Anti-human cytomegalovirus antibodies and uses thereof

Abstract

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

Description

[0001] This application is a divisional application of patent application CN201911226892.6, filed on December 4, 2019, with the invention name “Anti-human cytomegalovirus antibodies and their uses”.

[0002] The present invention relates to antibodies or antigen-binding fragments thereof that are specific for and bind with high affinity to human cytomegalovirus, and methods for producing such antibodies. The antibodies of the invention also have high potency in neutralizing infection. The invention also relates to the epitopes to which the antibodies bind, and the use of the antibodies for diagnosis, prevention, and treatment of infected individuals. Background Art

[0003] Human cytomegalovirus (HCMV) is widely distributed, affecting 40% to 100% of the population. It can cause severe complications and even death in immunocompromised individuals, such as organ and bone marrow transplant recipients, HIV patients, pregnant women, and fetuses, and is associated with malignancies and chronic diseases. In the United States and Europe, the prevalence of HCMV infection in live births is estimated to be 0.2% to 1.2%. HCMV seropositivity is more common in developing countries or low-income regions.

[0004] After primary infection with HCMV, the virus can remain latent, replicate, and spread within the host, evading the host's immune system. Complete HCMV clearance is rare. HCMV infection is typically asymptomatic in most individuals. In patients with underlying health conditions and impaired immune function (such as transplant recipients and HIV patients), primary infection or HCMV reactivation can lead to lesions in the urinary tract, central nervous system, liver, lungs, and circulatory system. HCMV can also cause secondary immunosuppression, increasing the risk of infection and leading to morbidity and mortality. Furthermore, HCMV is a leading cause of birth defects (including organ defects, developmental delay, intellectual disability, and visual and hearing loss) caused by vertical transmission during pregnancy. Currently, there are no therapies to prevent or treat fetal infection. Prevymis (MSD), used to prevent HCMV infection and related diseases, is the first new drug approved by the US FDA in nearly 15 years for HCMV infection and is intended for use only in adults. Cytogam and Cytotect are two marketed cytomegalovirus immune globulin preparations, but they have demonstrated significant side effects and uncertain therapeutic efficacy in clinical practice.

[0005] Because antibodies play an extremely important role in immune defense, especially in fighting infection, there are reasons to pursue further research on monoclonal antibodies against human cytomegalovirus, despite repeated failures. Based on current developments in antibody technology, highly neutralizing monoclonal antibodies derived from human B cells are more likely to be more effective.

[0006] The antibodies disclosed in the present invention are derived from human B cells, which make up for the shortcomings of the existing technology. The purpose of the present invention is to provide fully human monoclonal antibodies with strong specificity and high neutralizing titer, which are suitable for the medical treatment, prevention and / or diagnosis of HCMV infection, and can also be used to treat various disorders related to HCMV infection. Summary of the Invention

[0007] The present invention therefore provides a novel antibody that binds to human cytomegalovirus, as well as an antigen-binding fragment thereof, and a novel antigenic epitope bound by the antibody.

[0008] In one aspect, the present invention provides antibodies or antigen-binding fragments thereof that are highly specific and neutralize human cytomegalovirus infection with high titer. In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention bind to the envelope glycoprotein gB of human cytomegalovirus.

[0009] In an embodiment of the present invention, the present invention includes an antibody, or an antigen-binding fragment thereof, that is capable of specifically binding to the human cytomegalovirus gB glycoprotein fusion domain.

[0010] In some embodiments of the present invention, the present invention includes an antibody, or an antigen-binding fragment thereof, that binds to an epitope on a gB glycoprotein fusion domain comprising one or more amino acid residues selected from the group consisting of N208, L213, and Y226 of the gB glycoprotein fusion domain. In some embodiments, the antibody or antigen-binding fragment thereof of the present invention binds to an epitope on a gB glycoprotein fusion domain comprising amino acid residues L213 and Y226 of the gB glycoprotein fusion domain. In some embodiments, the antibody or antigen-binding fragment thereof of the present invention binds to an epitope on a gB glycoprotein fusion domain comprising amino acid residue N208 of the gB glycoprotein fusion domain.

[0011] In another aspect, the present invention provides antibodies or antigen-binding fragments thereof that are highly specific and neutralize HCMV infection with high potency. In some embodiments, the anti-human cytomegalovirus antibodies or antigen-binding fragments thereof of the present invention comprise at least one, at least two, or all three of the three complementarity determining regions (CDRs) contained in the heavy chain variable region (VH).

[0012] In some embodiments, the anti-human cytomegalovirus antibody or antigen-binding fragment thereof of the present invention comprises at least one, at least two, or all three of the three complementarity determining regions (CDRs) contained in the light chain variable region (VH).

[0013] In some embodiments, the anti-human cytomegalovirus antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH), wherein the VH comprises one, two or three complementarity determining regions (CDRs) contained in the VH shown in SEQ ID NO: 13, 14, 15 or 16.

[0014] In some embodiments, the anti-human cytomegalovirus antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region (VL), wherein the VL comprises one, two or three complementarity determining regions (CDRs) contained in the VL shown in SEQ ID NO: 33, 34, 35 or 36.

[0015] In some embodiments, the anti-human cytomegalovirus antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region VH and a light chain variable region VL, wherein

[0016] (a) the VH comprises three complementarity determining regions (CDRs) contained in the VH shown in SEQ ID NO: 13, 14, 15 or 16; and / or

[0017] (b) the VL comprises three complementarity determining regions (CDRs) contained in the VL shown in SEQ ID NO: 33, 34, 35 or 36.

[0018] In a preferred embodiment, VH comprises an amino acid sequence selected from SEQ ID NO: 13, 14, 15 or 16, or consists of said amino acid sequence.

[0019] In a preferred embodiment, VL comprises an amino acid sequence selected from SEQ ID NO: 33, 34, 35 or 36, or consists of said amino acid sequence.

[0020] In a preferred embodiment, the anti-human cytomegalovirus antibody or antigen-binding fragment thereof of the present invention comprises

[0021] The three complementarity determining regions HCDR of the heavy chain variable region are shown in SEQ ID NO: 13, 14, 15 or 16, and the three complementarity determining regions LCDR of the light chain variable region are shown in SEQ ID NO: 33, 34, 35 or 36.

[0022] In some embodiments, the anti-human cytomegalovirus antibodies or antigen-binding fragments thereof of the present invention comprise 1, 2, 3, 4, 5 or 6 of the exemplary combinations of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the antibodies or antigen-binding fragments thereof of the present invention shown in Table A.

[0023] In some embodiments, the anti-human cytomegalovirus antibody or antigen-binding fragment thereof of the present invention comprises a HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 9, 10, 11, or 12.

[0024] In some embodiments, the anti-human cytomegalovirus antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein

[0025] (i) the VH comprises complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4; HCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 5, 6, 7, or 8; and HCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 9, 10, 11, or 12;

[0026] and / or

[0027] (ii) wherein the VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 21, 22, 23, or 24; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 25, 26, 27, or 28; and LCDR3 comprises or consists of an amino acid sequence selected from SEQ ID NO: 29, 30, 31, or 32.

[0028] In a preferred embodiment, the present invention provides an anti-human cytomegalovirus antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0029] (a) the VH comprises

[0030] (i) HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 9; or

[0031] (ii) HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 10; or

[0032] (iii) HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 11; or

[0033] (iv) HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 12;

[0034] and / or

[0035] (b) the VL comprises

[0036] (i) LCDR1, LCDR2 and LCDR3 comprising or consisting of the following sequences: SEQ ID NO: 21, SEQ ID NO: 25 and SEQ ID NO: 29; or

[0037] (ii) LCDR1, LCDR2 and LCDR3 comprising or consisting of the following sequences: SEQ ID NO: 22, SEQ ID NO: 26 and SEQ ID NO: 30; or

[0038] (iii) LCDR1, LCDR2 and LCDR3 comprising or consisting of the following sequences: SEQ ID NO: 23, SEQ ID NO: 27 and SEQ ID NO: 31; or

[0039] (iv) LCDR1, LCDR2 and LCDR3 comprising or consisting of the following sequences: SEQ ID NO:24, SEQ ID NO:28 and SEQ ID NO:32.

[0040] In a preferred embodiment, the present invention provides an anti-human cytomegalovirus antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3 and the VL comprises (CDRs) LCDR1, LCDR2, and LCDR3, wherein the combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprised by the antibody or antigen-binding fragment thereof is shown in the following table (Table A):

[0041] Table A: Exemplary combinations of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 in the antibodies or antigen-binding fragments thereof of the invention

[0042]

[0043] In some embodiments, the anti-human cytomegalovirus antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region VH and / or a light chain variable region VL, wherein:

[0044] (a) Heavy chain variable region VH

[0045] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 13, 14, 15 or 16; or

[0046] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 13, 14, 15 or 16; or

[0047] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 13, 14, 15, or 16, preferably, the amino acid changes do not occur in the CDR regions;

[0048] and / or

[0049] (b) Light chain variable region VL

[0050] (i) comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 33, 34, 35 or 36;

[0051] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 33, 34, 35 or 36; or

[0052] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 33, 34, 35 or 36, preferably, the amino acid changes do not occur in the CDR regions.

[0053] In a preferred embodiment, the present invention provides an anti-human cytomegalovirus antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the combination of the heavy chain variable region VH and the light chain variable region VL comprised by the antibody or antigen-binding fragment thereof is shown in the following table (Table B):

[0054] Table B: Exemplary combinations of heavy chain variable regions VH and light chain variable regions VL in the antibodies or antigen-binding fragments thereof of the present invention

[0055]

[0056] In some embodiments, the antibodies of the present invention are antibodies in the form of IgG, IgM, IgA, IgD or IgE. In some embodiments, the antibodies of the present invention include a heavy chain constant region selected from, for example, a heavy chain constant region of a heavy chain constant region of IgG, IgM, IgA, IgD or IgE; in particular, a heavy chain constant region selected from the heavy chain constant region of IgG, more specifically the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, such as the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4. In one embodiment, the heavy chain constant region is a human IgG1 or human IgG3 heavy chain constant region. In one embodiment, the heavy chain constant region is a human IgG2 or human IgG4 heavy chain constant region. In one embodiment, the heavy chain constant region is a human IgG1 heavy chain constant region. In another embodiment, the antibody molecule of the present invention has a light chain constant region, for example, selected from a κ or λ light chain constant region, preferably a light chain constant region of κ (e.g., human κ).

[0057] In yet another embodiment, the antibody molecule of the present invention comprises a heavy chain constant region of IgG1 (e.g., human IgG1). In yet another embodiment, the antibody molecule of the present invention comprises a heavy chain constant region of IgG3 (e.g., human IgG3). In one embodiment, the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 47, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, or consists of said sequence.

[0058] In another embodiment, the antibody molecule of the invention comprises a kappa light chain constant region, e.g., a human kappa light chain constant region. In one embodiment, the light chain constant region comprises the amino acid sequence of SEQ ID NO: 46, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, or consists of said sequence.

[0059] In another embodiment, the antibody molecule of the invention comprises a lambda light chain constant region, e.g., a human lambda light chain constant region. In one embodiment, the light chain constant region comprises the amino acid sequence of SEQ ID NO: 45, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, or consists of said sequence.

[0060] In some embodiments, the anti-human cytomegalovirus antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and / or a light chain, wherein

[0061] (a) Heavy chain

[0062] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 37, 39, 41 or 43;

[0063] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 37, 39, 41 or 43; or

[0064] (iii) an amino acid sequence comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 37, 39, 41, or 43, preferably, the amino acid changes do not occur in the CDR region of the heavy chain, more preferably, the amino acid changes do not occur in the heavy chain variable region;

[0065] and / or

[0066] (b) Light chain

[0067] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 38, 40, 42 or 44;

[0068] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 38, 40, 42 or 44; or

[0069] (iii) comprising an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 38, 40, 42 or 44, preferably, the amino acid changes do not occur in the CDR region of the light chain, more preferably, the amino acid changes do not occur in the light chain variable region.

[0070] In a preferred embodiment, the present invention provides an anti-human cytomegalovirus antibody or an antigen-binding fragment thereof, which comprises a heavy chain and a light chain, wherein the combination of the heavy chain and the light chain comprised by the antibody or antigen-binding fragment thereof is shown in the following table (Table C):

[0071] Table C: Exemplary combinations of heavy and light chains in the antibodies or antigen-binding fragments thereof of the present invention

[0072]

[0073] In one embodiment of the present invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0074] In a preferred embodiment, the amino acid changes described herein occur in regions outside of the CDRs (e.g., in the FRs). More preferably, the amino acid changes described herein occur in regions outside of the heavy chain variable region and / or outside of the light chain variable region.

[0075] In some embodiments, the substitution is a conservative substitution. A conservative substitution refers to the substitution of one amino acid with another amino acid within the same class, for example, an acidic amino acid with another acidic amino acid, a basic amino acid with another basic amino acid, or a neutral amino acid with another neutral amino acid. Exemplary substitutions are shown in Table D below:

[0076] Table D

[0077]

[0078]

[0079] In certain embodiments, the substitution occurs in the CDR region of the antibody. Typically, the variant obtained has modifications (e.g., improvements) relative to the parent antibody in certain biological properties (e.g., increased affinity) and / or will have certain biological properties that are substantially retained by the parent antibody. An exemplary substitution variant is an affinity matured antibody.

[0080] In certain embodiments, the antibodies provided herein may be further modified to contain other non-proteinaceous moieties known in the art and readily available. Suitable portions for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. For example, the antibodies of the present invention, or their antigen-binding fragments, can be chemically modified by covalent coupling to a polymer, for example to increase its circulation half-life.

[0081] In certain embodiments, the antibody provided herein is through change to increase or reduce the degree of glycosylation of antibody. The addition or deletion of the glycosylation site of antibody can be easily achieved by changing the amino acid sequence to produce or remove one or more glycosylation sites. When the antibody comprises an Fc region, the carbohydrate attached thereto can be changed. In some applications, it can be useful to remove the modification of unwanted glycosylation sites, for example, to remove the fucose module to improve antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277: 26733). In other applications, galactosidation modification can be carried out to modify complement-dependent cytotoxicity (CDC).

[0082] In certain embodiments, it may be desirable to generate cysteine engineered antibodies, such as "thioMAbs," in which one or more residues of an antibody are replaced with cysteine residues. Cysteine engineered antibodies can be generated as described, for example, in U.S. Patent No. 7,521,541.

[0083] In some embodiments, the anti-human cytomegalovirus antibodies or antigen-binding fragments thereof of the present invention exhibit the same or similar binding affinity and / or specificity as the antibodies of the present invention for HCMV; and / or inhibit (e.g., competitively inhibit) the binding of the antibodies of the present invention to HCMV and / or bind to the same or overlapping epitopes as the antibodies of the present invention; and / or compete with the antibodies of the present invention for binding to HCMV; and / or have one or more biological properties of the antibodies of the present invention.

[0084] In some embodiments, the antibodies or fragments thereof of the present invention are capable of highly effective neutralization of HCMV. In some embodiments, the EC50 of the antibodies of the present invention for neutralizing HCMV is as low as 0.15 μg / ml or less. In some embodiments, the EC50 of the antibodies of the present invention for neutralizing HCMV is 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 μg / ml or less. In some embodiments, the antibodies of the present invention can neutralize HCMV in endothelial cells or fibroblasts with very low EC50.

[0085] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention can directly block viral invasion of host cells.

[0086] In some embodiments, the antibodies of the invention are capable of effectively binding to gB glycoprotein, for example, with an equilibrium dissociation constant (K D ) binds to gB glycoprotein, the K D Less than or equal to about 50 nM, preferably, less than or equal to about 40 nM, more preferably less than or equal to about 30 nM, more preferably less than or equal to about 20 nM, most preferably, the K D Less than or equal to about 5 nM, 4 nM, 3 nM, 2 nM, 1.5 nM, 1 nM, 0.9 nM, 0.8 nM, or 0.7 nM. In some embodiments, the antibody binding affinity is determined using a surface plasmon resonance (SPR) assay.

[0087] In some embodiments, at least a portion of the framework sequence of an antibody or antigen-binding fragment thereof of the invention is a human consensus framework sequence.

[0088] In some embodiments, the anti-human cytomegalovirus antibody of the present invention is an IgG-form antibody, such as an IgG1-form antibody, an IgG2-form antibody, an IgG3-form antibody, or an IgG4-form antibody.

[0089] In some embodiments, the anti-human cytomegalovirus antibody is a monoclonal antibody. In some embodiments, the anti-human cytomegalovirus antibody is humanized. In some embodiments, the anti-human cytomegalovirus antibody is a human antibody. In one embodiment, the anti-human cytomegalovirus antibody of the present invention also encompasses antibody fragments thereof, preferably selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv) or (Fab')2, single domain antibody, diabody (dAb) or linear antibody.

[0090] In certain embodiments, the anti-human cytomegalovirus antibody molecule is in the form of a bispecific or multispecific antibody molecule.

[0091] In some embodiments, the antibodies of the invention do not elicit an autoimmune response.

[0092] In some embodiments, the invention also encompasses antibodies conjugated to a label ("immunoconjugates").

[0093] In some embodiments, the antibodies of the present invention can be conjugated with a detectable label to facilitate imaging of sites containing target cells (e.g., cells infected with HCMV). Antibodies labeled with a variety of labels can be used in a variety of assays. Detection of the antibody-antigen complex formed between the antibody of the present invention and the target epitope (HCMV epitope) is facilitated by attaching a detectable substance to the antibody. Suitable detection methods include the use of labels such as radionuclides, enzymes, coenzymes, fluorescent agents, chemiluminescent substances, chromogens, enzyme substrates or cofactors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes, etc. Labeled antibodies can be used in various well-known tests, such as radioimmunoassays, enzyme immunoassays (e.g., ELISAs), fluorescent immunoassays, etc. Suitable labels and related methods are described, for example, in

[15] US 3,766,162, US 3,791,932, US 3,817,837, or US 4,233,402.

[0094] In some embodiments, the present invention provides nucleic acids encoding any of the antibodies or fragments thereof described herein, or any of their chains. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector, such as a eukaryotic vector. In one embodiment, a host cell comprising the nucleic acid or the vector is provided.

[0095] For example, a nucleic acid of the invention comprises:

[0096] A nucleic acid encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-16 and 33-44, or a nucleic acid encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-16 and 33-44 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-16 and 33-44.

[0097] The present invention also encompasses nucleic acids that hybridize under stringent conditions to the following nucleic acids, or nucleic acids that have one or more substitutions (e.g., conservative substitutions), deletions, or insertions with the following nucleic acids: a nucleic acid comprising a nucleic acid sequence encoding an amino acid sequence selected from any one of SEQ ID NOs: 13-16 and 33-44; or a nucleic acid comprising a nucleic acid sequence encoding an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 13-16 and 33-44.

[0098] In one embodiment, one or more vectors comprising the nucleic acid are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. In some embodiments, the vector contains an EF promoter and / or a CMV enhancer. For example, vectors include but are not limited to viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). In one embodiment, such as a pCDNA3.1 vector. Once an expression vector or DNA sequence for expression has been prepared, the expression vector can be transfected or introduced into a suitable host cell. Various techniques can be used to achieve this purpose, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene guns, lipid-based transfection or other conventional techniques. In the case of protoplast fusion, cells are cultivated in culture medium and screened for suitable activity. Methods and conditions for cultivating the transfected cells produced and for reclaiming the antibody molecules produced are known to those skilled in the art and can be based on methods known to the art, according to the specific expression vector and mammalian host cell used, change or optimize. In addition, one or more markers that allow selection of transfected host cells can be introduced to select cells that have stably incorporated DNA into their chromosomes. Markers can for example provide prototrophy, biocidal resistance (for example, antibiotic) or heavy metal (such as copper) resistance etc. to auxotrophic hosts.Selectable marker gene can be directly connected to the DNA sequence to be expressed or introduced into the same cell by co-transformation.Additional elements may also be needed for optimal synthesis of mRNA.These elements may include splicing signals, as well as transcription promoters, enhancers and termination signals.

[0099] In one embodiment, a host cell comprising one or more polynucleotides of the present invention is provided. In some embodiments, a host cell comprising an expression vector of the present invention is provided. In some embodiments, the host cell is preferably a cell that is universal in the expression of protein genes, etc., and is particularly suitable for the expression of antibody genes. Suitable host cells suitable for the present invention include prokaryotic microorganisms, such as Escherichia coli. The host cell can also be a eukaryotic microorganism such as a filamentous fungus or yeast, or various eukaryotic cells, such as insect cells or plant cells. Vertebrate cells can also be used as hosts. For example, a mammalian cell line adapted for suspension growth can be used. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK 293 or 293F cells), 293 cells, baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), Chinese hamster ovary cells (CHO cells), CHOK1SV cells, CHOK1SV GS-KO cells, CHOS cells, NSO cells, myeloma cell lines such as Y0, NS0, P3X63 and Sp2 / 0, and the like. For a review of mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In a preferred embodiment, the host cell is a CHO cell, such as a CHOS cell, CHOK1SV cell, or CHOK1SV GS-KO, or the host cell is a 293 cell, such as a HEK293 cell.

[0100] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells, e.g., HEK293 cells) or other cells suitable for preparing antibodies or antigen-binding fragments thereof. In another embodiment, the host cell is prokaryotic.

[0101] In some embodiments, the host cells are, for example, Escherichia coli cells, actinomycetes, yeast, insect cells (SF9, etc.), mammalian cells (293, HEK293, COS-1, CHO, myeloma cells, etc.).

[0102] For industrial production of recombinant antibodies, recombinant animal cell lines that stably and efficiently produce the antibodies are typically utilized, such as CHO cell lines. The preparation, cloning, gene amplification for high expression, and screening of such recombinant cell lines can be performed using known methods (e.g., see Omasa T.: J. Biosci. Bioeng., 94, 600-605, 2002).

[0103] In one embodiment, a method for preparing an antibody molecule of the invention is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody (e.g., any one polypeptide chain and / or multiple polypeptide chains) or an expression vector comprising the nucleic acid under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium or supernatant). In order to recombinantly produce an antibody molecule of the invention, nucleic acid encoding the antibody (e.g., the antibody described above, e.g., any one polypeptide chain and / or multiple polypeptide chains) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibody).

[0104] In a preferred embodiment of the present invention, the antibody of the present invention is a fully human antibody. Recombinant human antibodies can be obtained using known methods (see Nature, 312: 643, 1984, Nature, 321: 522, 1986, etc.). For example, the antibody of the present invention can be produced by culturing host cells into which the vector of the present invention has been introduced and purifying the produced antibody from the culture supernatant. More specifically, it can be produced by the following method: cDNAs encoding VH and VL are separately inserted into expression vectors for animal cells containing genes encoding human antibody CH and / or human antibody CL produced by the same cell or different human cells to construct a human antibody expression vector, and then introduced into animal cells for expression. In some embodiments, expression vectors recombinant with nucleic acids encoding VH or VL are generally prepared separately and co-transfected into host cells, but they can also be recombined into a single expression vector.

[0105] In some embodiments, polyclonal antibodies or monoclonal antibodies can be obtained using experimental animals such as mice, rabbits, and goats.

[0106] In some embodiments, the anti-human cytomegalovirus monoclonal antibodies or antigen-binding fragments thereof of the present invention can be obtained from antibody-producing cells derived from human blood and are fully human antibodies. Even when administered to humans as antibody drugs, these fully human antibodies are non-immunogenic and no immune response is observed. For example, the present monoclonal antibodies and antigen-binding fragments thereof can be obtained by the following method: isolating antibody-producing cell clones from healthy human blood through various steps, obtaining the antibodies from the culture supernatants of the antibody-producing cell clones, and affinity-purifying the resulting antibodies (see, for example, WO2010 / 114106).

[0107] The antibody molecules prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, and such factors, and these will be apparent to those skilled in the art. The purity of the antibody molecules of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0108] In some embodiments, the present invention also provides methods for identifying, screening or characterizing the physical / chemical properties and / or biological activities of the antibody molecules of the present invention.

[0109] In one aspect, the antibodies of the invention are tested for their antigen binding activity, for example, by known methods such as ELISA, Western blotting, etc. Binding to HCMV can be determined using methods known in the art, exemplary methods are disclosed herein. In some embodiments, surface plasmon resonance assays (e.g., affinity measurements) or ELISA assays are used.

[0110] It will be appreciated that any of the above assays can be performed using the immunoconjugates of the invention in place of or in addition to anti-human cytomegalovirus antibodies.

[0111] It will be appreciated that any of the above assays can be performed using anti-human cytomegalovirus antibodies in combination with additional active agents.

[0112] In some embodiments, the present invention provides pharmaceutical compositions comprising an antibody of the present invention.

[0113] In some embodiments, the present invention provides a composition comprising any antibody molecule described herein or a fragment thereof (preferably an antigen-binding fragment thereof) or an immunoconjugate thereof, preferably a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutical excipient.

[0114] The present invention also includes compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising antibodies of the present invention or their immunoconjugates and / or compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising polynucleotides encoding antibodies of the present invention. In certain embodiments, the compositions comprise one or more antibodies of the present invention or fragments thereof or one or more polynucleotides encoding one or more antibodies of the present invention or fragments thereof.

[0115] These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers or diluents known in the art.

[0116] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic agents, absorption delaying agents, and the like that are physiologically compatible. Pharmaceutical carriers suitable for use in the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions.

[0117] Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. For the use of excipients and their uses, see also "Handbook of Pharmaceutical Excipients", Fifth Edition, RC Rowe, PJ Eskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0118] If desired, the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0119] The compositions of the present invention can be in various forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), dispersions or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use. Common preferred compositions are in the form of injectable solutions or infusible solutions. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal (ip), intramuscular) injection. In a preferred embodiment, the antibody molecule is administered by intravenous infusion or injection. In another preferred embodiment, the antibody molecule is administered by intramuscular, intraperitoneal or subcutaneous injection.

[0120] The antibody specifically binding to human cytomegalovirus of the present invention can be lyophilized for storage and reconstituted in a suitable carrier before use. This technology has been proven to be effective for conventional protein preparations and can adopt well-known lyophilization and reconstitution techniques.

[0121] In some embodiments, the invention also provides kits comprising an antibody, pharmaceutical composition, or immunoconjugate of the invention, and optionally a package insert directing administration.

[0122] In some embodiments, the present invention also provides pharmaceutical products comprising the antibodies, pharmaceutical compositions, and immunoconjugates of the present invention, optionally further comprising a package insert for instructions for administration.

[0123] In some embodiments, the present invention also provides a method for preventing or treating human cytomegalovirus infection or human cytomegalovirus-related disease (HCMV-related disease), comprising administering an effective amount of the antibody or antigen-binding fragment thereof or immunoconjugate or pharmaceutical composition of the present invention.

[0124] "HCMV-related diseases" refers to any diseases associated with HCMV infection, including diseases in which HCMV has been shown or can be considered as the pathophysiological cause of the disease or one of the causes that exacerbate the disease. For example, various diseases caused by HCMV are also included within the scope of HCMV-related diseases. In some embodiments, HCMV-related diseases include but are not limited to, for example, (a) various diseases such as interstitial pneumonia, retinitis, gastroenteritis, encephalitis, etc. caused by the reactivation of HCMV in immunodeficiency states such as AIDS, cancer, after organ transplantation, after bone marrow transplantation, after hemodialysis, etc.; (b) congenital HCMV infection caused by the spread of HCMV infection from pregnant women to fetuses; (c) miscarriage, stillbirth, neonatal death caused by the above-mentioned congenital HCMV infection; (d) low birth weight, hepatosplenomegaly, jaundice, thrombocytopenic purpura, microcephaly, mental developmental disorder, mental retardation, chorioretinitis or hearing impairment caused by the above-mentioned congenital HCMV infection without causing death; (e) preventive or therapeutic drugs for abnormal liver function, interstitial pneumonia or mononucleosis, etc. that occur due to HCMV infection during the neonatal period or infancy. For exemplary diseases, also refer to Kazuko Taya, Infectious Diseases: Cytomegalovirus Infection, Infectious Diseases Weekly Report Japan. 2003; Week 15: 10-14; Griffiths, P.D., The treatment of cytomegalovirus infection. J. Anti. Chemo., 2002; 49: p243-253; Demmler, G.J., Congential cytomegalovirus infection and disease. Seminars in Pediatric Infection Diseases. Seminors in Pediatric Infectious Diseases, 1999; 10: p195-200.

[0125] In some embodiments, the present invention relates to methods for increasing, enhancing or stimulating the resistance of a human individual infected with human cytomegalovirus, including administering an effective amount of an antibody or an antigen-binding fragment thereof or an immunoconjugate or a pharmaceutical composition of the present invention.

[0126] In some embodiments, the individuals suitable for the methods of the present invention are HCMV-infected individuals.

[0127] In some embodiments, the individuals suitable for the methods of the present invention are transplant patients, pregnant women, neonates, HIV-infected individuals, cancer patients or patients with autoimmune diseases.

[0128] It will be appreciated that any treatment can be performed using the immunoconjugates or compositions or kits of the invention in place of or in addition to the antibodies of the invention.

[0129] The mode of administration of the antibodies of the present invention (and pharmaceutical compositions or immunoconjugates comprising the same) can be any suitable route, such as parenteral administration, for example, intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, mucosal (oral, intranasal, vaginal, rectal) or other means as known to those skilled in the art. In some embodiments, the anti-human cytomegalovirus antibodies of the present invention can be administered to a patient by any suitable route, for example, parenterally by intravenous (iv) infusion or bolus injection, intramuscularly or subcutaneously or intraperitoneally.

[0130] Various dosing schedules are contemplated herein, including, but not limited to, single dosing or multiple dosing over multiple time points, bolus dosing, and pulse infusion.

[0131] For the prevention or treatment of disease, the appropriate dosage of the antibody of the present invention will depend on the type of disease to be treated, the type of antibody, the severity and progression of the disease, whether the antibody is used for preventive purposes or for therapeutic purposes, previous treatment, the patient's clinical history and the response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient with a single treatment or through a series of treatments. Typically, the clinician administers the composition until the dosage for the desired effect is achieved. The antibody of the present invention can therefore be administered in a single dose, or administered within a certain timeframe with two or more doses (which may comprise the desired molecule of the same or different amounts), or administered by implantation or catheter continuous infusion. Appropriate dosage can be determined by using appropriate dose-response data. In certain embodiments, the antibody can be administered to the patient over an extended timeframe.

[0132] In certain embodiments, the anti-human cytomegalovirus antibodies or their antigen-binding fragments provided herein can be used to detect the presence of HCMV in a biological sample or for diagnosing HCMV infection. When the term "detection" is used herein, it includes quantitative or qualitative detection, and exemplary detection methods can relate to immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads of antibody molecule complexes, ELISA assays, PCR-technology (e.g., RT-PCR). In certain embodiments, the biological sample is other liquid samples of blood, serum or biological origin. In certain embodiments, the biological sample can be a tissue sample collected from, for example, salivary glands, lungs, liver, pancreas, kidneys, ears, eyes, placenta, digestive tract, heart, ovaries, pituitary glands, adrenal glands, thyroid glands, brain or skin.

[0133] The method of diagnosis may comprise contacting the antibody or antibody fragment with the sample. The method of diagnosis may also comprise detecting the antigen / antibody complex.

[0134] In some embodiments, the method comprises contacting a biological sample with an anti-human cytomegalovirus antibody or antigen-binding fragment thereof as described herein under conditions that allow binding of the anti-human cytomegalovirus antibody to HCMV, and detecting whether a complex forms between the anti-human cytomegalovirus antibody and HCMV. The formation of a complex indicates the presence of HCMV. The method can be an in vitro or in vivo method. In one embodiment, the anti-human cytomegalovirus antibody is used to select a subject suitable for treatment with the anti-human cytomegalovirus antibody, for example, where HCMV is a biomarker used to select the subject.

[0135] In one embodiment, the antibodies of the invention can be used to diagnose a disease described herein, e.g., to evaluate (e.g., monitor) treatment or progression of a disease described herein in an individual, diagnosis and / or staging thereof. In certain embodiments, an anti-human cytomegalovirus antibody conjugated to a marker is provided.

[0136] In some embodiments of any of the inventions provided herein, the sample is obtained prior to treatment with an anti-human cytomegalovirus antibody. In some embodiments, the sample is obtained prior to treatment with a pharmaceutical composition described herein. In some embodiments, the sample is formalin-fixed, paraffin-embedded (FFPE). In some embodiments, the sample is a biopsy (e.g., a core biopsy), a surgical specimen (e.g., a specimen from a surgical resection), or a fine needle aspirate.

[0137] In some embodiments, HCMV is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval.

[0138] In some embodiments, a detection kit comprising an antibody or antigen-binding fragment thereof of the present invention is provided for diagnosing a disease described herein, such as an HCMV-related disease or HCMV infection.

[0139] In some embodiments, a method of treating a disease described herein is provided, comprising: testing a subject (e.g., a sample) (e.g., a subject sample) for the presence of HCMV, thereby determining an HCMV value, comparing the HCMV value to a control value, and if the HCMV value is greater than the control value, administering a therapeutically effective amount of an anti-human cytomegalovirus antibody (e.g., an anti-human cytomegalovirus antibody described herein) or an antigen-binding fragment thereof to the subject, thereby treating the disease described herein.

[0140] In some embodiments, the antibodies of the invention can be used for passive immunization.

[0141] In some embodiments, the antibodies or fragments thereof of the present invention can also be used in kits for monitoring the production of vaccines with desired immunogenicity.

[0142] In some embodiments, the present invention also relates to a method for neutralizing human cytomegalovirus in an individual or sample, comprising contacting an antibody or antigen-binding fragment thereof of the present invention with the individual or sample, and testing the ability of the antibody or antigen-binding fragment thereof to bind to neutralize human cytomegalovirus.

[0143] The present invention therefore also relates to the use of the antibodies or antigen-binding fragments thereof of the present invention in the above-mentioned methods, and the use of the antibodies or antigen-binding fragments thereof of the present invention in the preparation of a medicament, composition or kit for use in the above-mentioned methods, and / or the use of the antibodies or antigen-binding fragments thereof of the present invention in the preparation of a kit for diagnosing the diseases described herein.

[0144] The methods and uses applicable to the antibodies or antigen-binding fragments thereof of the present invention are also applicable to immunoconjugates, compositions or kits comprising the antibodies or antigen-binding fragments thereof of the present invention.

[0145] In one aspect, the present invention relates to the following specific embodiments:

[0146] 1. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to human cytomegalovirus gB glycoprotein, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises three complementarity determining regions (HCDRs) of the heavy chain variable region as shown in SEQ ID NO: 13, 14, 15 or 16, and the VL comprises three complementarity determining regions (LCDRs) of the light chain variable region as shown in SEQ ID NO: 33, 34, 35 or 36.

[0147] 2. The antibody or antigen-binding fragment thereof according to embodiment 1, comprising

[0148] (1) the three CDRs of the heavy chain variable region set forth in SEQ ID NO: 13, and the three CDRs of the light chain variable region set forth in SEQ ID NO: 33;

[0149] (2) the three CDRs of the heavy chain variable region set forth in SEQ ID NO: 14, and the three CDRs of the light chain variable region set forth in SEQ ID NO: 34;

[0150] (3) the three CDRs of the heavy chain variable region set forth in SEQ ID NO: 15, and the three CDRs of the light chain variable region set forth in SEQ ID NO: 35; or

[0151] (4) The three CDRs of the heavy chain variable region shown in SEQ ID NO: 16, and the three CDRs of the light chain variable region shown in SEQ ID NO: 36.

[0152] 3. The antibody or antigen-binding fragment thereof according to embodiment 1, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0153] (i) the VH comprises complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4; HCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 5, 6, 7, or 8; and HCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 9, 10, 11, or 12;

[0154] and

[0155] (ii) wherein the VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 21, 22, 23, or 24; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 25, 26, 27, or 28; and LCDR3 comprises or consists of an amino acid sequence selected from SEQ ID NO: 29, 30, 31, or 32.

[0156] 4. The antibody or antigen-binding fragment thereof according to embodiment 1, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0157] (i) the VH comprises HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 9; and the VL comprises or consists of LCDR1, LCDR2 and LCDR3 comprising or consisting of the following sequences: SEQ ID NO: 21, SEQ ID NO: 25 and SEQ ID NO: 29; or

[0158] (ii) the VH comprises HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 10; and the VL comprises or consists of LCDR1, LCDR2 and LCDR3 comprising or consisting of the following sequences: SEQ ID NO: 22, SEQ ID NO: 26 and SEQ ID NO: 30; or

[0159] (iii) the VH comprises HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 11; and the VL comprises or consists of LCDR1, LCDR2 and LCDR3 comprising or consisting of the following sequences: SEQ ID NO: 23, SEQ ID NO: 27 and SEQ ID NO: 31; or

[0160] (iv) the VH comprises HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 12; and the VL comprises or consists of LCDR1, LCDR2 and LCDR3 comprising or consisting of the following sequences: SEQ ID NO: 24, SEQ ID NO: 28 and SEQ ID NO: 32.

[0161] 5. An antibody or antigen-binding fragment thereof that specifically binds to human cytomegalovirus gB glycoprotein, comprising a heavy chain variable region VH and a light chain variable region VL, wherein:

[0162] (a) Heavy chain variable region VH

[0163] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 13, 14, 15 or 16; or

[0164] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 13, 14, 15 or 16; or

[0165] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 13, 14, 15, or 16, wherein the amino acid changes do not occur in the CDR regions;

[0166] and

[0167] (b) Light chain variable region VL

[0168] (i) comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 33, 34, 35 or 36;

[0169] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 33, 34, 35 or 36; or

[0170] (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 33, 34, 35 or 36, wherein the amino acid changes do not occur in the CDR region.

[0171] 6. The antibody or antigen-binding fragment thereof of embodiment 5, wherein

[0172] (i) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 13; and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 33;

[0173] (ii) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 14; and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 34;

[0174] (iii) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 15; the VL comprises or consists of the amino acid sequence of SEQ ID NO: 35; or

[0175] (iv) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 16; and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 36.

[0176] 7. An antibody or antigen-binding fragment thereof that specifically binds to human cytomegalovirus gB glycoprotein, comprising a heavy chain and a light chain, wherein

[0177] (a) Heavy chain

[0178] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 37, 39, 41 or 43;

[0179] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 37, 39, 41 or 43; or

[0180] (iii) an amino acid sequence comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 37, 39, 41, or 43, preferably, the amino acid changes do not occur in the CDR region of the heavy chain, more preferably, the amino acid changes do not occur in the heavy chain variable region;

[0181] and / or

[0182] (b) Light chain

[0183] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 38, 40, 42 or 44;

[0184] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 38, 40, 42 or 44; or

[0185] (iii) comprising an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 38, 40, 42 or 44, preferably, the amino acid changes do not occur in the CDR region of the light chain, more preferably, the amino acid changes do not occur in the light chain variable region.

[0186] 8. An antibody or antigen-binding fragment thereof that specifically binds to human cytomegalovirus gB glycoprotein, wherein the antibody or antigen-binding fragment thereof binds to an epitope on the gB glycoprotein fusion domain comprising one or more amino acid residues selected from the group consisting of N208, L213 and Y226 of the gB glycoprotein fusion domain.

[0187] 9. The antibody or antigen-binding fragment thereof of embodiment 8, comprising one, two or all three CDRs in the HCDR of the heavy chain variable region shown in SEQ ID NO: 13 or SEQ ID NO: 15, and / or one, two or all three CDRs in the LCDR of the light chain variable region shown in SEQ ID NO: 33 or SEQ ID NO: 35.

[0188] 10. The antibody or antigen-binding fragment thereof of embodiment 8, comprising one, two or all three CDRs of HCDR1 shown in SEQ ID NO: 1 or 3, HCDR2 shown in SEQ ID NO: 5 or 7, or HCDR3 shown in SEQ ID NO: 9 or 11, and / or one, two or all three CDRs of HCDR1 shown in SEQ ID NO: 21 or 23, HCDR2 shown in SEQ ID NO: 25 or 27, or HCDR3 shown in SEQ ID NO: 29 or 31.

[0189] 11. The antibody or antigen-binding fragment thereof according to embodiment 8, comprising a heavy chain variable region represented by SEQ ID NO: 13 or SEQ ID NO: 15, and / or a light chain variable region represented by SEQ ID NO: 33 or SEQ ID NO: 35.

[0190] 12. The antibody or antigen-binding fragment thereof of any one of embodiments 1-11, wherein the antibody or antigen-binding fragment thereof has a K of about 50 nM or less. D Binds to human cytomegalovirus gB glycoprotein.

[0191] 13. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 11, wherein the antibody or antigen-binding fragment thereof is capable of binding to the fusion domain of human cytomegalovirus gB glycoprotein.

[0192] 14. The antibody or antigen-binding fragment thereof of any one of embodiments 1-11, wherein at least a portion of the framework sequence is a human consensus framework sequence.

[0193] 15. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-11, wherein the antibody is a human monoclonal antibody.

[0194] 16. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 11, wherein the antigen-binding fragment is selected from Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv) or (Fab')2, single-domain antibody, diabody (dAb) or linear antibody.

[0195] 17. The antibody according to any one of the preceding embodiments, wherein the antibody is an IgG class antibody, such as an IgG1 form antibody or an IgG2 form antibody or an IgG3 form antibody or an IgG4 form antibody.

[0196] 18. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of embodiments 1-17.

[0197] 19. A vector comprising the nucleic acid of embodiment 18, wherein the vector is an expression vector.

[0198] 20. A host cell comprising the vector of embodiment 19, wherein the host cell is a eukaryotic cell or a prokaryotic cell, such as an Escherichia coli cell, a yeast cell, a mammalian cell, or a plant cell. Preferably, the host cell is a CHO cell or a 293 cell, such as a HEK293 cell.

[0199] 21. A method of producing the antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, the method comprising culturing a host cell comprising a nucleic acid encoding the nucleic acid according to embodiment 18 or an expression vector according to embodiment 19.

[0200] 22. The method of embodiment 21, wherein the method further comprises recovering the antibody or antigen-binding fragment thereof from the host cell or culture medium.

[0201] 23. An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 17 and a label.

[0202] 24. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 17 or the immunoconjugate according to embodiment 23, and optionally one or more pharmaceutically acceptable excipients, such as pharmaceutically acceptable carriers, pharmaceutically acceptable excipients, including buffers or diluents.

[0203] 25. Use of the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 17 or the immunoconjugate according to embodiment 23 for preparing a pharmaceutical composition or kit for detecting, treating, preventing and / or alleviating HCMV infection or HCMV-related diseases.

[0204] 26. A method for preventing or treating HCMV infection or an HCMV-related disease in a human subject, comprising administering to a subject in need of such treatment an effective amount of the antibody or antigen-binding fragment thereof according to any one of embodiments 1-17, or the immunoconjugate of embodiment 23, or the pharmaceutical composition of embodiment 24.

[0205] 27. A method of improving, enhancing or stimulating resistance of a human subject to infection with HCMV, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof according to any one of embodiments 1-17, or the immunoconjugate of embodiment 23, or the pharmaceutical composition of embodiment 24.

[0206] 28. The method of embodiment 26 or 27, wherein the individual is infected with HCMV.

[0207] 29. The method of embodiment 26 or 27, wherein the individual is a transplant patient, a pregnant woman, a newborn, an AIDS patient, a cancer patient, or an autoimmune disease patient.

[0208] 30. A method of neutralizing HCMV in an individual or sample, comprising contacting the antibody or antigen-binding fragment thereof of any one of embodiments 1-17 with the individual or sample, and testing the ability of the antibody or antigen-binding fragment thereof to bind to neutralize HCMV.

[0209] The present invention is further illustrated in the following drawings. However, these drawings and the specific embodiments of the present invention should not be considered to limit the scope of the present invention, and changes that are easily conceivable to those skilled in the art will be included within the spirit of the present invention and the protection scope of the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0210] Figure 1 : Results of TRN1017, TRN1018, TRN1019 and TRN1020 neutralizing the standard HCMV virus strain Towne and virus strain TB40E in HFF cells.

[0211] Figure 1 A is the result of neutralization of the HCMV standard virus strain Towne in HFF cells infected with four antibodies, TRN1017, TRN1018, TRN1019 and TRN1020. Figure 1 B is the result of neutralization of HCMV strain TB40E in HFF cells infected with four antibodies, TRN1017, TRN1018, TRN1019 and TRN1020.

[0212] Figure 2 :The binding affinity of antibodies TRN1017, TRN1018, TRN1019 and TRN1020 to the antigen gB glycoprotein.

[0213] Figure 3 : Effect of single-point mutations at 12 glycosylation sites (N208, N281, N284, N302, N341, N383, N405, N409, N417, N447, N452 and N456) of gB glycoprotein on the binding activity of the antibody of the present invention.

[0214] Figure 4 :The linear structure of the extracellular segment of gB glycoprotein and its fusion domain (including three mutation positions).

[0215] definition

[0216] It should be understood that 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 will 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 those of ordinary skill in the art to which the present invention belongs.

[0217] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0218] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0219] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0220] As used herein, the terms "comprising" or "including" are intended to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of the stated elements, integers, or steps is also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0221] "Human cytomegalovirus" (HCMV) is a DNA double-helical virus of the genus Cytomegalovirus in the subfamily Herpesvirinae, also known as human herpersvirus 5 (HHV-5). As used herein, "human cytomegalovirus," "HCMV," "human herpesvirus 5," and "HHV-5" are all interchangeable.

[0222] "Human cytomegalovirus (HCMV) gB glycoprotein" (or "HCMV gB glycoprotein" or "HCMB-gB glycoprotein") is one of the main glycoproteins that constitute the HCMV coat and is known to contribute to virion cell invasion, cell fusion, and viral cell-to-cell infection. The amino acid sequence of the HCMV gB glycoprotein can be obtained from the publicly available sequence database NCBI GENE.

[0223] The “fusion domain of the human cytomegalovirus (HCMV) gB glycoprotein” or “HCMV gB glycoprotein fusion domain” refers to the region consisting of consecutive amino acid residues 150-250 in the amino acid sequence of the HCMV gB glycoprotein (Heidi G. Burke et al. Crystal Structure of the Human Cytomegalovirus Glycoprotein B. 2015).

[0224] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0225] For example, according to different CDR definition schemes, the residues of each CDR are as follows.

[0226]

[0227] A CDR can also be identified based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention).

[0228] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-mentioned ways.

[0229] Unless otherwise indicated, in the present invention, when referring to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues), the numbering refers to the position according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0230] In one embodiment, the CDRs of an antibody of the invention have boundaries defined by the IMGT rules, for example, using the IMGT database.

[0231] It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems may be different. Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0232] As used herein, the term "neutralize" refers to neutralizing the ability of a pathogen to initiate and / or maintain an infection in a host.

[0233] As used herein, the term "epitope" refers to a portion of an antigen (e.g., the gB glycoprotein of HCMV) that specifically interacts with an antibody molecule. An epitope within a protein antigen can be formed from continuous amino acids (typically linear epitopes) or discontinuous amino acids (typically conformational epitopes) juxtaposed by the tertiary folding of the protein. Epitopes formed by continuous amino acids are typically (but not always) exposed to denaturing solvents, while epitopes formed by tertiary folding are typically lost when treated with denaturing solvents.

[0234] An "antibody that binds to the same or overlapping epitope as a reference antibody" is an antibody that blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the reference antibody to its antigen in a competition assay, whereas conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the antibody to its antigen in a competition assay.

[0235] An antibody that competes with a reference antibody for binding to its antigen is one that blocks 50%, 60%, 70%, 80%, 90% or more of the binding of the reference antibody to its antigen in a competition assay. Conversely, a reference antibody blocks 50%, 60%, 70%, 80%, 90% or more of the binding of the antibody to its antigen in a competition assay. Numerous types of competition assays can be used to determine whether one antibody competes with another, such as ELISA, SPR, solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA) (see, e.g., Stahli et al., 1983, Methods in Enzymology 9: 242-253).

[0236] An antibody that inhibits (e.g., competitively inhibits) the binding of a reference antibody to its antigen is an antibody that inhibits the binding of the reference antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more. Conversely, the reference antibody inhibits the binding of the antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more. The binding of an antibody to its antigen can be measured by affinity (e.g., equilibrium dissociation constant). Methods for determining affinity are known in the art.

[0237] An antibody that exhibits the same or similar binding affinity and / or specificity as a reference antibody is an antibody that has at least 50%, 60%, 70%, 80%, 90% or more than 95% of the binding affinity and / or specificity of the reference antibody. This can be determined by any method known in the art for determining binding affinity and / or specificity.

[0238] An "IgG-type antibody" refers to an antibody whose heavy chain constant region belongs to the IgG type. All antibodies of the same type have the same heavy chain constant region, but different antibodies of different types have different heavy chain constant regions. For example, an IgG1-type antibody refers to an antibody whose heavy chain constant region Ig domain is the IgG3 domain of IgG1.

[0239] A "human" antibody (HuMAb) is an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences.

[0240] A "humanized" antibody refers to an antibody in which some, most or all of the amino acids outside the CDR domains of a non-human antibody (e.g., a mouse antibody) are replaced by corresponding amino acids derived from human immunoglobulins. In one embodiment of a humanized form of an antibody, some, most or all of the amino acids outside the CDR domains have been replaced by amino acids from human immunoglobulins, while some, most or all of the amino acids within one or more CDR regions have not changed. Small additions, deletions, insertions, substitutions or modifications of amino acids are permitted as long as they do not eliminate the ability of the antibody to bind to a specific antigen. A "humanized" antibody retains an antigenic specificity similar to that of the original antibody.

[0241] As used herein, a "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0242] As used herein, "antibody fragment" refers to a molecule different from an intact antibody, which comprises a portion of an intact antibody and binds to the antigen to which the intact antibody binds. As used herein, the term "antigen-binding fragment" refers to one or more fragments of an antibody that retains the ability to specifically bind to an antigen (e.g., gB glycoprotein of human HCMV). Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibodies (e.g., scFv), single-domain antibodies, bivalent or bispecific antibodies or fragments thereof, camelid antibodies, and bispecific antibodies or multispecific antibodies formed from antibody fragments.

[0243] As used herein, "multispecific" refers to an antibody that specifically binds to at least two different antigens or two different epitopes within an antigen, such as three, four, or five different antigens or epitopes.

[0244] As used herein, "bispecific" refers to an antibody that specifically binds to two different antigens or two different epitopes within the same antigen. A bispecific antibody may have cross-reactivity to other related antigens, or may bind to an epitope shared between two or more different antigens.

[0245] An "immunoconjugate" is an antibody conjugated to one or more additional substances, including but not limited to a label.

[0246] The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and promotes the detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., radioisotope labeling or fluorescent labeling) or can catalyze the chemical alteration of a detectable substrate compound or composition in the case of an enzymatic labeling. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically connecting) a detectable substance to the probe or antibody and indirect labeling of a probe or antibody by reacting with another reagent of direct labeling. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin.

[0247] "Vector" refers to a polynucleotide that can replicate within a biological system or can be moved between such systems. Vector polynucleotides typically contain elements such as an origin of replication, a polyadenylation signal, or a selectable marker, the function of which is to promote the replication or maintenance of these polynucleotides in a biological system. Examples of such biological systems can include cells, viruses, animals, plants, and biological systems reconstructed with biological components capable of replicating the vector. The polynucleotide comprising the vector can be a DNA or RNA molecule or a hybrid of these molecules. "Expression vector" refers to a vector that can be used in a biological system or a reconstructed biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0248] An "isolated" antibody is one that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0249] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0250] Calculation of sequence identity between sequences is performed as follows.

[0251] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

[0252] Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available at http: / / www.gcg.com), which has been integrated into the GAP program in the GCG software package. The Blossum 62 matrix or the PAM250 matrix is used, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 is used, and a length weight of 1, 2, 3, 4, 5, or 6 is used. In another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http: / / www.gcg.com). A particularly preferred parameter set (and the one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0253] The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller, (1989) CABIOS, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4.

[0254] Additionally or alternatively, one can further use the nucleic acid sequences and protein sequences described herein as a "query sequence" to perform searches against public databases to, for example, identify other family member sequences or related sequences.

[0255] As used herein, the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" describes hybridization and washing conditions. Guidance for conducting hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6, incorporated by reference. Aqueous and non-aqueous methods are described in the reference and either method can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions are 6X sodium chloride / sodium citrate (SSC) at about 45°C, followed by at least two washes in 0.2X SSC, 0.1% SDS at 50°C (the temperature of the washes can be increased to 55°C for low stringency conditions); 2) moderate stringency hybridization conditions are 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C; 3) high stringency hybridization conditions are 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C; and preferably 4) very high stringency hybridization conditions are 0.5 M sodium phosphate, 7% SDS at 65°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C. Very high stringency conditions (4) are the preferred conditions and the one that should be used unless otherwise specified. The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny screened or selected for the same function or biological activity as the initially transformed cell are included herein.

[0256] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0257] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0258] The term "effective amount" refers to an amount or dosage of an antibody, fragment, conjugate, or composition of the invention that produces the desired effect in a patient in need of treatment or prevention after administration to the patient in single or multiple doses. The effective amount can be readily determined by the attending physician, who is skilled in the art, by considering a variety of factors, such as the species of the mammal; its size, age, and general health; the specific disease involved; the extent or severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.

[0259] A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. A therapeutically effective amount of an antibody or antibody fragment, or conjugate or composition thereof, can vary depending on a variety of factors, such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment, or conjugate or composition thereof, are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%, and still more preferably at least about 80% or 90%, relative to an untreated individual. The ability of a compound to inhibit a measurable parameter can be evaluated in an animal model system that is predictive of efficacy in human autoimmune disease or inflammation.

[0260] A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result, at the required dosage and for the required period of time. Typically, a prophylactic amount will be less than a therapeutically effective amount because a prophylactic dose is used in an individual prior to or at an earlier stage of disease.

[0261] As used herein, "individual" or "subject" are used interchangeably and include mammals, such as humans.

[0262] As used herein, "treat," ...

[0263] As used herein, "prevention" includes the inhibition of the occurrence or development of a disease or condition or the symptoms of a particular disease or condition. In some embodiments, a subject or pregnant woman with a family history of an immune system disease (autoimmune disease or immunocompromise) is a candidate for a preventive regimen. Typically, in the context of an immune system disease (autoimmune disease or immunocompromise), the term "prevention" refers to the administration of a drug before the signs or symptoms of an immune system disease (autoimmune disease or immunocompromise) occur, particularly in a subject with an immune system disease (autoimmune disease or immunocompromise) risk.

[0264] "Subject / patient sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of a tissue or cell sample can be solid tissue, such as an organ or tissue sample or a biopsy sample or a puncture sample from a fresh, frozen and / or preserved organ; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during the subject's pregnancy or development. Tissue samples may contain compounds that are not naturally contaminated with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

[0265] These and other aspects and embodiments of the present invention are described in the accompanying drawings and the following detailed description of the invention and are exemplified in the following examples. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, however, it should be understood that the examples are described in an illustrative and non-limiting manner, and that various modifications may be made by those skilled in the art. Example

[0266] Example 1 Sequence Design of Specific Antigen HCMV Glycoprotein gB

[0267] Sequence information for the gB glycoprotein of 56 human cytomegalovirus (HCMV) strains (including experimental and clinical strains) was retrieved from the NCBI GENG database. Amino acid sequence alignment revealed that the gB glycoproteins shared over 70% homology. The gB glycoprotein of the Towne strain, whose sequence is shown in SEQ ID NO:48, was selected as a specific antigen (gB_Towne strain). A homologous sequence of the gB glycoproteins from all 56 strains was selected as another specific antigen (gB_Con. strain), whose sequence is shown in SEQ ID NO:49. These antigens were used to screen for broad-spectrum anti-HCMV monoclonal antibodies.

[0268] SEQ ID NO:48

[0269] MESRIWCLVVCVNLCIVCLGAAVSSSSTRGTSATHSHHSSHTTSAAHSRSGSVSQRVTSSQTVSHGVNETIYNTTLKYGD

[0270] VVGVNTTKYPYRVCSMAQGTDLIRFERNIVCTSMKPINEDLDEGIMVVYKRNIVAHTFKVRVYQKVLTFRRSYAYIHTTY

[0271] LLGSNTEYVAPPMWEIHHINSHSQCYSSYSRVIAGTVFVAYHRDSYENKTMQLMPDDYSNTHSTRYVTVKDQWHSRGSTW

[0272] LYRETCNLNCMVTITTARSKYPYHFFATSTGDVVDISPFYNGTNRNASYFGENADKFFIFPNYTIVSDFGRPNSALETHR

[0273] LVAFLERADSVISWDIQDEKNVTCQLTFWEASERTIRSEAEDSYHFSSAKMTATFLSKKQEVNMSDSALDCVRDEAINKL

[0274] QQIFNTSYNQTYEKYGNVSVFETTGGLVVFWQGIKQKSLVELERLANRSSLNLTHNETKESTDGNNATHLSNMESVHNLV

[0275] YAQLQFTYDTLRGYINRALAQIAEAWCVDQRRTLEVFKELSKINPSAILSAIYNKPIAARFMGDVLGLASCVTINQTSVK

[0276] VLRDMNVKESPGRCYSRPVVIFNFANSSYVQYGQLGEDNEILLGNHRTEECQLPSLKIFIAGNSAYEYVDYLFKRMIDLS

[0277] SISTVDSMIALDIDPLENTDFRVLELYSQKELRSINVFDLEEIMREFNSYKQRVKYVEDKGLNDIFEAQKIEWHELEVLFQGPGHHHHHHH。

[0278] SEQ ID NO:49

[0279] MESRIWCLVVCVNLCIVCLGAAVSSSSTSHATSSTHNGSHTSRTTSAQTRSVSQHVTSSEAVSHRANETIYNTTLKYGDVVGVNTTKYPYRVCSMAQGTDLIRFERNIVCTPMKPINEDLDEGIMVVYKRNIVAHTFKVRVYQKVLTFRRSYAYIHTTYLLGSNTEYVAPPMWEIHHINSHSQCYSSYSRVIAGTVFVAYHRDSYENKTMQLMPDDYSNTHSTRYVTVKDQWHSRGSTWLYRETCNLNCMVTITTARSKYPYHFFATSTGDVVDISPFYNGTNRNASYFGENADKFFIFPNYTIVSDFGRPNSAPETHRLVAFLERADSVISWDIQDEKNVTCQLTFWEASERTIRSEAEDSYHFSSAKMTATFLSKKQEVNMSDSALDCVRDEAINKLQQIFNTSYNQTYEKYGNVSVFETTGGLVVFWQGIKQKSLVELERLANRSSLNLTHETKESTDGNNTTHLSNMESVHNLVYAQLQFTYDTLRGYINRALAQIAEAWCVDQRRTLEVFKELSKINPSAILSAIYNKPIAARFMGDVLGLASCVTINQTSVKVLRDMNVKESPGRCYSRPVVIFNFANSSYVQYGQLGEDNEILLGNHRTEECQLPSLKIFIAGNSAYEYVDYLFKRMIDLSSISTVDSMIALDIDPLENTDFRVLELYSQKELRSSNVFDLEEIMREFNSYKQRVKYVEDKGLNDIFEAQKIEWHELEVLFQGPGHHHHHHH。

[0280] Example 2 Screening of Serum Antibodies against HCMV gB Glycoprotein

[0281] 1. Screening Process

[0282] Immunocompromised patients were recruited from various hospitals and a total of 272 whole blood samples were obtained from eligible patients. Human peripheral blood mononuclear cells (PBMCs) and plasma were separated using Ficoll lymphocyte separation medium (GE). Two designed HCMV gB glycoproteins were expressed and purified using conventional methods to a concentration of 2 μg / ml and then used for ELISA assays to measure antibody titers (using EC50 to measure serum antibody titers) in the 272 blood samples (and plasma after separation) and to classify cancer.

[0283] 2. Results

[0284] A total of 272 clinical blood samples from 17 different cancer types were collected, and the results showed that 268 samples were seropositive, a positivity rate of 98.53%, consistent with the reported prevalence of HCMV infection in the human population. These samples were from patients with glioblastoma, lung cancer, rectal cancer, liver cancer, gastric cancer, esophageal cancer, colon cancer, kidney cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, bladder cancer, gallbladder cancer, cervical cancer, and lupus erythematosus, indicating that HCMV is widely present in immunocompromised populations such as cancer patients and those with autoimmune diseases. Anti-human cytomegalovirus antibodies are present in these patients, and therefore anti-human cytomegalovirus monoclonal antibodies can be screened and obtained from their B cells.

[0285] Example 3 Isolation of anti-human cytomegalovirus monoclonal antibody gene

[0286] 1. Isolation of gB_Towne strain and gB_Con.Strain protein antigen-specific B cells

[0287] In order to improve the sorting efficiency of memory B cells, two HCMV gB glycoproteins (gB_Townestrain and gB_Con.Strain) were used simultaneously to perform specific sorting on the sample numbered Es0018 in Example 3. The sorting steps were as follows:

[0288] gB antigen-specific memory B lymphocytes from human peripheral blood can be isolated using a flow cytometer (BD FACSAria) according to the manufacturer's instructions. Single memory B lymphocytes are isolated in single cell sorting mode using a logic gate using the CD3- / CD14- / CD16- / CD235a- / CD19+ / SIgD- / labeled gB_Towne and gB_Con.+. To increase sorting specificity, both gB_Towne and gB_Con are labeled with dual-color fluorescence (PE-Cy7 and BV421). The cells sorted are double-positive cells located on the diagonal line and single-positive cells within the logic gate.

[0289] 3. Single cell antibody variable region gene amplification steps

[0290] The steps are as follows: A complete set of RT-PCR / PCR primers and experimental conditions have been successfully designed and established for amplifying antibody variable region genes of all 7 known human antibody heavy chain families and 13 light chain families, including IgG, IgA, IgM, IgD and IgE (see CN 107760690 B).

[0291] 4. Results

[0292] Multiple memory B cells were isolated from the sample. Designed primers were used to amplify the antibody variable region genes in single cells to obtain antibody gene sequences. The somatic mutation rate of the antibody heavy chain VH gene segment ranged from 10% to 18.5%. Single-cell antibody variable region gene amplification yielded 103 heavy chain variable region gene sequences and 109 light chain variable region gene sequences. After paired testing, the antibody positivity rate was 32.4%. The somatic mutation rates of the heavy chain and light chain of the positive antibodies ranged from 7% to 16.5%, and 2% to 15%, respectively.

[0293] Example 4 Expression of anti-human cytomegalovirus monoclonal antibodies

[0294] 1. Construction of expression vector

[0295] Linear expression vectors were constructed for the heavy chain variable region gene sequence and light chain variable region gene sequence of the above-mentioned successfully paired antibodies, and the isolated heavy chain and light chain variable region genes were combined with their respective constant region sequences using the Overlapping PCR method (Crystal Structure of the Human Cytomegalovirus Glycoprotein B, Heidi G. Burke and Ekaterina E. Heldwein, PLoS Pathog. 2015 Oct; 11(10): e1005227) to form full-length heavy chain and light chain genes, and the recombinant antibodies were expressed for antibody screening and identification.

[0296] 2. Screening for positive antibodies

[0297] The expression vector (pcDNA3.1) containing the heavy and light chain genes obtained above was transfected into HEK293 cells using a transfection reagent (Quigen) and cultured in 12-well plates in DMEM medium containing FBS for 48 hours. After 48 hours, the cell culture supernatant was collected and ELISA was used to detect antibody expression and binding to the antigen HCMV gB glycoprotein (gB_Towne strain and gB_Con.Strain). The standard was TRN006 (CN 103910796 B) with a starting concentration of 0.5 μg / mL; the cell culture supernatant had a starting concentration of the stock solution and was diluted 10-fold in four gradients; commercially available SuperBlock mouse anti-human IgG Fab-HRP (Abcam) was diluted 1:10,000. After terminating the reaction, dual-wavelength readings at 450 nm (sample) and 630 nm (plate wells) were measured using a multifunctional microplate reader, and the final calculated value was OD450-OD630. Points where the TRN006 OD450-OD630 values had a good linear relationship (R2>0.99) with the corresponding concentrations were selected to establish a linear regression equation, and the antibody concentration was calculated using the sample values falling within the linear interval.

[0298] 3. Identification results

[0299] Finally, linear expression vectors were constructed for the heavy and light chains of the 134 successfully paired antibodies, and co-transfected into HEK293 cells. 42 binding-positive antibodies were screened, with a positive rate of 32.3%. Among them, 36 strains showed strong positivity for gB_Townestrain, and 24 strains showed strong positivity for both gB_Con.strain and gB_Towne strain.

[0300] Among the four antibodies screened according to the present invention, the heavy chain may have CDR1s of SEQ ID NOs: 1-4, and / or CDR2s of SEQ ID NOs: 5-8, and / or CDR3s of SEQ ID NOs: 9-12. In specific embodiments, the light chain may have CDR1s of SEQ ID NOs: 21-24, and / or CDR2s of SEQ ID NOs: 25-28, and / or CDR3s of SEQ ID NOs: 29-32.

[0301] TRN1017

[0302] DNA sequencing (Invitrogen) of the corresponding DNA preparations identified 24 strains that showed strong positive antibodies to both the gB_Con.strain and the gB_Towne strain. One of the antibodies was identified as TRN1017, which had:

[0303] the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 13, and the light chain variable region amino acid sequence set forth in SEQ ID NO: 33;

[0304] The heavy chain amino acid sequence is shown in SEQ ID NO: 37, and the light chain amino acid sequence is shown in SEQ ID NO: 38.

[0305] TRN1018

[0306] DNA sequencing of the corresponding DNA preparations identified 24 strains that showed strong positive antibodies to both the gB_Con.strain and the gB_Towne strain. One of the antibodies was identified as TRN1018, which had:

[0307] the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 14, and the light chain variable region amino acid sequence set forth in SEQ ID NO: 34;

[0308] The heavy chain amino acid sequence is shown in SEQ ID NO: 39, and the light chain amino acid sequence is shown in SEQ ID NO: 40.

[0309] TRN1019

[0310] DNA sequencing of the corresponding DNA preparations identified 24 strains that showed strong positive antibodies against both the gB_Con.strain and the gB_Towne strain. One of the antibodies was identified as TRN1019, which had:

[0311] the heavy chain variable region amino acid sequence shown in SEQ ID NO: 15, and the light chain variable region amino acid sequence shown in SEQ ID NO: 35;

[0312] The heavy chain amino acid sequence is shown in SEQ ID NO: 41, and the light chain amino acid sequence is shown in SEQ ID NO: 42.

[0313] TRN1020

[0314] DNA sequencing of the corresponding DNA preparations identified 24 strains that showed strong positive antibodies against both the gB_Con.strain and the gB_Towne strain. One of the antibodies was identified as TRN1020, which had:

[0315] the heavy chain variable region amino acid sequence shown in SEQ ID NO: 16, and the light chain variable region amino acid sequence shown in SEQ ID NO: 36;

[0316] The heavy chain amino acid sequence is shown in SEQ ID NO: 43, and the light chain amino acid sequence is shown in SEQ ID NO: 44.

[0317] The amino acid sequences of the variable regions were analyzed and the CDR sequences of the four antibodies were determined using the IMGT numbering rules as follows:

[0318]

[0319] Example 5 Evaluation of the antiviral neutralization ability of anti-human cytomegalovirus monoclonal antibodies

[0320] 1. Evaluation of Antiviral Neutralizing Ability of Antibodies Using HFF Cells

[0321] The cell model HFF, as well as the standard HCMV virus strain Towne and virus strain TB40E (all purchased from ATCC) were selected to evaluate the neutralizing ability of antibodies. The controls were the reported HCMV-positive antibodies SM5_1 and SM10 (Crystal Structure of the Human Cytomegalovirus Glycoprotein B, Heidi G. Burke and Ekaterina E. Heldwein, PLoS Pathog. 2015 Oct; 11(10): e1005227). Before infection, 5.00E+03 HFF cells were plated per well in a 96-well plate, and the cell density should reach about 90%. The starting concentration of the antibody was 100 μg / mL, and 8 gradients of 3-fold dilution were made in PBS. 10 μL of DMEM medium was added to each well. The virus control group was added with 10 μL of DMEM medium. The cells were infected with a virus at an MOI of 0.96, that is, 40 μL of virus stock solution was added per well. After mixing, the cells were incubated in a 37°C incubator for 1 hour as the virus infection solution. Discard the cell supernatant from the 96-well plate and rinse twice with 100 μL PBS. Add 50 μL of viral infection solution containing different antibody dilutions to the 96-well plate and incubate at 37°C for 4 hours. Aspirate the viral infection solution and rinse once with 100 μL PBS. Add 100 μL of DMEM medium to each well and incubate in a 37°C, 5% CO2 incubator for 96 hours. Observe and record the cell status daily during this period. Discard the cell culture supernatant and rinse twice with PBS. Add 50 μL of 4% paraformaldehyde to each well and fix the cells at room temperature for 30 minutes. Rinse twice with PBS. Stain the cell nuclei with 100 μL of 10 μg / mL Hoechst for 15 minutes. Rinse three times with 200 μL PBS (gently shaking the 96-well culture plate). Observe the cell nuclear staining using blue light and the viral infection using green light. The fluorescence intensity of the emitted light at 535 nm was measured under 485 nm excitation light. The relative fluorescence intensity was used to describe the degree of cell infection with the virus and the antiviral effect of the antibody. The results were calculated (formula: antiviral effect = 100 - fluorescence intensity of the antibody-virus complex group / fluorescence intensity of the virus group * 100).

[0322] 2. Evaluation of Antiviral Neutralizing Ability of Antibodies in Primary HUVEC Cells

[0323] The ability of antibodies to inhibit Towne's infection was evaluated in primary HUVEC cells. Before infection, 4.00E+04 HUVEC cells were plated per well in a 96-well plate, reaching a cell density of approximately 80%. Using a starting concentration of 100 μg / mL, the antibody was diluted three-fold in eight steps, with 10 μL added to each well. For the virus control, 10 μL of culture medium was added. The cells were infected with virus at an MOI of 5 by adding 40 μL of the virus stock solution per well, mixing, and incubating in a 37°C incubator for 1 hour. The supernatant from the 96-well plate was discarded, and the cells were rinsed twice with 100 μL of PBS. 50 μL of the virus infection solution at various antibody dilutions was added to the 96-well plate and infected for 4 hours at 37°C. The infection solution was aspirated, and 100 μL of DMEM medium was added to each well. The cells were cultured at 37°C in a 5% CO2 incubator for 7 days, with daily observation and recording of cell status. The cell culture supernatant was discarded, and the cells were rinsed twice with PBS. Add 50 μL of 4% paraformaldehyde to each well and incubate at room temperature for 30 minutes to fix the cells. Rinse twice with PBS. Stain the cell nuclei with 100 μL of 10 μg / mL Hoechst for 15 minutes. Rinse three times with 200 μL of PBS (gently shaking the 96-well culture plate). Observe the cell nuclei staining with blue light and the virus infection with green light. Measure the fluorescence intensity of the 535 nm emission light under 485 nm excitation light. Use relative fluorescence intensity to describe the degree of cell infection with the virus and the antiviral effect of the antibody. Calculate the results (formula: antiviral infection effect = 100 - fluorescence intensity of the antibody-virus complex group / fluorescence intensity of the virus group * 100)

[0324] 3. Results

[0325] SM5_1 and SM10 in the result graph are reported anti-cytomegalovirus antibodies, used as positive controls; TRN079 is a rabies virus antibody (CN201611069303.4), HCV0082 is an HCV antibody, and two unrelated antibodies are used as negative controls. Figure 1 As shown in Figure A, the EC50 values of the four antibodies, TRN1017, TRN1018, TRN1019, and TRN1020, against the Towne strain of HCMV in the HFF cell line ranged from 0.017 to 0.074 μg / ml. EC50 stands for median effective concentration, which is the concentration that elicits a specific response in 50% of the test animals or inhibits a specific response by half. A lower EC50 indicates a stronger neutralizing antibody. Figure 1As shown in Figure B, in the HFF cell line, the EC50 of antibodies from strains TRN1017, TRN1018, TRN1019, and TRN10204 against the clinical HCMV strain TB40E was 0.015-0.091 ug / ml. These results demonstrate that the antibodies of the present invention have extremely high neutralizing activity against both the standard HCMV strain Towne and the HCMV strain TB40E.

[0326] At the same time, the ability of two of the antibodies, TRN1017 and TRN1020, to resist Towne virus infection was evaluated in the primary HUVEC cell line, and the antibodies were also shown to have extremely high neutralizing activity, with EC50 values of 0.089ug / ml and 0.041ug / mL, respectively.

[0327] HUVEC stands for human umbilical vein endothelial cells, and HFF stands for human foreskin fibroblasts (HFF). These results demonstrate that the antibodies of the present invention have a high ability to neutralize different HCMV strains. Furthermore, the antibodies of the present invention have neutralizing abilities against HCMV infected with different cell types, suggesting that the antibodies of the present invention have the potential to neutralize HCMV in different tissue locations within an individual.

[0328] Example 6 Antibody Affinity Determination

[0329] The binding ability of antibodies to gB glycoprotein (gB_Towne strain) was determined in vitro using Biacore (SPR principle). Anti-human IgG (Fc) was coupled to two channels of a CM5 chip via amino coupling, with channel 1 coupled to 5485.4RU and channel 2 coupled to 5622.4RU. The capture antibody concentration was 1ug / ml and the binding time was 45s. The concentrations of the bound antigen protein gB0049 were 1.25ug / ml, 2.5ug / ml, 5ug / ml, 10ug / ml, and 20ug / ml, with an association time of 90s and a dissociation time of 600s. The regeneration solution was 3M MgCl2, and the regeneration time was 30s.

[0330] The results are as follows Figure 2 As shown, the K of the antibody binding to gB glycoprotein D The values are all in the nM range, among which the K D The value is 0.60 nM, and the K D The value was 27.9 nM and the K D The value is 1.13 nM, indicating that the antibody of the present invention has a high binding affinity to the antigen gB glycoprotein.

[0331] Example 7 Antibody Antigen Binding Epitope Study

[0332] 1. Effect of antigen deglycosylation on antibody binding ability

[0333] The present invention studies the effect of gB glycosylation on antibodies. First, a kit is used to perform single-point mutations on 12 glycosylation sites (N208, N281, N284, N302, N341, N383, N405, N409, N417, N447, N452 and N456) of the gB_Towne protein. After the mutations are converted to glutamine (the physicochemical properties of asparagine and glutamine are similar, and the effect of the mutation on the spatial structure is minimally changed), indirect ELISA is used to detect the binding activity of the gB glycoprotein mutants with three antibodies, SM10, TRN1017 and TRN1019.

[0334] like Figure 3 The results show that after the gB glycoprotein N208, N281, N284, N302, N341, N383, N405, N409, N417, N447, N452 and N456 glycosylation sites were mutated to glutamine, the binding ability of the TRN1017 and TRN1019 antibodies of the present invention to the gB glycoprotein N208Q mutant was significantly reduced, and the binding change with other gB glycoprotein mutants was not obvious. At the same time, considering that the binding epitope of the control antibody SM10 is adjacent to the N208 site in spatial structure, and the binding change of SM10 to the gB glycoprotein N208Q mutant is not significant, it shows that the mutation of N208 does not change the adjacent spatial structure too much, thereby proving that the N208 site on the gB glycoprotein is the key antigen binding epitope of the antibody of the present invention.

[0335] 3. Site-directed mutagenesis of amino acid sites of gB glycoprotein

[0336] To further characterize the epitopes of these antibodies, we performed an alanine scan (186aa-228aa) centered on the N208 site, ultimately identifying sites L213 and Y226. Using SPR, we determined the affinity constants of TRN1017, TRN1018, TRN1019, and TRN1020 antibodies for mutants targeting these two sites. The results showed a significant decrease in binding capacity for these mutants (no significant change in binding was observed for the control antibody 1G2), indicating that L213 and Y226 are key epitopes for the antibodies described herein.

[0337] Thus, the information of three epitope amino acids of these antibodies was determined (N208, L213 and Y226), and these three amino acids are structurally located in the fusion domain of gB glycoprotein (amino acids R150-C250, this region is the structural basis for the membrane fusion function of gB glycoprotein). Antibodies targeting this domain can directly block the virus from invading host cells ( Figure 4 ).

[0338] In summary, different from the antibody (Structure of HCMV glycoprotein B in the postfusion conformation bound to aneutralizing human antibody, Sumana Chandramouli et al., Nature Communications volume 6, Article number: 8176 (2015)) reported for AD-1, AD-2, AD-4 and AD-5 domains; Germline V-genes sculpt the binding site of a family of antibodies neutralizing human cytomegalovirus, Christy AThomson et al., The EMBO Journal (2008) 27, 2592-2602), the gB glycoprotein fusion domain that the antibody epitope of the present invention is directed to. And these antibodies all show very high virus neutralizing activity, have very high drug potential. Simultaneously, the identification of antibody epitope information provides a theoretical basis for the design of HCMV-gB glycoprotein structure vaccine.

[0339] Example 8 Antibody Antinuclear Resistance Detection

[0340] Hep-2 cells are human laryngeal cancer epithelial cells. Due to their rich antigenic spectrum, strong antigenic specificity, and high antigen content, the indirect immunofluorescence assay (IFA) using Hep-2 cells as a substrate is commonly used internationally as the standard method for detecting antinuclear antibodies (ANAs). This method was used to test the antinuclear resistance of four strains, TRN1017, TRN1018, TRN1019, and TRN1020, according to the instructions for the antinuclear antibody (ANA) detection kit.

[0341] The results showed that the four antibodies TRN1017, TRN1018, TRN1019 and TRN1020 had no immunofluorescence reaction to Hep-2 cells at a concentration of 100ug / ml, indicating that the antibodies had no autoimmune reaction to Hep-2 cells.

[0342] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

[0343] Sequence Listing:

[0344] Heavy chain complementary determining region CDR sequence information table

[0345] serial number CDR Sequence information SEQ ID NO: 1 CDR1 GFTFRHYW SEQ ID NO:2 CDR1 GFTFSNHG SEQ ID NO:3 CDR1 GYTFTNYW SEQ ID NO:4 CDR1 GFSFSTYA SEQ ID NO:5 CDR2 IHGSGTTT SEQ ID NO:6 CDR2 ISSDGTDT SEQ ID NO:7 CDR2 IFPRDSYS SEQ ID NO:8 CDR2 VSGRGTST SEQ ID NO:9 CDR3 VRDDYTSGYN SEQ ID NO: 10 CDR3 ARDGRCGDERCYSGLPDV SEQ ID NO:11 CDR3 AIYNDLRSGNS SEQ ID NO:12 CDR3 AKDSYVGGSFDWLPRPDYFDH

[0346] Light chain complementary determining region CDR sequence information table

[0347] serial number CDR Sequence information SEQ ID NO:21 CDR1 RSDVGGYNY SEQ ID NO:22 CDR1 QSVGGY SEQ ID NO:23 CDR1 QSITKY SEQ ID NO:24 CDR1 NSNIGKNY SEQ ID NO:25 CDR2 DVS SEQ ID NO:26 CDR2 DAS SEQ ID NO:27 CDR2 TTS SEQ ID NO:28 CDR2 NNN SEQ ID NO:29 CDR3 SSYTTKSTLYV SEQ ID NO:30 CDR3 QQRSNWPPLT SEQ ID NO:31 CDR3 QQSFSTLWT SEQ ID NO:32 CDR3 ATWDKTLNFWV

[0348] Antibody heavy chain variable region (VH) and light chain variable region (VL) amino acid sequence information table

[0349]

[0350] Antibody heavy chain (HC) and light chain (LC) amino acid sequences

[0351]

[0352]

[0353]

[0354] Light chain lambda constant region:

[0355] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWK SHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO:45)

[0356] Light chain kappa (κ) constant region:

[0357] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:46)

[0358] Heavy chain constant region:

[0359] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT

[0360] YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW

[0361] YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE

[0362] MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT

[0363] QKSLSLSPGK(SEQ ID NO:47)

Claims

1. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to human cytomegalovirus gB glycoprotein for use in the preparation of a pharmaceutical composition or kit for detecting, treating, preventing and / or alleviating HCMV infection; wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein (i) the VH comprises HCDR1, HCDR2 and HCDR3 consisting of the following sequences: SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 9; and the VL comprises LCDR1, LCDR2 and LCDR3 consisting of the following sequences: SEQ ID NO: 21, SEQ ID NO: 25 and SEQ ID NO: 29; or (ii) the VH comprises HCDR1, HCDR2 and HCDR3 consisting of the following sequences: SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 10; and the VL comprises LCDR1, LCDR2 and LCDR3 consisting of the following sequences: SEQ ID NO: 22, SEQ ID NO: 26 and SEQ ID NO: 30; or (iii) the VH comprises HCDR1, HCDR2 and HCDR3 consisting of the following sequences: SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 11; and the VL comprises LCDR1, LCDR2 and LCDR3 consisting of the following sequences: SEQ ID NO: 23, SEQ ID NO: 27 and SEQ ID NO: 31; or (iv) the VH comprises HCDR1, HCDR2 and HCDR3 consisting of the following sequences: SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 12; and the VL comprises LCDR1, LCDR2 and LCDR3 consisting of the following sequences: SEQ ID NO: 24, SEQ ID NO: 28 and SEQ ID NO:

32.

2. Use of a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human cytomegalovirus gB glycoprotein, or a pharmaceutical composition comprising said antibody or antigen-binding fragment, in the preparation of a medicament for preventing or treating HCMV infection in a human individual; wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein (i) the VH comprises HCDR1, HCDR2 and HCDR3 consisting of the following sequences: SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 9; and the VL comprises LCDR1, LCDR2 and LCDR3 consisting of the following sequences: SEQ ID NO: 21, SEQ ID NO: 25 and SEQ ID NO: 29; or (ii) the VH comprises HCDR1, HCDR2 and HCDR3 consisting of the following sequences: SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 10; and the VL comprises LCDR1, LCDR2 and LCDR3 consisting of the following sequences: SEQ ID NO: 22, SEQ ID NO: 26 and SEQ ID NO: 30; or (iii) the VH comprises HCDR1, HCDR2 and HCDR3 consisting of the following sequences: SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 11; and the VL comprises LCDR1, LCDR2 and LCDR3 consisting of the following sequences: SEQ ID NO: 23, SEQ ID NO: 27 and SEQ ID NO: 31; or (iv) the VH comprises HCDR1, HCDR2 and HCDR3 consisting of the following sequences: SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 12; and the VL comprises LCDR1, LCDR2 and LCDR3 consisting of the following sequences: SEQ ID NO: 24, SEQ ID NO: 28 and SEQ ID NO:

32.

3. Use of a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human cytomegalovirus gB glycoprotein, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, in the preparation of a medicament for improving, enhancing, or stimulating a human subject's resistance to infection with HCMV; wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein (i) the VH comprises HCDR1, HCDR2 and HCDR3 consisting of the following sequences: SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 9; and the VL comprises LCDR1, LCDR2 and LCDR3 consisting of the following sequences: SEQ ID NO: 21, SEQ ID NO: 25 and SEQ ID NO: 29; or (ii) the VH comprises HCDR1, HCDR2 and HCDR3 consisting of the following sequences: SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 10; and the VL comprises LCDR1, LCDR2 and LCDR3 consisting of the following sequences: SEQ ID NO: 22, SEQ ID NO: 26 and SEQ ID NO: 30; or (iii) the VH comprises HCDR1, HCDR2 and HCDR3 consisting of the following sequences: SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 11; and the VL comprises LCDR1, LCDR2 and LCDR3 consisting of the following sequences: SEQ ID NO: 23, SEQ ID NO: 27 and SEQ ID NO: 31; or (iv) the VH comprises HCDR1, HCDR2 and HCDR3 consisting of the following sequences: SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 12; and the VL comprises LCDR1, LCDR2 and LCDR3 consisting of the following sequences: SEQ ID NO: 24, SEQ ID NO: 28 and SEQ ID NO:

32. The use according to claim 2 or 3, wherein the individual is HCMV-infected.

5. The use according to claim 2 or 3, wherein the individual is a transplant patient, a pregnant woman, a newborn, an AIDS patient, a cancer patient or an autoimmune disease patient.

6. The method of any one of claims 1 to 3, wherein the heavy chain variable region VH comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 13, 14, 15 or 16.

7. The method of claim 6, wherein the heavy chain variable region VH comprises or consists of an amino acid sequence selected from SEQ ID NO: 13, 14, 15 or 16.

8. The method of any one of claims 1 to 3, wherein the light chain variable region (VL) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 33, 34, 35 or 36.

9. The method of claim 8, wherein the light chain variable region VL comprises or consists of an amino acid sequence selected from SEQ ID NO: 33, 34, 35 or 36.

10. The method of claim 1, wherein (i) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 13; and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 33; (ii) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 14; and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 34; (iii) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 15; the VL comprises or consists of the amino acid sequence of SEQ ID NO: 35; or (iv) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 16; and the VL comprises or consists of the amino acid sequence of SEQ ID NO:

36.

11. The method of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 37, 39, 41 or 43.

12. The use of claim 11, wherein the heavy chain comprises or consists of an amino acid sequence selected from SEQ ID NO: 37, 39, 41 or 43.

13. The use of any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 38, 40, 42 or 44.

14. The use of claim 11, wherein the light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 38, 40, 42 or 44.

15. The use of claim 14, wherein the light chain comprises or consists of an amino acid sequence selected from SEQ ID NO: 38, 40, 42 or 44.

16. The use of claim 13, wherein the light chain comprises or consists of an amino acid sequence selected from SEQ ID NO: 38, 40, 42 or 44.

17. The method of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 37; and the light chain comprises or consists of the amino acid sequence of SEQ ID NO: 38; (ii) the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 39; and the light chain comprises or consists of the amino acid sequence of SEQ ID NO: 40; (iii) the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 41; and the light chain comprises or consists of the amino acid sequence of SEQ ID NO: 42; or (iv) the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 43; and the light chain comprises or consists of the amino acid sequence of SEQ ID NO:

44.

18. The use of any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof has a K of 50 nM or less. D Binds to human cytomegalovirus gB glycoprotein.

19. The use according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof is capable of binding to the fusion domain of human cytomegalovirus gB glycoprotein.

20. The use of any one of claims 1 to 3, wherein at least a portion of the framework sequence of the antibody or antigen-binding fragment thereof is a human consensus framework sequence.

21. The use of any one of claims 1-3, wherein the antibody is a human monoclonal antibody.

22. The use of any one of claims 1-3, wherein the antigen-binding fragment is selected from Fab, Fab', Fab'-SH, Fv, single-chain antibody, (Fab')2, or diabody.

23. The use of claim 22, wherein the single-chain antibody is a scFv.

24. The use according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof is an IgG class antibody or antigen-binding fragment thereof.

25. The use according to claim 24, wherein the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof in the form of IgG1, IgG2, IgG3 or IgG4.

26. The use according to any one of claims 1 to 3, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

27. The method according to claim 26, wherein the pharmaceutical excipient is selected from the group consisting of pharmaceutical carriers and pharmaceutical excipients.

28. The use of claim 27, wherein the pharmaceutically acceptable excipient is selected from a buffer or a diluent.

29. A non-diagnostic method for neutralizing HCMV in a sample in vitro, comprising contacting the sample with the antibody or antigen-binding fragment thereof as defined in any one of claims 1 to 25, and testing the ability of the antibody or antigen-binding fragment thereof to bind to neutralize HCMV.

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