Shingrix® vaccine does not have a His tag, we were unable to use a Ni-NTA sensor. Instead, we immobilized XB22-14 using a FAB sensor. Results showed that XB22-14 binds to the active ingredient in the Shingrix® vaccine with high affinity (i.e., KD in the range of 0.1–7 nM).

[0056] Example 3: 4.1.3 XB22-14 specifically binds to gE on the cell membrane of VZV-infected ARPE19 cells. VZV Oka strain (obtained from ATCC) was used to infect ARPE-19 cells in T75 cell culture flasks in the presence of advanced DMEM / F12 + 50 mM sucrose + 2 mM glutamine + 10% ultra-low IgG FBS for 3 days. At the end of the 3-day period, infected cells were scraped and resuspended in 10 ml of cell culture medium, and the supernatant was transferred to confluent ARPE-19 cells growing in a new T175 flask in advanced DMEM / F12 + 50 mM sucrose + 2 mM glutamine + 10% ultra-low IgG FBS. Cell infection in the T175 flask was followed for 3 days, at the end of which >90% of the cells were expanded and infected. Infection occurred primarily by cell-to-cell spread between individual ARPE-19 cells, rather than via syncytia formation in the presence of 10% ultra-low IgG FBS. At the end of the 3-day period, the cell culture supernatant was removed, and ARPE-19 cells infected with VZV Oka were harvested with a cell scraper and fixed in 10% neutral-buffered formalin. The cells were washed with DPBS and used for staining with XB22-14 (VZ27). As shown in Figure 1, XB-22-14 can specifically bind to gE present on the surface of infected ARPE19 cells.

[0057] Example 4 - Effector function of XB22-14. Biolayer interferometry was used to evaluate the affinity of XB22-14, which contains an IgG1 kappa constant region, for FcγR1 (CD64), FcγRIIa / b (CD32a and CD32b), FcγRIIIa / b (CD16a and CD16b), and FcαR1 (CD89). The Octet RED96 BLI system was used to evaluate the kinetics of XB22-14 antibody binding to Fcg receptors. Figure 2 shows representative Octet BLI sensorgrams and 2:1 heterogeneous fitting curves of XB22-14 antibody binding to various receptors. Kinetic analysis demonstrated that the XB22-14 antibody binds to CD64 with a high Octet response range (approximately 0.5–0.9 nM). The Octet sensorgrams were fitted using a 2:1 heterogeneous ligand binding model, yielding a 1.1 x 10 -10 M KD1 and 3.2 x 10 -9 Two binding affinity constants with KD2 of 1.0 M were obtained. The Octet kinetics of XB22-14 antibody binding to CD32a, CD32b, and CD16a revealed a lower Octet response range (approximately 0.05–0.30 nM) and a weaker binding affinity range (approximately 1.4 × 10) compared to XB22-14 antibody binding to CD64. -5 ~1.8×10 -8 M Kd1 and approximately 1.2 x 10 -7 ~1.5×10 -8 The Octet kinetics of XB22-14 antibody binding to CD16b and CD89 revealed no measurable Octet response. Figure 2 shows Octet sensorgrams of XB22-14 binding to the FcRn receptor at pH 6.0 and 7.2. Fitting these sensorgrams using a 2:1 heterologous ligand binding model revealed a higher binding affinity range at pH 6.0 (KD1 = 1.3 × 10 -8 M and KD2 = 2.8 × 10 -8 M) and a lower binding affinity range at pH 7.2 (KD1 = 1.1 × 10 -6 M and KD2 = 1.7 × 10 -7 M) was revealed.

[0058] Example 5 - XB22-14 can mediate antibody-dependent cellular cytotoxicity (ADCC) of cells expressing gE on their surface. This study used Promega's ADCC Reporter Bioassay. The target cells used in this study were the 293F cell line, engineered to express gE / gl on the cell surface. The effector cells used were Jurkat cells stably expressing the human FcγRIIIa receptor and NFAT-inducible luciferase. The target cells were incubated with 50 μg / mL antibody and serial 2-fold dilutions of effector cells in a 96-well plate. Considering the ease of contact accessibility between the antigen-antibody complex on the target cells and FcγRIIIa on the effector cell surface, the 293F target cells were mixed with the effector cells at a 1:1 ratio. A human IgG1κ isotype control was used as a negative control for XB22-14. All experimental samples were run in duplicate. Luminescence from the plate was quantified using a Tecan 200 Pro reader. As shown in Figure 3, XB22-14 can mediate ADCC of cells expressing gE on their surface.

[0059] Example 6 - Antibody-dependent cellular phagocytosis (ADCP) assay. This study used Promega's ADCP Reporter Bioassay. The target cells used in this study were the 293F cell line, engineered to express gE / gl on the cell surface. The effector cells used were Jurkat cells stably expressing the human FcγRIIa receptor and NFAT-inducible luciferase. The target cells were incubated with 50 μg / mL antibody and serial 2-fold dilutions of effector cells in a 96-well plate. Considering the ease of contact accessibility between the antigen-antibody complex on the target cells and FcγRIIa on the effector cell surface, the 293F target cells were mixed with the effector cells at a 4:1 ratio. A human IgG1κ isotype control was used as a negative control for XB22-14. All experimental samples were run in duplicate. Luminescence from the plate was quantified using a Tecan 200 Pro reader. As shown in Figure 4, XB22-14 can mediate ADCP of cells expressing gE on their surface.

[0060] Example 7 - XB22-14 can prevent VZV infection of MeWo cells. MeWo cells, a fibroblast cell line isolated from human skin, were used for VZV infection studies. The ability of XB22-14 to prevent VZV infection of human skin cells was tested in a cell-based in vitro assay. Briefly, VZV virus stock (Oka strain) was propagated and harvested from ARPE-19 cells without serum supplementation. 1,000 individual cell foci of the VZV stock were mixed with XB22-14 at concentrations ranging from 15 to 0.02 μg / mL and then cultured at 1 × 10 in the presence of advanced DMEM-F12 medium containing 0.5% sucrose and 2 mM glutamine in a 96-well plate. -5The virus-antibody mixture was added to confluent MeWo cells that had been grown with XB22-14 for 1 day. The infected cells were cultured for 24 hours at 35°C and 5% CO2. At the end of the 24 hours, the virus-antibody mixture was removed, and the antibody alone was added back to the infected cells. The cells were further incubated for an additional 2 days, after which VZV-infected cells were visualized by immunostaining with a VZV-specific monoclonal antibody mixture from GenTex (catalog no. GTX38720) (Figure 5A). VZV-infected cells were counted using microscopic images at 100x magnification. The number of spots in the presence and absence of XB22-14 is plotted in Figure 5B. The neutralization rate was calculated relative to the total number of infected cells in the absence of antibody XB22-14 (Figure 5C). Figure 5D shows the effect of 0.3 to 1.2 μM acyclovir on inhibiting VZV replication in MeWo cells over a 5-day period. Acyclovir was able to prevent VZV infection by up to 60%. In comparison, XB22-14VZV at concentrations ranging from 1.5 to 1 μg / mL was able to prevent 100% VZV infection of MeWo cells. At lower concentrations (0.56 to 0.02 μg / mL), XB22-14 showed partial VZV neutralization in a concentration-dependent manner (Figure 5.1B, Figure 5.1C).

[0061] Example 8 - VZV microneutralization assay in ARPE19 cells. Reactivation of latent VZV in the ophthalmic branch of the trigeminal nerve can cause ophthalmic herpes zoster (UVZ), which can lead to ocular complications such as uveitis. Uveitis is defined as severe inflammation of intraocular tissues, including the iris, retina, ciliary body, and choroid. Uveitis involves inflammatory cells in these tissues as well as in the aqueous and vitreous humors. Herpesviruses are common infectious causes of uveitis, and VZV is responsible for the majority of ARN cases worldwide. This can lead to retinal detachment, chronic inflammation, and potentially blindness.

[0062] Retinal pigment epithelial (RPE) cells are a layer of resident antigen-presenting cells that play a fundamental role in maintaining immune privilege within the eye through several mechanisms. ARPE-19 is a naturally established retinal pigment epithelial (RPE) cell line derived from a normal eye. To test the ability of XB22-14 to prevent viral infection of ARPE-19 cells, we performed a viral microneutralization assay. ARPE-19 is a naturally established retinal pigment epithelial (RPE) cell line derived from a normal eye.

[0063] The ability of XB22-14 to prevent VZV infection of ARPE-19 cells was tested in vitro using the following experiment. Briefly, 1000 individual cell foci of infection from a VZV stock were transferred to trypsinized ARPE-19 cells (1.25 × 10 cells) in the presence or absence of 3.75 to 0.002 μg / mL of XB22-14 or an IgG1κ isotype control. 4 The virus and cells were co-incubated with 1000 x 1000 cells (100 x 1000 cells) in a non-binding 96-well plate with shaking at 25°C for 1 hour. The cells were then transferred to a cell culture-treated 96-well plate in the presence of 1% ultra-low IgG fetal bovine serum and incubated at 35°C and 5% CO2. The cells were incubated for 5 days, then fixed with 5% formaldehyde, stained with a VZV-specific monoclonal antibody mixture from GeneTex (catalog no. GTX38720), and visualized at 40x magnification (Figure 6A). Microscopic images were processed using the Image J program, and the immunostained areas were integrated and compared between XB22-14-treated and isotype control-treated cells. The IC50 of XB22-14 to prevent VZV infection of ARPE-19 cells was found to be 0.015 μg / mL (Figure 6B). However, the IgG1κ isotype control had no effect on viral infection.

[0064] Other embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the above description is intended to illustrate, but not to limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. Varicella-zoster virus (VZV) glycoprotein A pharmaceutical composition comprising a purified monoclonal antibody that specifically binds to gene encoding glutamate E (gE) and comprises a heavy chain variable region amino acid sequence comprising the CDRs of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, and a light chain variable region amino acid sequence comprising the CDRs of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:

8.

2. The pharmaceutical composition of claim 1 , wherein the heavy chain of the antibody is gamma 1.

3. 2. The pharmaceutical composition of claim 1, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO: 1 and the light chain variable region has the amino acid sequence of SEQ ID NO:

2.

4. Varicella-zoster virus (VZV) glycoprotein for the treatment of shingles Use of purified monoclonal antibodies that specifically bind to gE.

5. The use of claim 4, wherein the monoclonal antibody comprises a heavy chain variable region amino acid sequence comprising the CDRs of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, and a light chain variable region amino acid sequence comprising the CDRs of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:

8.

6. The use according to claim 4, wherein the heavy chain of the antibody is gamma 1.

7. The use according to claim 5, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO: 1 and the light chain variable region has the amino acid sequence of SEQ ID NO:

2.

8. A method of treating a mammal with shingles, comprising administering a therapeutically effective amount of varicella zoster virus (VZV) glycoprotein to a mammal for the treatment of shingles. administering to said mammal a purified monoclonal antibody that specifically binds to gE.

9. The method of claim 8, wherein the monoclonal antibody comprises a heavy chain variable region amino acid sequence comprising the CDRs of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, and a light chain variable region amino acid sequence comprising the CDRs of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:

8.

10. The method of claim 8 , wherein the heavy chain of the antibody is gamma 1.

11. 10. The method of claim 9, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO: 1 and the light chain variable region has the amino acid sequence of SEQ ID NO:

2.

12. 10. The method of claim 8, further comprising administering to said mammalian subject an agent that inhibits the function of IL-1 alpha.

13. 13. The method of claim 12, wherein the agent that inhibits IL-1 alpha function is an anti-IL-1 alpha antibody.

14. 13. The method of claim 12, wherein the agent that binds to IL-1 alpha is a protein that binds to the extracellular portion of the interleukin-1 receptor component (IL-1R1).

15. 13. The method of claim 12, wherein the agent that binds to IL-1 alpha is a protein that binds to IL-1 receptor accessory protein (IL-1RAcP).

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