ANTIBODY AGAINST HUMAN CYTOMEGALOVIRUS AND USE THEREOF

Abstract

Disclosed are a monoclonal antibody that is specific to human cytomegalovirus and binds to human cytomegalovirus with a high affinity, or an antigen-binding fragment thereof, and a method for preparing the antibody. The antibody is also highly effective in neutralizing infections. Also disclosed are an epitope to which the antibody binds, and the use of the antibody in the diagnosis, prevention and treatment of an infected individual.

Claims

1.-30. (canceled)

31. An antibody or an antigen-binding fragment thereof specifically binding 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 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 shown in SEQ ID NO: 33, 34, 35, or 36; preferably, the antibody or antigen-binding fragment thereof comprises: (1) three CDRs of the heavy chain variable region shown in SEQ ID NO: 13, and three CDRs of the light chain variable region shown in SEQ ID NO: 33; (2) three CDRs of the heavy chain variable region shown in SEQ ID NO: 14, and three CDRs of the light chain variable region shown in SEQ ID NO: 34; (3) three CDRs of the heavy chain variable region shown in SEQ ID NO: 15, and three CDRs of the light chain variable region shown in SEQ ID NO: 35; or (4) three CDRs of the heavy chain variable region shown in SEQ ID NO: 16, and three CDRs of the light chain variable region shown in SEQ ID NO: 36; preferably, 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 the complementarity determining regions (CDR) 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; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 9, 10, 11, or 12; and, (ii) wherein the VL comprises the complementarity determining regions (CDR) 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; LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 29, 30, 31, or 32; preferably, 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 comprising or consisting of the following sequences of SEQ ID NO: 1, SEQ IDNO: 5, and SEQ ID NO: 9; and the VL comprises LCDR1, LCDR2, and LCDR3 comprising or consisting of the following sequences of SEQ ID NO: 21, SEQ IDNO: 25, and SEQ ID NO: 29; or (ii) the VH comprises HCDR1, HCDR2, and HCDR3 comprising or consisting of the following sequences of SEQ ID NO: 2, SEQ IDNO: 6, and SEQ ID NO: 10; and the VL comprises LCDR1, LCDR2, and LCDR3 comprising or consisting of the following sequences of SEQ ID NO: 22, SEQ IDNO: 26, and SEQ ID NO: 30; or (iii) the VH comprises HCDR1, HCDR2, and HCDR3 comprising or consisting of the following sequences of SEQ ID NO: 3, SEQ IDNO: 7, and SEQ ID NO: 11; and the VL comprises LCDR1, LCDR2, and LCDR3 comprising or consisting of the following sequences of SEQ ID NO: 23, SEQ IDNO: 27, and SEQ ID NO: 31; or (iv) the VH comprises HCDR1, HCDR2, and HCDR3 comprising or consisting of the following sequences of SEQ ID NO: 4, SEQ IDNO: 8, and SEQ ID NO: 12; and the VL comprises LCDR1, LCDR2, and LCDR3 comprising or consisting of the following sequences of SEQ ID NO: 24, SEQ IDNO: 28, and SEQ ID NO: 32.

32. The antibody or antigen-binding fragment thereof according to claim 31, comprising a heavy chain variable region VH and a light chain variable region VL, wherein (a) the heavy chain variable region VH (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 the group consisting of SEQ ID NOs: 13, 14, 15, or 16; or (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 13, 14, 15, or 16; or (iii) comprises an amino acid sequence having 1 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 amino acid conservative 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; and, (b) the light chain variable region VL (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 the group consisting of SEQ ID NO: 33, 34, 35, or 36; (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 33, 34, 35, or 36; or (iii) comprises an amino acid sequence having 1 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 amino acid conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO 33, 34, 35 or 36, wherein the amino acid changes do not occur in the CDR regions; preferably, the antibody or antigen-binding fragment thereof, wherein (i) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 13: 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; 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.

33. The antibody or antigen-binding fragment thereof according to claim 31, comprising a heavy chain and a light chain, wherein (a) the heavy chain (i) 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 the group consisting of SEQ ID NOs: 37, 39, 41, or 43; or (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 37, 39, 41, or 43; or (iii) comprises an amino acid sequence having 1 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 amino acid conservative 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 regions of the heavy chain, more preferably the amino acid changes do not occur in the variable region of the heavy chain; and/or (b) the light chain (i) 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 the group consisting of SEQ ID NOs: 38, 40, 42, or 44; or (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 38, 40, 42, or 44; or (iii) comprises an amino acid sequence having 1 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 amino acid conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO 38, 40, 42 or 44, preferably the amino acid changes do not occur in the CDR regions of the light chain, more preferably the amino acid changes do not occur in the variable region of the light chain.

34. The antibody or antigen-binding fragment thereof according to claim 31, 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; preferably, 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; preferably, comprising one, two, or all three CDRs in HCDR1 shown in SEQ ID NO: 1 or 3, HCDR2 shown in SEQ ID NO: 5 or 7, HCDR3 shown in SEQ ID NO: 9 or 11 and/or one, two, or all three CDRs in LCDR1 shown in SEQ ID NO: 21 or 23, LCDR2 shown in SEQ ID NO: 25 or 27, LCDR3 shown in SEQ ID NO: 29 or 31; preferably, comprising the heavy chain variable region shown in SEQ ID NO: 13 or SEQ ID NO: 15, and/or the light chain variable region shown in SEQ ID NO: 33 or SEQ ID NO: 35.

35. The antibody or antigen-binding fragment thereof according to claim 31, wherein the antibody or antigen-binding fragment thereof binds to human cytomegalovirus gB glycoprotein with a KD of about 50 nM or less.

36. The antibody or antigen-binding fragment thereof according to claim 31, wherein the antibody or antigen-binding fragment thereof is capable of binding to the fusion domain of the human cytomegalovirus gB glycoprotein.

37. The antibody or antigen-binding fragment thereof according to claim 31, wherein at least a portion of the framework sequences are a human consensus framework sequences.

38. The antibody or antigen-binding fragment thereof of according to claim 31, wherein the antibody is a human monoclonal antibody.

39. The antibody or antigen-binding fragment thereof according to claim 31, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab′, Fab′-SH, Fv, a single chain antibody (e.g., Scfv) or (Fab′)2, a single domain antibody, a diabody (dAb), or a linear antibody.

40. The antibody or antigen-binding fragment thereof according to claim 31, wherein the antibody is an IgG-like antibody, such as an antibody in the form of IgG1 or an antibody in the form of IgG2 or an antibody in the form of IgG3 or an antibody in the form of IgG4.

41. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof according to claim 31.

42. A vector comprising the nucleic acid according to claim 41, the vector being an expression vector.

43. A host cell comprising a vector according to claim 42, wherein the host cell is a eukaryotic or prokaryotic cell, such as an E. 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.

44. A method of producing an antibody or antigen-binding fragment thereof, the method comprising culturing a host cell comprising the nucleic acid according to claim 41; preferably, wherein the method further comprises recovering the antibody or antigen-binding fragment thereof from the host cell or culture medium.

45. An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to claim 31 and a label.

46. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to claim 31 or an immunoconjugate comprising the antibody or antigen-binding fragment thereof and a label, and optionally one or more pharmaceutically acceptable auxiliary materials, such as a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, including a buffer or diluent.

47. A method of detecting, treating, preventing and/or alleviating of a HCMV infection or a HCMV-related disease in a human subject, comprising administering to a subject in need of an effective amount of the antibody or antigen-binding fragment thereof according to claim 31 or an immunoconjugate comprising the antibody or antigen-binding fragment thereof and a label.

48. A method of preventing or treating a HCMV infection or a 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 claim 31, or an immunoconjugate comprising the antibody or antigen-binding fragment thereof and a label, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to claim 31 or an immunoconjugate comprising the antibody or antigen-binding fragment thereof and a label, and optionally one or more pharmaceutically acceptable auxiliary materials, such as a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, including a buffer or diluent.

49. A method of increasing, enhancing, or stimulating resistance in a human subject infected with HCMV, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof according to claim 31, or an immunoconjugate comprising the antibody or antigen-binding fragment thereof and a label, or the pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to claim 31 or an immunoconjugate comprising the antibody or antigen-binding fragment thereof and a label, and optionally one or more pharmaceutically acceptable auxiliary materials, such as a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, including a buffer or diluent; preferably, wherein the subject is a HCMV-infected subject; preferably, wherein the subject is a transplant patient, a pregnant woman, a newborn, an AIDS patient, a cancer patient, or a patient with autoimmune diseases.

50. A method of neutralizing HCMV in an individual or sample, comprising contacting the antibody or antigen-binding fragment thereof according to claim 31 with the individual or sample and testing the capacity of the antibody or antigen-binding fragment thereof to bind to neutralize HCMV.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0129] FIG. 1 shows the results of neutralization of the HCMV standard strains Towne and TB40E in HFF cells with TRN1017, TRN1018, TRN1019, and TRN1020.

[0130] FIG. 1A shows the results of neutralization of the HCMV standard strain Towne in HFF cells with four antibodies TRN1017, TRN1018, TRN1019, and TRN1020. FIG. 1B shows the results of neutralization of HCMV strain TB40E infected in HFF cells with four antibodies TRN1017, TRN1018, TRN1019, and TRN1020.

[0131] FIG. 2 shows the binding affinity of antibodies TRN1017, TRN1018, TRN1019, and TRN1020, respectively, to the antigen gB glycoprotein.

[0132] FIG. 3 shows the effects of single point mutations at the 12 glycosylation sites (N208, N281, N284, N302, N341, N383, N405, N409, N417, N447, N452, and N456) of the gB glycoprotein on the binding activity of the antibodies of the present invention.

[0133] FIG. 4 shows linear structure of the extracellular segment of the gB glycoprotein and fusion domain thereof (including three mutation positions).

DEFINITIONS

[0134] It is to be understood that the terms used herein are for the purpose of describing particular 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 specified, all technical and scientific terms used herein have the same meanings as those generally understood by a person of ordinary skill in the art to which the present invention belongs.

[0135] For purposes of interpreting this specification, the following definitions will be used and whenever appropriate, terms used in the singular may also include the plural and vice versa. It is to be understood that the terms used herein are for the purpose of describing particular embodiments only, and are not intended to be limiting.

[0136] The term “about” when used in conjunction with a numerical value is intended to encompass numerical values within a range having a lower limit that is 5% less than the numerical value specified, and an upper limit that is 5% greater than the numerical value specified.

[0137] As used herein, the term “and/or” means any one of the alternatives or two or more of the alternatives.

[0138] As used herein, the terms “comprising” or “including” means including the recited elements, integers or steps but not excluding any other elements, integers or steps. As used herein, the terms “comprising” or “including” when used herein, unless otherwise indicated, encompass the stated elements, integers, or steps. For example, reference to an antibody variable region “comprising” a particular sequence is also intended to encompass antibody variable regions consisting of that particular sequence.

[0139] “Human Cytomegalovirus” (HCMV) is a DNA double-helix virus of the genus Cytomegalovirus in the subfamily of Herpesviruses, also known as human herpesvirus type 5 (HHV-5). As used herein, “human cytomegalovirus”, “HCMV”, “human herpes virus type 5”, “HHV-5” are all generic.

[0140] The “gB glycoprotein of human cytomegalovirus (HCMV)” (or “HCMV gB glycoprotein” or “HCMB-gB glycoprotein”) is one of the major glycoproteins constituting the outer shell of HCMV, which is known to contribute to the invasion of viral particles into cells, cell fusion, and intercellular infection of viruses. The amino acid sequence of the HCMV gB glycoprotein can be obtained from the publicly available sequence database NCBI GENE.

[0141] “Fusion domain of the gB glycoprotein of human cytomegalovirus (HCMV)” or “HCMV gB glycoprotein fusion domain” refers to a region consisting of contiguous amino acid residues from position 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).

[0142] A “complementarity determining region” or “CDR region” or “CDR” is a region of an antibody variable domain that is highly variable in sequence and forms a structurally defined loop (“hypervariable loop”) and/or includes an antigen-contacting residue (“antigen-contacting point”). The CDRs are primarily responsible for the binding to antigenic epitopes. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus. CDRs located within the variable domain of the heavy chain of an antibody are referred to as HCDR1, HCDR2, and HCDR3, while CDRs located within the variable domain of the light chain of an antibody are referred to as LCDR1, LCDR2, and LCDR3. Within a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundary for each CDR can be determined using any one or 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 Ed. U.S. 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 North CDR definitions based on affinity propagation clustering using a large number of crystal structures.

[0143] For example, according to different CDR determination schemes, the residues of each CDR are defined as follows.

TABLE-US-00005 Kabat AbM Chothia Contact CDR Scheme Scheme Scheme Scheme LCDR1 L24-L34 L24-L34 L26-L32 L30-L36 LCDR2 L50-L56 L50-L56 L50-L52 L46-L55 LCDR3 L89-L97 L89-L97 L91-L96 L89-L96 HCDR1 H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering system) HCDR1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia Numbering system) HCDR2 H50-H65 H50-H58 H53-H55 H47-H58 HCDR3 H95-H102 H95-H102 H96-H101 H93-H101 (Kabat Numbering system)

[0144] CDRs can also be determined based on the same Kabat numbering positions as the reference CDR sequences (any of the exemplary CDRs in the present invention).

[0145] Unless otherwise indicated, in the present invention, the term “CDR” or “CDR sequence” encompasses CDR sequences determined in any of the ways described above.

[0146] Unless otherwise stated, in the present invention, reference to residue positions in an antibody variable region, including heavy chain variable region residues and light chain variable region residues, refers to numbering positions 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)).

[0147] In one embodiment, the CDR boundary of the antibody of the present invention is determined by IMGT rules, for example using the IMGT database.

[0148] It should be noted that the boundaries of the CDRs of variable regions of the same antibody obtained based on different assignment systems may differ. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. Thus, in reference to defining an antibody with a specific CDR sequence as defined herein, the scope of the antibody also encompasses antibodies whose variable region sequences comprise the specific CDR sequence, but whose claimed CDR boundaries differ from the specific CDR boundaries as defined herein due to the application of different schemes (e.g., different assignment system rules or combinations).

[0149] As used herein, the term “neutralization” refers to the ability of neutralizing a pathogen to initiate and/or maintain infection in a host.

[0150] 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. Epitopes within a protein antigen can be formed from contiguous amino acids (usually linear epitopes) or discrete amino acids (usually conformational epitopes) juxtaposed by the tertiary folding of the protein. Epitopes formed from contiguous amino acids are typically (but not always) remain exposed to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents.

[0151] “An antibody that binds to the same or an overlapping epitope” as a reference antibody refers to 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, and conversely, a 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.

[0152] An antibody that competes with a reference antibody for binding to its antigen refers to 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. Conversely, a 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. 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)).

[0153] An antibody that inhibits (e.g., competitively inhibits) the binding of a reference antibody to its antigen refers to an antibody that inhibits 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen. Conversely, a reference antibody inhibits 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the antibody to its antigen. 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.

[0154] An antibody that exhibits the same or similar binding affinity and/or specificity as a reference antibody refers to an antibody that is capable of having a binding affinity and/or specificity of at least 50%, 60%, 70%, 80%, 90% or 95% or more of that of the reference antibody. This can be determined by any method known in the art for determining binding affinity and/or specificity.

[0155] “An antibody in the form of IgG” refers to the IgG form to which the heavy chain constant region of an antibody belongs. The heavy chain constant regions are the same for all antibodies of the same type, and differ between antibodies of different types. For example, an antibody in the form of IgG1 refers to a IgG3 domain whose heavy chain constant region Ig domain is IgG1.

[0156] A “human” antibody (HuMAb) refers to an antibody having a variable region in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains constant regions, the constant regions are also derived from human germline immunoglobulin sequences.

[0157] A “humanized” antibody refers to an antibody in which some, most, or all of the amino acids outside the CDR domain of a non-human antibody (e.g., a mouse antibody) have been replaced with the corresponding amino acids from a human immunoglobulin. In one embodiment of the humanized form of the antibody, some, most or all of the amino acids outside of the CDR domain have been replaced with amino acids from a human immunoglobulin, while some, most or all of the amino acids within one or more of the CDR regions have not been altered. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible so long as they do not eliminate the ability of the antibody to bind a particular antigen. A “humanized” antibody retains similar antigen specificity as the original antibody.

[0158] 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.

[0159] As used herein, an “antibody fragment” refers to a molecule that differs from an intact antibody, which comprises a portion of the intact antibody and binds to the antigen to which the intact antibody binds. As used herein, the term “antigen-binding fragment” as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., the gB glycoprotein of human HCMV). Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab′, Fab′-SH, F(ab′).sub.2, diabodies, linear antibodies, single chain antibodies (e.g., scFv), single domain antibodies, bivalent antibodies or bispecific antibodies or fragments thereof, camelid antibodies, and bispecific or multispecific antibodies formed from antibody fragments.

[0160] As used herein, “multispecific” refers to an antibody that specifically binds to at least two different antigens or two different epitopes within an antigen, e.g., three, four, or five different antigens or epitopes.

[0161] As used herein, “bispecific” refers to an antibody that specifically binds to two different antigens or two different epitopes within the same antigen. Bispecific antibodies may be cross-reactive with other related antigens, or can bind to epitopes shared between two or more different antigens.

[0162] An “immunoconjugate” is an antibody conjugated to one or more other substances, including but not limited to labels.

[0163] As used herein, the term “label” refers to a compound or composition that is directly or indirectly conjugated or fused to an agent, such as a polynucleotide probe or antibody, and facilitates the detection of the agent to which it is conjugated or fused. The label may be detectable by itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze chemical alteration of the detectable substrate compound or composition. The term is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reaction with another reagent that is directly labeled. Examples of indirect labeling include the detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.

[0164] A “vector” refers to a polynucleotide that is capable of replicating within a biological system or is moveable between such systems. Vector polynucleotides typically contain elements such as an origin of replication, a polyadenylation signal, or a selectable marker of which the function is to facilitate the replication or maintenance of the polynucleotide in a biological system. Examples of such biological systems may include cells, viruses, animals, plants, and biological systems reconstituted with biological components capable of replicating vectors. The polynucleotide comprising the vector may be a DNA or RNA molecule or a hybrid molecule of these molecules. An “expression vector” refers to a vector that can be used in a biological system or a reconstituted biological system to direct translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0165] An “isolated” antibody is an antibody that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined, for example, by electrophoresis (e.g., SDS-PAGE, IEF, capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al. J. Chromatogr. B848: 79-87 (2007)).

[0166] An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location other than its natural chromosomal location.

[0167] Calculation of sequence identity between sequences was performed as follows.

[0168] 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 may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the aligned reference sequence 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 as the corresponding position in the second sequence, then the molecules are identical at that position.

[0169] Sequence comparison between two sequences and calculation of percent identity can be accomplished using mathematical algorithms. In a preferred embodiment, the percent identity between two amino acid sequences was determined using the Needlema and Wunsch ((1970) J. Mol. Biol 48:444-453) algorithms (available at http://www.gcg.com) that have been integrated into the GAP program of the GCG software package, using either the Blossum 62 matrix or the PAM250 matrix and the GAP weights 16, 14, 12, 10, 8, 6 or 4 and the length weights 1, 2, 3, 4, 5 or 6. In yet another preferred embodiment, the percent identity between two amino acid sequences was determined using the GAP program in the GCG software package (available at http://www.gcg.com), using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and one set of parameters 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.

[0170] 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 the PAM120 weighted remainder table, a gap length penalty of 12 and a gap penalty of 4.

[0171] Additionally or alternatively, the nucleic acid sequences and protein sequences described herein can be further used as “query sequences” to perform searches against public databases, e.g., to identify other family member sequences or related sequences.

[0172] As used herein, the term “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes the hybridization and wash conditions. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, incorporated by reference. Both aqueous and non-aqueous methods are described in the references and either method can be used. Specific hybridization conditions referred to herein are as follows: 1) the low stringency hybridization condition including washing twice in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by in 0.2×SSC, 0.1% SDS at least 50° C. (for low stringency conditions, the temperature for washing can be increased to 55° C.); 2) the medium stringency hybridization condition including washing once or more times in 6×SSC at about 45° C., followed by in 0.2×SSC, 0.1% SDS at 60° C.; 3) the high stringency hybridization condition including washing once or more times in 6×SSC at about 45° C., followed by in 0.2×SSC, 0.1% SDS at 65° C.; and preferably 4) the very high stringency hybridization condition including washing once or more times in 0.5 M sodium phosphate, 7% SDS at 65° C., followed by 0.2×SSC, 0.1% SDS at 65° C. The very high stringency condition (4) is the preferred condition and the one that should be used unless otherwise stated. The terms “host cell”, “host cell line” and “host cell culture” are used interchangeably and refer to a cell into which an exogenous nucleic acid is introduced, including progeny of such a cell. Host cells include “transformants” and “transformed cells”, which include primarily transformed cells and progeny derived therefrom, regardless of the number of passages. The progeny may not be exactly the same as the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected in the initially transformed cell are included herein.

[0173] The term “pharmaceutical auxiliary material” refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, carrier or stabilizer, etc., with which the active substance is administered.

[0174] The term “pharmaceutical composition” refers to a composition that is present in a form that allows the biological activity of the active ingredients contained therein to be effective, and which does not contain additional ingredients having unacceptable toxicity to the subject to which the composition is administered.

[0175] The term “effective amount” refers to an amount or dose of an antibody or fragment or conjugate or composition of the present invention which, upon administration to a patient in a single or multiple dose, produces the desired effect in the patient in need of treatment or prevention. An effective amount can be readily determined by the attending physician, as one skilled in the art, by considering the following factors: such as species of mammals: size, age and general health; specific diseases involved; the extent or severity of the disease; response of an individual patient; the specific antibody administered; modes of administration; bioavailability characteristics of the administered formulation; a selected dosing regimen; and the use of any concomitant therapy.

[0176] A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time required, to achieve the desired therapeutic result. The therapeutically effective amount of the antibody or antibody fragment or conjugate or composition thereof can vary depending on factors such as the disease state, the age, sex, and weight of the subject, and the ability of the antibody or antibody portion to elicit a desired response in the subject. A therapeutically effective amount is also one in which any toxic or adverse effect of the antibody or antibody fragment or conjugate or composition thereof is less than a therapeutically beneficial effect. A “therapeutically effective amount” preferably inhibits a measurable parameter by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 50%, 60%, or 70% and still more preferably by 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 predictive of efficacy in human autoimmune disease or inflammation.

[0177] A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time required, to achieve the desired prophylactic result. Generally, a prophylactically effective 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 the disease.

[0178] As used herein, “individual” or “subject” is used interchangeably and includes mammals, such as human.

[0179] As used herein, “treating” refers to slowing, interrupting, arresting, relieving, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

[0180] As used herein, “preventing” includes the inhibition of the occurrence or development of a disease or disorder or the symptoms of a particular disease or disorder. In some embodiments, a subject or pregnant woman with a family history of an immune system disease (autoimmune disease or immune compromised) is a candidate for a prophylactic regimen. In general, in the context of an immune system disease (autoimmune disease or Immunocompromisation), the term “prevention” refers to the administration of a drug prior to the onset of a sign or symptom of an immune system disease (autoimmune disease or Immunocompromisation), particularly in a subject at risk for an immune system disease (autoimmune disease or Immunocompromisation).

[0181] A “subject/patient sample” refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample may be a solid tissue, such as fresh, frozen and/or preserved organs or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid (ascites fluid), or interstitial fluid; cells from a subject at any time of gestation or development. Tissue samples may contain compounds that are not naturally intermixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0182] These and other aspects and embodiments of the present invention are described in the accompanying drawings and the following detailed description of the present 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 have been described by way of illustration and not limitation, and that various modifications may be made by those skilled in the art.

Examples

Example 1 Design of Sequences of the Specific Antigen HCMV Glycoprotein gB

[0183] Information on sequences of glycoprotein gB of 56 human cytomegalovirus strains (including experimental and clinical strains) was retrieved from NCBI GENG database, and the homology of gB glycoprotein was more than 70% through amino acid sequence alignment. The gB glycoprotein of standard strain Towne was selected as one specific antigen (gB_Towne strain), with the sequence shown in SEQ ID NO: 19, while the homologous sequence of gB glycoprotein of 56 virus strains was selected as another specific antigen (gB_Con. Strain), with the sequence shown in SEQ ID NO: 20, for screening a broad-spectrum monoclonal antibody against human cytomegalovirus.

TABLE-US-00006 SEQ ID NO: 19 MESRIWCLVVCVNLCIVCLGAAVSSSSTRGTSATHSHHSSHTTSAAHSRS GSVSQRVTSSQTVSHGVNETIYNTTLKYGDVVGVNTTKYPYRVCSMAQGT DLIRFERNIVCTSMKPINEDLDEGIMVVYKRNIVAHTFKVRVYQKVLTFR RSYAYIHTTYLLGSNTEYVAPPMWEIHHINSHSQCYSSYSRVIAGTVFVA YHRDSYENKTMQLMPDDYSNTHSTRYVTVKDQWHSRGSTWLYRETCNLNC MVTITTARSKYPYHFFATSTGDVVDISPFYNGTNRNASYFGENADKFFIF PNYTIVSDFGRPNSALETHRLVAFLERADSVISWDIQDEKNVTCQLTFWE ASERTIRSEAEDSYHFSSAKMTATFLSKKQEVNMSDSALDCVRDEAINKL QQIFNTSYNQTYEKYGNVSVFETTGGLVVFWQGIKQKSLVELERLANRSS LNLTHNETKESTDGNNATHLSNMESVHNLVYAQLQFTYDTLRGYINRALA QIAEAWCVDQRRTLEVFKELSKINPSAILSAIYNKPIAARFMGDVLGLAS CVTINQTSVKVLRDMNVKESPGRCYSRPVVIFNFANSSYVQYGQLGEDNE ILLGNHRTEECQLPSLKIFIAGNSAYEYVDYLFKRMIDLSSISTVDSMIA LDIDPLENTDFRVLELYSQKELRSINVFDLEEIMREFNSYKQRVKYVEDK GLNDIFEAQKIEWHELEVLFQGPGHHHHHHH SEQ ID NO: 20 MESRIWCLVVCVNLCIVCLGAAVSSSSTSHATSSTHNGSHTSRTTSAQTR SVSQHVTSSEAVSHRANETIYNTTLKYGDVVGVNTTKYPYRVCSMAQGTD LIRFERNIVCTPMKPINEDLDEGIMVVYKRNIVAHTFKVRVYQKVLTFRR SYAYIHTTYLLGSNTEYVAPPMWEIHHINSHSQCYSSYSRVIAGTVFVAY HRDSYENKTMQLMPDDYSNTHSTRYVTVKDQWHSRGSTWLYRETCNLNCM VTITTARSKYPYHFFATSTGDVVDISPFYNGTNRNASYFGENADKFFIFP NYTIVSDFGRPNSAPETHRLVAFLERADSVISWDIQDEKNVTCQLTFWEA SERTIRSEAEDSYHFSSAKMTATFLSKKQEVNMSDSALDCVRDEAINKLQ QIFNTSYNQTYEKYGNVSVFETTGGLVVFWQGIKQKSLVELERLANRSSL NLTHETKESTDGNNTTHLSNMESVHNLVYAQLQFTYDTLRGYINRALAQI AEAWCVDQRRTLEVFKELSKINPSAILSAIYNKPIAARFMGDVLGLASCV TINQTSVKVLRDMNVKESPGRCYSRPVVIFNFANSSYVQYGQLGEDNEIL LGNHRTEECQLPSLKIFIAGNSAYEYVDYLFKRMIDLSSISTVDSMIALD IDPLENTDFRVLELYSQKELRSSNVFDLEEIMREFNSYKQRVKYVEDKGL NDIFEAQKIEWHELEVLFQGPGHHHHHHH.

Example 2 Screening of HCMV gB Glycoprotein Serum Antibody

[0184] 1. Screening Process

[0185] Immune-compromised volunteers were recruited from different hospitals, and 272 whole blood samples were obtained from eligible patients; human peripheral blood mononuclear cells (PBMC) and plasma were isolated with Ficoll lymphocyte separation medium (GE). The two designed HCMV gB glycoproteins were expressed and purified by conventional methods at a concentration of 2 μg/mL for later use. 272 blood samples (separated plasma) were tested for antibody titer (serum antibody titer measured by EC50) by ELISA assay and cancer was classified.

[0186] 2. Results

[0187] 272 clinical blood samples belong to 17 different cancer disease types, and the result showed that 268 samples were seropositive, with a positive rate as high as 98.53%, which was consistent with the conclusion reported by the study on the prevalence of HCMV infection in human population. These samples were classified into 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, etc, respectively, indicating that HCMV widely exists in immune-compromised populations such as cancer patients and patients with autoimmune diseases. Anti-human cytomegalovirus antibodies exist in these patients, so anti-human cytomegalovirus monoclonal antibodies can be screened from their B cells.

Example 3 Isolation of Anti-Human Cytomegalovirus Monoclonal Antibody Gene

[0188] 1. Sorting of gB_Towne strain and gB_Con. Strain protein antigen-specific B cells

[0189] To improve the sorting yield of memory B cells, the sample numbered Es0018 in Example 3 was selected for specific sorting with two HCMV gB glycoproteins (gB_Towne strain and gB_Con. Strain) at the same time. The steps for screening are as follows:

[0190] Single memory B lymphocytes can be obtained from the gB antigen-specific memory B lymphocytes in human peripheral blood in a single cell's sorting mode by using flow cytometry (BD FACSAria) according to the manufacturer's instructions, applying the logic gates set up according to CD3−/CD14−/CD16−/CD235a−/CD19+/SIgD−/−labeled gB_Towne and gB_Con. +. To increase the specificity of the sorting, the labeled gB_Towne and gB_Con were both labeled with two-color fluorescence (PE-Cy7 and Bv421), resulting in double-positive cells on the diagonal and single-positive cells within the logic gate.

[0191] 3. Amplification of Single Cell Antibody Variable Region Gene

[0192] The steps are as follows: A complete set of RT-PCR/PCR primers and experimental conditions have been successfully designed and established to amplify all known seven human-derived antibody heavy chain families and thirteen light chain families, including antibody variable region genes from IgG, IgA, IgM, IgD, and IgE (see CN107760690B).

[0193] 4. Results

[0194] A plurality of memory B cells were isolated from a sample, and antibody gene sequences were obtained after amplification of antibody variable region genes in a single cell using the designed primers, with a somatic mutation rate of antibody heavy chain VH gene fragment ranging from 10% to 18.5%. After the gene amplification of the variable region of the single cell antibody, 103 gene sequences of the heavy chain variable region of the antibody and 109 gene sequences of the light chain variable region of the antibody were obtained respectively, after the pairing, the positive rate of the detected antibody was 32.4%, the somatic mutation rate of the heavy chain of the positive antibody was 7% to 16.5%, and the light chain was 2% and 15%.

Example 4 Expression of Anti-Human Cytomegalovirus Monoclonal Antibody

[0195] 1. Construction of Expression Vectors

[0196] Linear expression vectors were constructed for the heavy chain variable region gene sequence and the light chain variable region gene sequence of the above-mentioned successfully paired antibody, respectively, and the isolated heavy chain and light chain variable region genes were respectively combined with their respective constant region sequences using the method of Overlapping PCR (Crystal Structure of the Human Cytomegalovirus Glycoprotein B, Heidi G. Burke and Ekaterina E. Heldwein, PLoS Pathog. 2015 October; 11(10): e1005227) into full length heavy and light chain genes, and recombinant antibodies were expressed for antibody screening and identification.

[0197] 2. Screening Positive Antibodies

[0198] The above-mentioned expression vector (PCDNA 3.1) containing the heavy and light chain genes was transfected into HEK293 cells using a transfection reagent (Quigen) and cultured in a 12-well plate with DMEM medium containing FBS for 48 hours. After 48 hours, the cell culture supernatant was collected and tested for antibody expression and binding to the antigen HCMV gB glycoprotein (gB_Towne strain and gB_Con. Strain) by ELISA. The standard is TRN006 (CN 103910796 B), and the initial concentration is 0.5 μg/mL; the initial concentration of cell culture supernatant is the stock solution, 10 fold dilution in 4 series; mouse anti-human IgG Fab-HRP (Abcam) was diluted with commercially available SuperBlock in 1:10000. After the reaction is terminated, the dual-wavelength readings of 450 nm (sample) and 630 nm (microplate well) were detected with a multi-purpose microplate reader, and the final calculated values were OD450-OD630. The points with good linear relationship (R2>0.99) between the value of TRN006 OD450-OD630 and the corresponding concentration were selected to establish the linear regression equation, and the antibody concentration was calculated with the sample value falling within the linear interval.

[0199] 3. Identification Results

[0200] Finally, the linear expression vectors were constructed for the heavy and light chains of 134 antibodies, respectively, which were successfully paired, and were co-transfected into HEK293 cells; 42 positive binding antibodies were screened, with a positive rate of 32.3%, wherein 36 antibodies showed strongly positive for gB_Towne strain, and 24 antibodies showed strongly positive for gB_Con. Strain and gB_Towne strain.

[0201] Of the four antibodies of the present invention screened: the heavy chain may have CDR1 of SEQ ID NO: 1-4, and/or CDR2 of SEQ ID NO: 5-8, and/or CDR3 of SEQ ID NO: 9-12. In particular examples, the light chain has CDR1 of SEQ ID NO: 21-24, and/or CDR2 of SEQ ID NO: 25-28, and/or CDR3 of SEQ ID NO: 29-32.

[0202] TRN1017

[0203] 24 antibodies showing strong positive for both gB_Con. Strain and gB_Towne strain were identified by DNA sequencing (Invitrogen) of the corresponding DNA preparations. One of the antibodies was identified as TRN1017 having:

[0204] a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 13, and a light chain variable region amino acid sequence as shown in SEQ ID NO: 33;

[0205] a heavy chain amino acid sequence as shown in SEQ ID NO: 37, and a light chain amino acid sequence as shown in SEQ ID NO: 38.

[0206] TRN1018

[0207] 24 antibodies showing strong positive for both gB_Con. Strain and gB_Towne strain were identified by DNA sequencing of the corresponding DNA preparations. One of the antibodies was identified as TRN1018 having:

[0208] a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 14, and a light chain variable region amino acid sequence as shown in SEQ ID NO: 34;

[0209] a heavy chain amino acid sequence as shown in SEQ ID NO: 39, and a light chain amino acid sequence as shown in SEQ ID NO: 40.

[0210] TRN1019

[0211] 24 antibodies showing strong positive for both gB_Con. Strain and gB_Towne strain were identified by DNA sequencing of the corresponding DNA preparations. One of the antibodies was identified as TRN1019 having:

[0212] a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 15, and a light chain variable region amino acid sequence as shown in SEQ ID NO: 35;

[0213] a heavy chain amino acid sequence as shown in SEQ ID NO: 41, and a light chain amino acid sequence as shown in SEQ ID NO: 42.

[0214] TRN1020

[0215] 24 antibodies showing strong positive for both gB_Con. strain and gB_Towne strain were identified by DNA sequencing of the corresponding DNA preparations. One of the antibodies was identified as TRN1020 having:

[0216] a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 16, and a light chain variable region amino acid sequence as shown in SEQ ID NO: 36;

[0217] a heavy chain amino acid sequence as shown in SEQ ID NO: 43, and a light chain amino acid sequence as shown in SEQ ID NO: 44.

[0218] The amino acid sequences of the above variable regions were analyzed, and the CDR sequences of these four antibodies were determined as follows using the IMGT numbering rule:

TABLE-US-00007 Heavychain Antibody variable region VH CDR1 VH CDR2 VH CDR3 TRN1017 SEQ ID NO: 13 SEQ ID NO: 1 SEQ ID NO: 5 SEQ ID NO: 9 TRN1018 SEQ ID NO: 14 SEQ ID NO: 2 SEQ ID NO: 6 SEQ ID NO: 10 TRN1019 SEQ ID NO: 15 SEQ ID NO: 3 SEQ ID NO: 7 SEQ ID NO: 11 TRN1020 SEQ ID NO: 16 SEQ ID NO: 4 SEQ ID NO: 8 SEQ ID NO: 12 Lightchain Antibody variable region VL CDR1 VL CDR2 VL CDR3 TRN1017 SEQ ID NO: 33 SEQ ID NO: 21 SEQ ID NO: 25 SEQ ID NO: 29 TRN1018 SEQ ID NO: 34 SEQ ID NO: 22 SEQ ID NO: 26 SEQ ID NO: 30 TRN1019 SEQ ID NO: 35 SEQ ID NO: 23 SEQ ID NO: 27 SEQ ID NO: 31 TRN1020 SEQ ID NO: 36 SEQ ID NO: 24 SEQ ID NO: 28 SEQ ID NO: 32

Example 5 Evaluation of the Antiviral Neutralizing Capacity of Monoclonal Antibodies Against Human Cytomegalovirus

[0219] 1. Evaluation of Antiviral Neutralizing Capacity of Antibodies in HFF Cells

[0220] The cellular model HFF, as well as the standard strains Towne and TB40E of HCMV (both purchased from ATCC) were chosen for the evaluation of the antibody neutralizing capacity, the controls were the already reported antibodies SM5_1 and SM10, respectively (Crystal Structure of the Human Cytomegalovirus Glycoprotein B, Heidi G Burke and Ekaterina E. Heldwein, PLoS Pathog. 2015 Oct.: 11(10): e1005227). Prior to infection, 5.00E+03 HFF cells per well were plated in a 96-well plate, with cell density being about 90%. The antibody with an initial concentration of 100 μg/mL was diluted 3-fold in 8 series in PBS, 10 μL dilution was added to each well, and 10 μL DMEM medium was added to virus control group. The cells were infected with a virus dose of MOI=0.96, i.e. 40 μL/well of virus stock solution was added, the mixture was incubated in a 37° C. incubator for 1 hour as virus infection solution. The cell supernatant was discarded from the 96-well plate, followed by rinsing twice with 100 μL PBS. 50 μL of virus infection solutions with different antibody dilutions were added to the 96-well plate and the mixture was infected for 4 hours in a 37° C. incubator. The virus infection solution was aspirated and rinsed once with 100 μL PBS. 100 μL DMEM medium was added to each well, and cultured in an incubator containing 5% CO.sub.2 at 37° C. for 96 hours, during which the cell status was observed and recorded every day. The cell culture supernatant was discarded and rinsed twice with PBS. 50 μL of 4% paraformaldehyde was added to each well; the plate was placed at room temperature for 30 minutes to fix the cells, and rinsed twice with PBS. Cell nucleus were stained 15 minutes with 100 μL of 10 μg/mL Hoechst and rinsed 3 times with 200 μL of PBS (the 96-well plate was gently shaken), the nuclei staining was observed with blue light and viral infection was observed with green light. The fluorescence intensity of the emitted light at 535 nm was measured under excitation light at 485 nm, and the relative fluorescence intensity was used to describe the degree of viral infection of cells and the effect of antibodies against viral infection. Calculation results (Formula: anti-viral infection effect=100−fluorescence intensity of antibody virus complex group/fluorescence intensity of virus group×100)

[0221] 2. Evaluation of Antiviral Neutralizing Capacity of Antibodies in Primary HUVEC Cells

[0222] Antibodies were evaluated for their capacity to resist Towne infection in primary HUVEC cell lines. Prior to infection, 4.00E+04 HUVEC cells per well were plated in a 96-well plate, with cell density being about 80%. The antibody with an initial concentration of 100 μg/mL was diluted 3-fold in 8 series, 10 μL dilution was added to each well, and 10 μL medium was added to virus control group. The cells were infected with a virus dose of MOI=5, i.e. 40 μL/well of virus stock solution was added, the mixture was incubated in a 37° C. incubator for 1 hour as virus infection solution. The cell supernatant was discarded from the 96-well plate, followed by rinsing twice with 100 μL PBS. 50 μL of virus infection solutions with different antibody dilutions were added to the 96-well plate and the mixture was infected for 4 hours in a 37° C. incubator. The virus infection solution was aspirated and supplemented with 100 μL DMEM medium per well, the mixture was cultured in an incubator containing 5% CO.sub.2 at 37° C. for 7 days, during which the cell status was observed and recorded every day. The cell culture supernatant was discarded and rinsed twice with PBS. 50 μL of 4% paraformaldehyde was added to each well; the plate was placed at room temperature for 30 minutes to fix the cells, and rinsed twice with PBS. Cell nucleus were stained 15 minutes with 100 μL of 10 μg/mL Hoechst and rinsed 3 times with 200 μL of PBS (the 96-well plate was gently shaken), the nuclei staining was observed with blue light and viral infection was observed with green light. The fluorescence intensity of the emitted light at 535 nm was measured under excitation light at 485 nm, and the relative fluorescence intensity was used to describe the degree of viral infection of cells and the effect of antibodies against viral infection. Calculation results (Formula: anti-viral infection effect=100−fluorescence intensity of antibody virus complex group/fluorescence intensity of virus group×100)

[0223] 3. Results

[0224] In the results plot, SM5_1 and SM10 are anti-cytomegalovirus antibodies already reported as positive controls; TRNO79 is a rabies virus antibody (CN201611069303.4), HCV0082 is an HCV antibody, and the two unrelated antibodies are used as negative controls. As shown in FIG. 1A, the EC50 of the four strains antibodies TRN1017, TRN1018, TRN1019, and TRN1020 on HCMV standard strain Towne is 0.017-0.074 μg/mL. EC50 refers to half the effective concentration, and refers to the concentration at which 50% of the test animals are induced to produce a specific response, or at which a response index is half inhibited. The smaller EC50 indicates a stronger neutralizing capacity of the antibody. As shown in FIG. 1B, the EC50 of the four strains antibodies TRN1017, TRN1018, TRN1019, and TRN1020 on HCMV clinical strain TB40E is 0.015-0.091 μg/mL. The above results show that the antibodies of the present invention have a very high neutralizing activity against both the standard strain of HCMV Towne, and the strain TB40E.

[0225] Meanwhile, two of the antibodies, TRN1017 and TRN1020, were also evaluated in primary HUVEC cell lines for the capability of resisting Towne virus infection, and the antibodies also have extremely high neutralizing activity, with the EC50 being 0.089 μg/mL and 0.041 μg/mL, respectively.

[0226] HUVEC is a human umbilical vein endothelial cell and HFF is a human foreskin fibroblast (HFF) cell. The above results demonstrate that the antibodies of the present invention have a higher ability to neutralize virus for different HCMV virus strains; further, the antibodies of the present invention have the ability to neutralize HCMV infected with different cell types, and thus the antibodies of the present invention have the potential to neutralize HCMV at different tissue sites in an individual.

Example 6 Determination of Antibodies Affinity

[0227] The ability of the antibody to bind to the gB glycoprotein (gB_Towne strain) was determined in vitro using Biacore (SPR principle). The anti-human-IgG (Fc) was coupled to two channels of the CM5 chip by amino coupling, finally channel 1 was coupled with 5485.4RU and channel 2 was coupled with 5622.4RU. The capture antibody concentration was 1 μg/mL and the binding time was 45 s. The concentration of bound antigen protein gB0049 was 1.25 μg/mL, 2.5 μg/mL, 5 μg/mL, 10 μg/mL, and 20 μg/mL, with a binding time of 90s, and a dissociation time of 600s. The regeneration solution was 3M MgCl.sub.2 and the regeneration time was 30 s. The result was shown in FIG. 2, the K.sub.D values of the antibody binding to gB glycoprotein were all on the nM level, wherein the K.sub.D value of TRN1020 was 0.60 nM, the K.sub.D value of TRN1018 was 27.9 nM, and the K.sub.D value of TRN1017 was 1.13 nM, indicating that the antibody of the present invention has a higher binding capacity to the antigen gB glycoprotein.

Example 7 Studies on Antigen-Binding Epitopes of Antibodies

[0228] 1. Effect of Antigen Deglycosylation on Antibody-Binding Capacity

[0229] In the present invention, the effect of gB glycoprotein glycosylation on the antibody was investigated. Firstly, 12 glycosylation sites (N208, N281, N284, N302, N341, N383, N405, N409, N417, N447, N452, and N456) of gB_Towne protein were mutated into glutamine by single point mutation using the kit (the physical and chemical properties of asparagine and glutamine are similar, and the effect of mutation on the spatial structure is minimally changed), then, the binding activity of the mutant of gB glycoprotein to SM10, TRN1017 and TRN1019 antibodies was detected by indirect ELISA, respectively.

[0230] As shown in the results of FIG. 3, after the glycosylation sites of gB glycoproteins N208, N281, N284, N302, N341, N383, N405, N409, N417, N447, N452, and N456 were mutated to glutamine, the binding ability of the TRN1017 and TRN1019 antibodies of the present invention to the N208Q mutant of gB glycoprotein was significantly reduced, and the binding change to other gB glycoprotein mutants was not significant. Meanwhile, considering that the binding epitope of the control antibody SM10 is spatially adjacent to the N208 site, whereas the binding change of SM10 to the gB glycoprotein N208Q mutant is not significant, indicating that the mutation of N208 does not excessively alter the adjacent spatial structure, thus demonstrating the N208 site on the gB glycoprotein to be a key antigen binding epitope for the antibodies of the present invention.

[0231] 3. Site-Directed Mutation of Amino Acid Site of gB Glycoprotein

[0232] In order to further identify the epitope information of these antibodies, we performed alanine scanning (186aa-228aa) on the upstream and downstream amino acids centered at N208, and finally screened L213 and Y226 sites. The affinity constants of the TRN1017, TRN1018, TRN1019 and TRN1020 antibodies to the mutants at these two sites were determined using the SPR principle, and the results showed that the binding ability of the antibodies to these two mutants was significantly reduced (no significant change in the binding of the control antibody 1G2), indicating that L213 and Y226 are critical epitopes for the antibodies of the present invention. Thus, information on the three epitope amino acids of these antibodies (N208, L213, and Y226) was determined, which are structurally located in the fusion domain of the gB glycoprotein (amino acids R150-C250, which is the structural basis for the membrane fusion function of the gB glycoprotein), and antibodies targeting this domain could directly block virus entry into host cells (FIG. 4).

[0233] In conclusion, unlike antibodies that have been reported against AD-1, AD-2, AD-4, and AD-5 domains (Structure of HCMV glycoprotein B in the postfusion conformation bound to a neutralizing human antibody, Sumana Chandramouli et al. Nature Communications volume 6, Article number: 8176 (2015)); Germline V-genes sculpt the binding site of a family of antibodies neutralizing human cytomegalovirus, Christy A Thomson et al. The EMBO Journal (2008) 27, 2592-2602), the antibody epitopes of the present invention are directed against the gB glycoprotein fusion domain. All these antibodies showed high virus neutralization activity and high drug-forming potential. At the same time, the identification of antibody epitope information provides a theoretical basis for the design of HCMV_gB glycoprotein structural vaccine.

Example 8 Assay for Antinuclear Resistance of Antibody

[0234] Hep-2 cell is a human laryngeal cancer epithelial cell, which is internationally generally selected as a substrate for an indirect immunofluorescence method as a detection standard method of the antinuclear antibody because of the characteristics of rich antigen spectrum, strong antigen specificity, and high antigen content of Hep-2. This method was used in the present invention to detect the antinuclear resistance of 4 antibodies of TRN1017, TRN1018, TRN1019, and TRN1020, and the detection was performed according to the instructions of antinuclear antibody (ANA) detection kit.

[0235] The results show that the antibodies of TRN1017, TRN1018, TRN1019, and TRN1020 have no immunofluorescence reaction to Hep-2 cells at the concentration of 100 μg/mL, indicating that the antibodies have no autoimmune reaction to Hep-2 cells.

[0236] The description of the above Examples is only used to understand the method and core idea of the present invention. It should be noted that, several improvements and modifications may be made by persons of ordinary skill in the art without departing from the principle of the present invention, and these improvements and modifications should also be considered within the protection scope of the present invention.

SEQUENCE LISTING

[0237] Listing of heavy chain CDR sequence information

TABLE-US-00008 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

[0238] Listing of light chain CDR sequence information

TABLE-US-00009 Serial  Sequence  number CDR 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

[0239] Listing of amino acid sequence information of antibody heavy chain variable region (VH) and light chain variable region (VL)

TABLE-US-00010 SEQ ID  Variable NO region Sequence information TRN101 SEQ ID  VH QVLLVQSGGGLVQPGGSLRLAC 7 NO: 13 ETSGFTFRHYWMHWVRQAPGKG LVWLSSIHGSGTTTRYADSVKG RFTISRDNAKNILFLQINSPRP EDTATYYCVRDDYTSGYNWGQG AFVTVSS SEQ ID  VL QSALTQPASVSGSRGQSITIPC NO: 33 TGSRSDVGGYNYVSWYQQRPGK APKLILYDVSHRPSGISDRFSG SKLGDTASLTISGLRDEDEADY YCSSYTTKSTLYVFGTGTTVSV L TRN101 SEQ ID  VH QVQLVQSGGGVVQPGRSLRLSC 8 NO: 14 AASGFTFSNHGIHWVRQAPGKG LEWVAVISSDGTDTRDADSVRG RFTISRDNSRNTVYLDMNSLRA EDTAVYYCARDGRCGDERCYSG LPDVWGRGTRVSVSS SEQ ID  VL EIVLTQSPATLSLSPGERATLS NO: 34 CRASQSVGGYLAWYQQKVGQAP RLLIYDASNRATGIPARFSGSG SGTDFTLTISSLEPEDFAVYYC QQRSNWPPLTFGGGTKVEIK TRN101 SEQ ID  VH QVQLVQSGAEVKKPGESLKISC 9 NO: 15 KGSGYTFTNYWIGWVRQMPGAG LEWMAIIFPRDSYSAYSPSFQG RVTISVDKSISTAYLHWSSLEA SDTAVYYCAIYNDLRSGNSWGQ GTPLIVSS SEQ ID  VL DIQMTQSPSSLSASVGDRVTIT NO: 35 CRASQSITKYLNWYQQKPGRAP KLLIHTTSTLQSGVPSRFSGSG SGTDFTLTISSLQLEDFGTYYC QQSFSTLWTFGQGTKLDIKX TRN102 SEQ ID  VH EVQLVESGGGLAQPGGSLRLSC 0 NO: 16 EASGFSFSTYAMSWVRQAPGKG LEWVAAVSGRGTSTFYLDSVKG RFTVSRDNSRTTVFLQMNSLKV EDTAFYYCAKDSYVGGSFDWLP RPDYFDHWGRGVLVTVSS SEQ ID  VL QSVLTQPPSLSATPGQTVTISC NO: 36 SGSNSNIGKNYVSWYQQLPGAA PKLLLYNNNLRPSGIPARFSGS KSGTSATLGITGLQAGDEATYS CATWDKTLNFWVFGGGTKVTV

[0240] Amino acid sequences of antibody heavy chain (HC) and light chain (LC)

TABLE-US-00011 TRN101 SEQ ID  HC QVLLVQSGGGLVQPGGSLRLACETSGFTFR 7 NO: 37 HYWMHWVRQAPGKGLVWLSSIHGSGTTTRY ADSVKGRFTISRDNAKNILFLQINSPRPED TATYYCVRDDYTSGYNWGQGAFVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID  LC QSALTQPASVSGSRGQSITIPCTGSRSDVG NO: 38 GYNYVSWYQQRPGKAPKLILYDVSHRPSGI SDRFSGSKLGDTASLTISGLRDEDEADYYC SSYTTKSTLYVFGTGTTVSVLGQPKAAPSV TLFPPSSEELQANKATLVCLISDFYPGAVT VAWKADSSPVKAGVETTTPSKQSNNKYAAS SYLSLTPEQWKSHRSYSCQVTHEGSTVEKT VAPTECS TRN101 SEQ ID  HC QVQLVQSGGGVVQPGRSLRLSCAASGFTFS 8 NO: 39 NHGIHWVRQAPGKGLEWVAVISSDGTDTRD ADSVRGRFTISRDNSRNTVYLDMNSLRAED TAVYYCARDGRCGDERCYSGLPDVWGRGTR VSVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK SEQ ID  LC EIVLTQSPATLSLSPGERATLSCRASQSVG NO: 40 GYLAWYQQKVGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQ RSNWPPLTFGGGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC TRN101 SEQ ID  HC QVQLVQSGAEVKKPGESLKISCKGSGYTFT 9 NO: 41 NYWIGWVRQMPGAGLEWMAIIFPRDSYSAY SPSFQGRVTISVDKSISTAYLHWSSLEASD TAVYYCAIYNDLRSGNSWGQGTPLIVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID  LC DIQMTQSPSSLSASVGDRVTITCRASQSIT NO: 42 KYLNWYQQKPGRAPKLLIHTTSTLQSGVPS RFSGSGSGTDFTLTISSLQLEDFGTYYCQQ SFSTLWTFGQGTKLDIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC TRN102 SEQ ID  HC EVQLVESGGGLAQPGGSLRLSCEASGFSFS 0 NO: 43 TYAMSWVRQAPGKGLEWVAAVSGRGTSTFY LDSVKGRFTVSRDNSRTTVFLQMNSLKVED TAFYYCAKDSYVGGSFDWLPRPDYFDHWGR GVLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK SEQ ID  LC QSVLTQPPSLSATPGQTVTISCSGSNSNIG NO: 44 KNYVSWYQQLPGAAPKLLLYNNNLRPSGIP ARFSGSKSGTSATLGITGLQAGDEATYSCA TWDKTLNFWVFGGGTKVTVLGQPKAAPSVT LFPPSSEELQANKATLVCLISDFYPGAVTV AWKADSSPVKAGVETTTPSKQSNNKYAASS YLSLTPEQWKSHRSYSCQVTHEGSTVEKTV APTECS

TABLE-US-00012 Light chain λ constant region: (SEQ ID NO: 17) GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVK AGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV APTECS Light chain κ constant region: (SEQ ID NO: 18) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC Heavy chain constant region: (SEQ ID NO: 45) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK