Anti-human cytomegalovirus antibody and its uses

Abstract

The present disclosure relates to an anti-human cytomegalovirus antibody and its use. The anti-human cytomegalovirus antibody can bind to CMV trimer and / or pentamer complexes, especially an antibody that completely targets the binding region of the pentamer to the cell receptor, which can completely block the binding of the virus to the cell and has important clinical significance.

Description

[0001] This application is a divisional application. The filing date of the original application is October 27, 2022, the application number is 2022113262365, and the invention title is "Anti-human cytomegalovirus antibody and its uses". Technical Field

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

[0003] Human cytomegalovirus (HCMV) is a ubiquitous pathogen belonging to the β-herpesvirus subfamily of the Herpesviridae family, also known as human herpesvirus 5 (HHV5). It usually mainly presents as asymptomatic infection in immunocompetent individuals and only causes severe complications in immunocompromised individuals. The incidence of CMV infection varies greatly among adults, accounting for 36% to 77% of the population in developed countries, while it is generally higher than 90% in developing countries (1). In immunocompromised individuals, reinfection or reactivation of CMV can cause severe complications and even endanger life (2, 3). In addition, congenital HCMV infection is the main cause of 0.7% of neonatal vision / hearing defects or mental retardation (4, 5).

[0004] Membrane fusion of human herpesviruses is a complex process that requires highly conserved envelope glycoproteins gB and gH / gL as well as multiple glycoprotein complexes to mediate virus attachment, fusion, or endocytosis of host cells (6, 7). Currently, the function of HCMV glycoprotein gB is considered to be the main fusogen that mediates virus membrane fusion with infected cells, which is triggered after the trimeric or pentameric complex binds to the receptor (8 - 10). Two key gH / gL-containing complexes encoded by HCMV are required for entry into cells. The HCMV trimeric complex is formed by gH / gL / gO, in which gL-Cys144 is linked to gO-Cys351 by a disulfide bond. The HCMV pentameric complex contains five different subunits gH / gL / UL128 / UL130 / UL131A, and gL-Cys144 is bound to UL128-Cys162 by a disulfide bond. Because gO-Cys351 in the trimer and UL128-Cys162 in the pentamer have the same binding site to gL-Cys144, the formation of pentamers and trimers is mutually exclusive (11). In addition, the ratio of pentamers and trimers on the virus membrane surface is regulated by UL148 and US16 (12, 13), and recently it has been shown that UL116 acts as a gH chaperone during the assembly and maturation of the gH complex in infected cells and also affects the formation of the gH complex (14, 15). Genetic studies have clearly demonstrated that pentamers are required for entry into epithelial and endothelial cells but not for entry into fibroblasts, and more importantly, UL128 / UL130 / UL131A in the pentamer can maintain the tropism of HCMV for non-fibroblast cells (16, 17). In contrast, trimers are considered sufficient to mediate CMV entry into fibroblasts. Some studies have shown that gO is required to maintain the infectivity of cell-free virus, and viruses lacking gO do not infect fibroblasts, epithelial, or endothelial cells (18 - 20).

[0005] Human cytomegalovirus (HCMV) was first isolated in 1956 (21). Although experimental vaccines and therapeutic monoclonal antibodies (mAbs) have entered clinical trials, there are still no approved preventive vaccines or therapeutic monoclonal antibodies for clinical use. So far, the CMV gB protein as an immunogen and MF59 as an adjuvant (gB / MF59) are the best CMV candidate vaccines in clinical trials, with a protection rate of 43% to 50% in phase II clinical trials in solid organ transplant recipients (SOTs) (22). Chemical drugs such as ganciclovir (GCV), letermovir, maribavir, and CMV hyperimmune globulin (CMVIG) have shown efficacy against HCMV (23 - 25). CMVIG has shown good effects after SOT or allogeneic hematopoietic cell transplantation (26 - 28). After primary infection in the first trimester of pregnancy, CMVIG administered every two weeks effectively prevented mother-to-child HCMV transmission (29). However, current treatment methods are severely limited by toxicity or resistance to chemical drugs and the drawbacks of blood-derived CMVIG products (30, 31). MAb-based therapies have the advantage of overcoming the limitations of current available drugs and could become an effective clinical alternative. Pentamer-specific mAbs can block HCMV infection of trophoblast progenitor cells (32), and increased antibody levels targeting pUL128L within 30 days of HCMV infection in pregnant women are associated with a reduced risk of virus transmission to the fetus (33). In addition, mAbs with the ability to neutralize CMV in epithelial cells can protect solid organ transplant recipients (34). Meanwhile, gH-specific mAbs have shown broad-spectrum properties in inhibiting virus infection and transmission (35). A recent phase II clinical trial showed that vaccination with Genentech's RG7667 (including pentamer-specific and anti-gH antibodies) in high-risk kidney transplant recipients could delay the onset of CMV virus and result in fewer CMV diseases compared to placebo (36). Therefore, antibody cocktail therapies composed of pentamer-specific and gH-specific mAbs may lead to a breakthrough in CMV immunotherapy. However, there are still no approved preventive vaccines or therapeutic monoclonal antibodies (mAbs) for clinical prevention and treatment of HCMV infection.

[0006] The HCMV pentamer mainly mediates virus infection of non-fibroblast cells such as epithelial cells, endothelial cells, and immune cells. Neutralizing antibodies against the pentamer are more effective in inhibiting virus infection of non-fibroblast cells than neutralizing antibodies against the gB protein. Therefore, in the future, combining neutralizing antibodies against the pentamer with neutralizing antibodies against the gB protein for drug use can efficiently block virus infection of multiple cell types and has greater potential clinical value.

[0007] MSL-109 is a monoclonal antibody against the HCMV gH / gL complex that failed in phase II clinical trials. This antibody cannot bind to free virus and cannot relieve CMV viremia clinically (37). The binding epitope of antibody 8I21 on the pentamer only partially overlaps with the region where the pentamer binds to cellular receptors (43). 8I21 cannot completely block the binding of the virus to cellular receptors, and there is a possibility of breakthrough viral infection. Therefore, developing an antibody that completely targets the region where the pentamer binds to cellular receptors and completely blocks the binding of the virus to cells has important clinical significance. SUMMARY OF THE INVENTION

[0008] The present disclosure identified and characterized a group of 8 antibodies reactive with HCMV pentamers, which can be divided into 2 groups according to their reactivity with CMV trimers and pentamers. Antibodies PC0004, PC0010, PC0012, PC0014, PC0035, and PC0037 in the first group bind to both trimers and pentamers simultaneously. Experiments have shown that 3 of the 6 antibodies in group 1 (PC0012, PC0014, and PC0035) can neutralize HCMV and recognize a highly conserved domain on the gH / gL protein in both the trimer and pentamer. These antibodies neutralize HCMV not by blocking the binding of CMV to host cells, but by inhibiting the post-attachment process of virus entry into host cells. Antibodies PC0031 and PC0034 in the second group only bind to pentamers. The inventors found that one of the two pentamer-specific antibodies (PC0034) neutralizes HCMV by blocking the adsorption of the virus to host cells. Further analysis showed that among the 214 CMV genomic sequences currently collected in the NCBI database, the antigenic site targeted by antibody PC0034 is 100% conserved in the UL128 and UL131A proteins.

[0009] The present disclosure reveals the key residues on the antigenic epitopes targeted by these two groups of potent neutralizing antibodies and provides important information about the neutralizing epitopes on the trimer or pentamer complex for the design and development of trimers and / or pentamers as CMV vaccines. The potent neutralizing mAbs provided by the present disclosure can be attractive candidate antibodies for developing "cocktail" antibody therapies for the prevention and treatment of HCMV infection.

[0010] In one aspect, the present disclosure provides an antibody against human cytomegalovirus or an antigen-binding fragment thereof, comprising a CDR combination of a heavy chain and a light chain selected from the following:

[0011] (1) respectively comprising the heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO.3 - 5, and respectively comprising the light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO.8 - 10;

[0012] (2) Comprising the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO.13 - 15 respectively, and the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO.18 - 20 respectively;

[0013] (3) Comprising the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO.23 - 25 respectively, and the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO.28 - 30 respectively;

[0014] (4) Comprising the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO.33 - 35 respectively, and the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO.38 - 40 respectively;

[0015] (5) Comprising the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO.43 - 45 respectively, and the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO.48 - 50 respectively;

[0016] (6) Comprising the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO.53 - 55 respectively, and the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO.58 - 60 respectively;

[0017] (7) Comprising the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO.63 - 65 respectively, and the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO.68 - 70 respectively; and

[0018] (8) Comprising the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO.73 - 75 respectively, and the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO.78 - 80 respectively.

[0019] In another aspect, the present disclosure provides a nucleic acid encoding the aforementioned antibody or its antigen - binding portion.

[0020] In another aspect, the present disclosure provides a vector comprising the aforementioned nucleic acid.

[0021] In another aspect, the present disclosure provides a host cell comprising the aforementioned nucleic acid or vector.

[0022] In another aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned antibody or its antigen - binding portion, nucleic acid, vector and / or cell.

[0023] In another aspect, the present disclosure provides an immunoconjugate comprising the aforementioned antibody or an antigen-binding fragment thereof and a label.

[0024] In another aspect, the present disclosure provides a method for generating the aforementioned antibody or an antigen-binding fragment thereof, the method comprising culturing a host cell comprising the aforementioned nucleic acid or expression vector.

[0025] In another aspect, the present disclosure provides the use of the aforementioned antibody or an antigen-binding portion thereof, nucleic acid, vector, host cell, or immunoconjugate in the preparation of a pharmaceutical composition or kit for detecting, treating, preventing, and / or alleviating CMV infection or CMV-related diseases.

[0026] In another aspect, the present disclosure provides a method for preventing or treating HCMV infection or HCMV-related diseases in a human individual, comprising administering to an individual in need of treatment an effective amount of the aforementioned antibody or an antigen-binding portion thereof, nucleic acid, vector, host cell, or immunoconjugate.

[0027] In another aspect, the present disclosure provides a method for enhancing, boosting, or stimulating the resistance of a human individual infected with HCMV, comprising administering to an individual in need an effective amount of the aforementioned antibody or an antigen-binding portion thereof, nucleic acid, vector, host cell, or immunoconjugate.

[0028] In another aspect, the present disclosure provides a method for neutralizing HCMV in an individual or a sample, which comprises contacting the aforementioned antibody or an antigen-binding fragment thereof with the individual or the sample, and testing the ability of the aforementioned antibody or an antigen-binding fragment thereof to bind and neutralize HCMV. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The drawings herein are incorporated into the specification and constitute a part of this specification, showing embodiments consistent with this specification, and are used together with the specification to explain the principles of this specification.

[0030] Figure 1 Shows the detection results of CMV trimeric complex and pentameric complex detected by high performance liquid chromatography and polyacrylamide gel electrophoresis (SDS-PAGE), wherein, A. Detection results by high performance liquid chromatography; B. Detection results by polyacrylamide gel electrophoresis.

[0031] Figure 2 Shows the ELISA detection results of antibodies specifically binding to the pentameric complex.

[0032] Figure 3 Shows the binding results of antibodies binding to CMV trimer and pentameric complex.

[0033] Figure 4Shows the neutralizing activity of antibodies that specifically bind to CMV pentamers, where; A. Results of antibody neutralization of HCMV virus strain Towne in human embryonic lung fibroblasts (MRC-5); B. A. Results of antibody neutralization of HCMV virus strain BE13 / 2012 in human embryonic lung fibroblasts (MRC-5); C. Neutralization of HCMV virus strains VR1814, NR, and AD169 FIX in ARPE-19-based epithelial cells.

[0034] Figure 5 Shows the binding results of antibodies that specifically bind to CMV trimer and pentamer complexes, where A. Results of the binding of neutralizing antibodies to pentamers; B. Results of the binding of neutralizing antibodies to trimers. In Figure A, the curve with the highest response value corresponds to a pentamer concentration of 6 μg / mL or 4 μg / mL, the curve with the lowest response value corresponds to a pentamer concentration of 0.375 μg / mL or 0.25 μg / mL, and the intermediate response value curves correspond to pentamer concentrations diluted two-fold. In Figure B, the curve with the highest response value corresponds to a trimer concentration of 4 μg / mL, the curve with the lowest response value corresponds to a trimer concentration of 0.25 μg / mL, and the intermediate response value curves correspond to trimer concentrations diluted two-fold.

[0035] Figure 6 Shows the results of cross-competitive blocking binding of neutralizing antibodies to CMV pentamers.

[0036] Figure 7 Shows the binding results of neutralizing antibodies to pentamer mutants, where A. Results of the binding of neutralizing antibodies to pentamer mutants; B. Effects of neutralizing antibodies on CMV virus adhesion to host cells; C. Effects of neutralizing antibodies on CMV-infected cells; D. Effects of neutralizing antibodies on CMV spread and transmission.

[0037] Figure 8 Shows that antibody PC0034 blocks the binding of HCMV pentamers to epithelial and endothelial cells, where A. Binding results of antibody PC0034 and 9I6 to HCMV pentamers. In the figure, the curve with the highest response value corresponds to a pentamer concentration of 4 μg / mL, the curve with the lowest response value corresponds to a pentamer concentration of 0.25 μg / mL, and the intermediate response value curves correspond to pentamer concentrations diluted two-fold. B. SPR analysis results of antibody PC0034 and 9I6; C. Binding of HCMV pentamers to epithelial cells (APRE-19) and endothelial cells (HUVEC); D. Antibody PC0034 can inhibit the binding of pentamers to epithelial and endothelial cells.

[0038] Figure 9Shows the binding results of antibodies with HCMV pentamer mutants. Among them, A. Binding results of antibody PC0034 with pentamer mutant UL131A_E23A. In the figure, the curve with the highest response value corresponds to a pentamer mutant concentration of 80 μg / mL, the curve with the lowest response value corresponds to a pentamer mutant concentration of 5 μg / mL, and the middle response value curve corresponds to the pentamer mutant concentration with a two-fold serial dilution. B. Binding results of antibody PC0034 with pentamer mutant UL131A_K27A. In the figure, the curve with the highest response value corresponds to a pentamer mutant concentration of 40 μg / mL, the curve with the lowest response value corresponds to a pentamer mutant concentration of 2.5 μg / mL, and the middle response value curve corresponds to the pentamer mutant concentration with a two-fold serial dilution. C. Binding results of antibody PC0034 with pentamer mutant UL128_K47A. In the figure, the curve with the highest response value corresponds to a pentamer mutant concentration of 60 μg / mL, the curve with the lowest response value corresponds to a pentamer mutant concentration of 3.75 μg / mL, and the middle response value curve corresponds to the pentamer mutant concentration with a two-fold serial dilution. D. Binding results of antibody PC0034 with pentamer mutant UL128_T94A. In the figure, the curve with the highest response value corresponds to a pentamer mutant concentration of 40 μg / mL, the curve with the lowest response value corresponds to a pentamer mutant concentration of 2.5 μg / mL, and the middle response value curve corresponds to the pentamer mutant concentration with a two-fold serial dilution. E. Binding results of antibody PC0034 with pentamers. In the figure, the curve with the highest response value corresponds to a pentamer concentration of 20 μg / mL, the curve with the lowest response value corresponds to a pentamer concentration of 1.25 μg / mL, and the middle response value curve corresponds to the pentamer concentration with a two-fold serial dilution. F. Binding results of pentamer mutants with epithelial cells. In the figure, the curve with the highest response value corresponds to a pentamer concentration of 20 μg / mL, the curve with the lowest response value corresponds to a pentamer concentration of 1.25 μg / mL, and the middle response value curve corresponds to the pentamer concentration with a two-fold serial dilution. G. Binding results of pentamer mutants with epithelial cells; H and I. Distribution of the K27 site on subunit UL131A and the T94 and K47 sites on subunit UL128 on the pentamer complex. Detailed implementation manners

[0039] I. Definitions

[0040] In this disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory operation procedures used herein are all widely used terms and conventional procedures in the corresponding fields. At the same time, to better understand this disclosure, the definitions and explanations of relevant terms are provided below.

[0041] To explain this specification, the following definitions will be used, and terms used in the singular may also include the plural where appropriate, 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.

[0042] The term "about", when used in conjunction with a numerical value, means a numerical value that encompasses 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.

[0043] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0044] As used herein, the term "comprising" or "including" means including the recited element, integer, or step, but not excluding any other element, integer, or step. In this context, when the term "comprising" or "including" is used, the case consisting of the recited element, integer, or step is also covered, unless otherwise specified. For example, when referring to an antibody variable region "comprising" a specific sequence, an antibody variable region consisting of that specific sequence is also intended to be covered.

[0045] "Human Cytomegalovirus" (HCMV) is a DNA double-stranded virus of the genus Cytomegalovirus in the subfamily Betaherpesvirinae, also known as Human herpesvirus 5 (HHV-5). As used herein, "Human Cytomegalovirus", "HCMV", "Human herpesvirus 5", and "HHV-5" are all interchangeable.

[0046] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. A complete antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but in some cases may include fewer chains, such as antibodies that naturally occur in camels which may contain only heavy chains. Antibodies can be humanized or human antibodies and single domain antibodies, such as VH, VHH, or VL. Examples of antibody fragments include but are not limited to Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p3-25, Kipriyanov). The foregoing fragments can include multiple chains joined together, such as by disulfide bonds and / or by peptide linkers. Antibody fragments generally comprise at least or about 50 amino acids and typically at least or about 200 amino acids.

[0047] The term "complementary determining region" or "CDR region" or "CDR" or "hypervariable region" refers to the amino acid regions in the variable regions of antibodies that are primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus.

[0048] There are various well-known schemes in the art for determining the CDR sequences in a given VH or VL amino acid sequence: Kabat complementarity-determining regions (CDRs) are determined based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), while Chothia refers to the positions of structural loops (Chothia et al., (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:877-883), AbM CDRs are a compromise between Kabat CDRs and Chothia structural loops and are used by the AbM antibody modeling software of Oxford Molecular, and "Contact" CDRs are based on the analysis of available complex crystal structures. The residues of each of these CDRs are shown below according to different CDR determination schemes.

[0049] Table 1 CDR residue schemes

[0050]

[0051]

[0052] CDRs can also be determined based on having the same Kabat numbering positions as a reference CDR sequence (such as any of the exemplary CDRs of the present invention).

[0053] Unless otherwise specified, in the present invention, the terms "CDR" or "CDR sequence" cover CDR sequences determined in any of the above ways.

[0054] Unless otherwise specified, in the present invention, when referring to the residue positions in the antibody variable regions (including heavy chain variable region residues and light chain variable region residues), it refers to the 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)).

[0055] In one embodiment, the CDRs of the antibodies of the present invention are delimited by IMGT rules, for example, by using the IMGT database to delimit the boundaries.

[0056] 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 vary. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. Therefore, when referring to an antibody defined by the specific CDR sequences of the present invention, the scope of the antibody also encompasses such antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined by the present invention due to the application of different schemes (such as different assignment system rules or combinations).

[0057] The term "variant" related to an antibody as used herein refers to an antibody that contains an amino acid change in a target antibody region (such as the heavy chain variable region or the light chain variable region or the heavy chain CDR region or the light chain CDR region) that has been substituted, deleted, and / or inserted by at least 1 amino acid residue (such as 1 - 30, or 1 - 20, or 1 - 10 amino acid residues, such as 1 or 2 or 3 or 4 or 5 amino acid residues), or by chemical derivatization of one or more amino acid residues, wherein the variant substantially retains the biological properties of the antibody molecule before the change. In one aspect, the present disclosure encompasses variants of any antibody mentioned herein. In one embodiment, the antibody variant retains at least 60%, 70%, 80%, 90%, or 100% of the biological activity (such as antigen - binding ability) of the antibody before the change. It can be understood that the heavy chain variable region or the light chain variable region, or each CDR region of the antibody can be changed individually or in combination. In some embodiments, the amino acid change in one or more or all three heavy chain CDRs does not exceed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Preferably, the above - mentioned amino acid change is an amino acid substitution, preferably a conservative substitution. In some embodiments, the antibody variant has at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with the parental antibody in the target antibody sequence region.

[0058] The term "conservative substitution" refers to the substitution of one amino acid by another amino acid within the same category, for example, an acidic amino acid is substituted by another acidic amino acid, a basic amino acid is substituted by another basic amino acid, or a neutral amino acid is substituted by another neutral amino acid. Exemplary substitutions are shown in Table 2 below:

[0059] Table 2 Amino Acid Conservative Substitutions

[0060]

[0061]

[0062] As used herein, the term "neutralize" refers to the ability to neutralize the ability of a pathogen to initiate and / or maintain an infection in a host.

[0063] As used herein, the term "epitope" refers to the 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 from non-contiguous amino acids juxtaposed by the tertiary folding of the protein (usually conformational epitopes). Epitopes formed from contiguous amino acids are usually (but not always) exposed to denaturing solvents, while epitopes formed by tertiary folding are usually lost upon treatment with denaturing solvents.

[0064] An "antibody that binds the same or overlapping epitopes as a reference antibody" refers to an antibody that blocks 50%, 60%, 70%, 80%, 90%, or more than 95% of the binding of the reference antibody to its antigen in a competition assay. Conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90%, or more than 95% of the binding of this antibody to its antigen in a competition assay.

[0065] 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 more than 95% of the binding of the reference antibody to its antigen in a competition assay. Conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90%, or more than 95% of the binding of this 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 assays as: ELISA, SPR, solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA) (see, for example, Stahli et al., 1983, Methods in Enzymology 9:242-253).

[0066] 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 more than 95% of the binding of the reference antibody to its antigen. Conversely, the reference antibody inhibits 50%, 60%, 70%, 80%, 90%, or more than 95% of the binding of this antibody to its antigen. The binding of an antibody to its antigen can be measured by affinity (e.g., equilibrium dissociation constant). Methods for measuring affinity are known in the art.

[0067] An antibody that exhibits the same or similar binding affinity and / or specificity as a reference antibody refers to an antibody that can have 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 measuring binding affinity and / or specificity.

[0068] "Antibody in IgG form" refers to the IgG form to which the constant region of the heavy chain of the antibody belongs. The constant regions of the heavy chains of all antibodies of the same isotype are the same, and the constant regions of the heavy chains are different between antibodies of different isotypes. For example, an antibody in IgG1 form refers to an IgG3 domain of IgG1 in which the Ig domain of the constant region of its heavy chain is IgG1.

[0069] "Human" antibody (HuMAb) refers to an antibody having a variable region in which both the framework region and the CDR region 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.

[0070] "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 (such as a mouse antibody) are replaced by the corresponding amino acids derived from a human immunoglobulin. In one embodiment of the humanized form of the antibody, some, most, or all of the amino acids outside the CDR domains have been replaced by amino acids from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions have not been altered. Small additions, deletions, insertions, substitutions, or modifications of amino acids are tolerated as long as they do not eliminate the ability of the antibody to bind a specific antigen. A "humanized" antibody retains an antigen specificity similar to that of the original antibody.

[0071] 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.

[0072] As used herein, an "antibody fragment" refers to a molecule different from a full-length antibody that comprises a portion of the full-length antibody and binds to the antigen to which the full-length antibody binds. As used herein, the term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind 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’)2, diabodies, linear antibodies, single-chain antibodies (e.g., scFv), single-domain antibodies, bivalent or bispecific antibodies or fragments thereof, camelid antibodies, and bispecific or multispecific antibodies formed from antibody fragments.

[0073] 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.

[0074] 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 have cross-reactivity to other related antigens or may bind to epitopes shared between two or more different antigens.

[0075] An "immunoconjugate" is an antibody conjugated to one or more other substances, including but not limited to labels.

[0076] 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 an antibody, and facilitates the detection of the reagent to which it is conjugated or fused. The label itself may be detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze a chemical change in a detectable substrate compound or composition. The term is intended to cover both the direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody and the indirect labeling of a probe or antibody by reaction with another directly labeled reagent. Examples of indirect labeling include the detection of a primary antibody using a fluorescently labeled secondary antibody and the end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.

[0077] The term "isolated" antibody refers to an antibody 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, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (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).

[0078] The term "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from the components of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

[0079] The term "affinity" or "binding affinity" refers to the intrinsic binding affinity reflecting the interaction between the members of a binding pair, such as an antibody and an antigen. The affinity of molecule X for its ligand Y is usually expressed by the equilibrium dissociation constant (KD). The equilibrium dissociation constant is the ratio of the dissociation rate constant and the association rate constant (kdis and kon, respectively). The smaller the KD, the less dissociation, representing a stronger affinity between the antibody and the antigen. Affinity can be measured by common methods known in the art, for example, the KD measured using surface plasmon resonance (SPR) in a BIACORE instrument. Generally, an antibody (e.g., the neutralizing antibody TRN1021 of the present disclosure) dissociates from an antigen with an equilibrium dissociation constant (KD) of no higher than 1×10 -5 M, such as less than about 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M or 1×10 -10 M or less.

[0080] The term "immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, including but not limited to a carrier. The term "pharmaceutical composition" refers to a preparation that exists in a form that allows the biological activity of the active ingredient contained therein to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the preparation is administered.

[0081] The term "pharmaceutical carrier" refers to one or more non-toxic materials that are administered together with a therapeutic agent and do not interfere with the biological activity of the active ingredient, including but not limited to buffers, preservatives, compatible carriers, diluents, adjuvants (such as Freund's adjuvant (complete and incomplete)), excipients, vehicles, and optionally other additives or encapsulating substances. The pharmaceutical carriers suitable for the present disclosure can be conventional pharmaceutical formulation excipients; and compositions and formulations suitable for delivering the disclosed neutralizing antibodies.

[0082] The term "pharmaceutical composition" refers to a composition that exists in a form that allows the biological activity of the active ingredient contained therein to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered.

[0083] The term "effective amount" refers to such an amount or dose of an antibody or fragment or conjugate or composition of the present invention that, when administered to a patient in a single or multiple doses, produces the desired effect in a patient in need of treatment or prevention. The effective amount can be readily determined by an attending physician, who is a person skilled in the art, by considering a variety of factors such as the species of 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 dosing regimen selected; and the use of any concomitant therapies.

[0084] "Therapeutically effective amount" refers to an amount that, at the required dose and for the required period of time, effectively achieves the desired therapeutic result. The therapeutically effective amount of an antibody or antibody fragment or its conjugate or composition can vary depending on various factors such as the disease state, the age, sex, and weight of the individual, and the ability of the antibody or antibody moiety to elicit the desired response in the individual. The therapeutically effective amount is also an amount in which any toxic or harmful effects of the antibody or antibody fragment or its conjugate or composition are less than the therapeutic beneficial effects. Relative to an untreated individual, the "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%. The ability of a compound to inhibit a measurable parameter can be evaluated in an animal model system predictive of efficacy in human autoimmune diseases or inflammation.

[0085] "Prophylactically effective amount" refers to an amount that, at the required dose and for the required period of time, effectively achieves the desired prophylactic result. Generally, since prophylactic doses are used in an individual either before or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0086] As used herein, the terms "individual" or "subject" are used interchangeably and include mammals, such as humans.

[0087] As used herein, "treatment" refers to slowing, interrupting, arresting, alleviating, halting, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

[0088] The term "vaccine" or "vaccine composition" refers to a composition comprising at least one immunogenic composition that induces an immune response in an animal.

[0089] The term "subject" or "individual" is a primate (e.g., humans and non-human primates such as monkeys). In certain embodiments, the individual or subject is a human.

[0090] II. DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

[0091] In one aspect, the present disclosure provides an anti-human cytomegalovirus antibody or an antigen-binding fragment thereof, which comprises a CDR combination of a heavy chain and a light chain selected from the following:

[0092] (1) a heavy chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO. 3-5, and a light chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO. 8-10;

[0093] (2) a heavy chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO. 13-15, and a light chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO. 18-20;

[0094] (3) a heavy chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO. 23-25, and a light chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO. 28-30;

[0095] (4) a heavy chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO. 33-35, and a light chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO. 38-40;

[0096] (5) a heavy chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO. 43-45, and a light chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO. 48-50;

[0097] (6) a heavy chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO. 53-55, and a light chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO. 58-60;

[0098] (7) a heavy chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO. 63-65, and a light chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO. 68-70; and

[0099] (8) a heavy chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO. 73-75, and a light chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO. 78-80.

[0100] In some embodiments, the aforementioned antibody or its antigen-binding portion comprises a combination of a heavy chain variable region and a light chain variable region selected from the following:

[0101] (1) The heavy chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO.1, and the light chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO.6;

[0102] (2) The heavy chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO.11, and the light chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO.16;

[0103] (3) The heavy chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO.21, and the light chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO.26;

[0104] (4) The heavy chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO.31, and the light chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO.36;

[0105] (5) The heavy chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO.41, and the light chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO.46;

[0106] (6) The heavy chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 51, and the light chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 56;

[0107] (7) The heavy chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 61, and the light chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 66;

[0108] (8) The heavy chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 71, and the light chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 76.

[0109] In some embodiments, the foregoing antibody is an antibody of the IgG1, IgG2, IgG3 or IgG4 type.

[0110] In some embodiments, the foregoing antibody is an IgG1 type antibody.

[0111] In some embodiments, the constant region of the foregoing heavy chain comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 81.

[0112] In some embodiments, the constant region of the foregoing light chain comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 89 or 91.

[0113] In some embodiments, the foregoing nucleic acid molecule comprises a nucleic acid sequence of an antibody heavy chain variable region selected from SEQ ID NO.2, 12, 22, 32, 42, 52, 62, 72 or any variant thereof, and a nucleic acid sequence of an antibody light chain variable region selected from SEQ ID NO.7, 17, 27, 37, 47, 57, 67, 77 or any variant thereof.

[0114] In another aspect, the present disclosure provides a nucleic acid encoding the foregoing antibody or an antigen-binding portion thereof.

[0115] In another aspect, the present disclosure provides a vector comprising the foregoing nucleic acid.

[0116] In another aspect, the present disclosure provides a host cell comprising the foregoing nucleic acid or vector.

[0117] In another aspect, the present disclosure provides a pharmaceutical composition comprising the foregoing antibody or an antigen-binding portion thereof, nucleic acid, vector, and / or cell.

[0118] In another aspect, the present disclosure provides an immunoconjugate comprising the foregoing antibody or an antigen-binding fragment thereof and a label.

[0119] In another aspect, the present disclosure provides a method for generating the foregoing antibody or an antigen-binding fragment thereof, the method comprising culturing a host cell comprising a nucleic acid encoding the foregoing nucleic acid or expression vector.

[0120] In another aspect, the present disclosure provides the use of the foregoing antibody or an antigen-binding portion thereof, nucleic acid, vector, host cell, or immunoconjugate in the preparation of a pharmaceutical composition or kit for detecting, treating, preventing, and / or alleviating CMV infection or CMV-related diseases.

[0121] In some embodiments, the CMV is HCMV.

[0122] In another aspect, the present disclosure provides a method for preventing or treating HCMV infection or HCMV-related diseases in a human individual, comprising administering to an individual in need of treatment an effective amount of the foregoing antibody or an antigen-binding portion thereof, nucleic acid, vector, host cell, or immunoconjugate.

[0123] In another aspect, the present disclosure provides a method for enhancing, strengthening, or stimulating the resistance of a human individual infected with HCMV, comprising administering to an individual in need an effective amount of the foregoing antibody or an antigen-binding portion thereof, nucleic acid, vector, host cell, or immunoconjugate.

[0124] In some embodiments, the individual is an HCMV-infected individual.

[0125] In another aspect, the present disclosure provides a method for neutralizing HCMV in an individual or a sample, which includes contacting the aforementioned antibody or its antigen-binding fragment with the individual or the sample, and testing the ability of the aforementioned antibody or its antigen-binding fragment to bind and neutralize HCMV.

[0126] The aforementioned anti-human cytomegalovirus antibody can bind to CMV trimer and / or pentamer complexes, especially antibodies that completely target the pentamer-cell receptor binding region, which can completely block the binding of the virus to cells, and have important clinical significance.

[0127] For the purpose of clear and concise description, features are described herein as part of the same or separate embodiments. However, it will be understood that the scope of the present disclosure may include some embodiments having combinations of all or some of the described features.

[0128] Examples

[0129] Example 1 Expression, purification and identification of CMV recombinant antigen protein

[0130] The gene sequences of UL74 (gO), UL75 (gH), UL115 (gL), UL128, UL130, and UL131A of the VR1814 (GU179289.1) strain in NCBI GenBank were selected and codon-optimized. Among them, the UL75 (gH) gene was truncated to 1-715 amino acids to remove the transmembrane region and intracellular segment for expressing soluble pentamer protein. Among them, the amino acid sequence of the UL74 (gO) antigen is shown in SEQ ID NO.83; the amino acid sequence of the truncated UL75 (gH) antigen protein is shown in SEQ ID NO.84; the amino acid sequence of the UL115 (gL) antigen is shown in SEQ ID NO.85; the amino acid sequence of the UL128 antigen is shown in SEQ ID NO.86; the amino acid sequence of the UL130 antigen is shown in SEQ ID NO.87; the amino acid sequence of the UL131A antigen is shown in SEQ ID NO.88.

[0131] All antigen proteins carry 6 histidine tags at the carboxyl terminus and are separately constructed into the pcDNA3.1 eukaryotic expression vector. The coding genes of each subunit of the gH / gL / UL128 / UL130 / UL131A pentamer complex (the mass ratio of each subunit plasmid is 1:0.8:0.6:0.6:0.6) and the gH / gL / gO trimer complex (the mass ratio of each subunit plasmid is 1:1:1) are co-transfected into 293i cells transiently. After culturing for 5 days, the cell supernatant was collected and purified by the Ni-NTA method, and detected by high performance liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Figure 1A and 1B), it was determined that the obtained recombinant protein complex had a complete pentameric and trimeric structure.

[0132] Example 2 Obtaining CMV pentamer- and trimer-reactive antibodies

[0133] After obtaining the recombinant antigen protein prepared in Example 1, blood specimens of healthy volunteers were screened by ELISA to identify samples with high antibody titers against the purified recombinant pentamer complex. PBMCs of blood samples with high antibody titers were sorted by flow cytometry using pentamers labeled with two-color fluorescence as probes to obtain single pentamer-specific memory B lymphocytes.

[0134] The immunoglobulin (Ig) variable regions (VHDJH and VLJL) of the heavy and light chain gene segments were amplified from the sorted single B cells by RT / nested PCR, and a linear expression vector of the antibody was constructed by overlapping PCR. The recombinant expressed antibody (38) was obtained by transient transfection of 293T cells. A total of 16 antibodies specifically binding to the pentamer were identified by ELISA binding experiments ( Figure 2 ). The somatic mutation ranges of these antibodies were 2.01 - 15.38% for the VH gene and 6.47 - 19.93% for the VL gene, respectively. These 16 antibodies were from 11 different Ig gene clone lines, and 8 of them (PC0004, PC0031, PC0010, PC0012, PC0014, PC0034, PC0035, and PC0037), representing 8 different clone lines, were selected for production and purification of antibodies for further characterization (Table 1). It can be seen that in this example, antibodies specifically binding to HCMV pentamers and trimers were isolated from volunteer blood samples.

[0135] Table 1. V(D)J rearrangements of genes of antibodies specifically binding to HCMV pentamers and trimers

[0136]

[0137] Table 2 Variable regions of anti-human cytomegalovirus antibodies

[0138]

[0139] PC0004

[0140] The VH amino acid sequence is as shown in SEQ ID NO.1, its encoding nucleic acid is as shown in SEQ ID NO.2, and its HCDR1, HCDR2, and HCDR3 are as shown in SEQ ID NO.3, 4, and 5, respectively.

[0141] VH amino acid sequence:

[0142] EVQLVESGGAMIQPGGSLRLSCAAS GFSFDDYT MYWVRQTPGTGLEWVAL ITWNGVTT RYADSVQGRFTISRDNRKNSLSLQMNSLRPGDSGLYYC ARDIGPLRDSDYYYYGVGV WGLGTTVTVSS

[0143] VH nucleic acid sequence:

[0144] GAGGTTCAGCTGGTGGAGTCTGGGGGAGCCATGATACAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCAGTTTTGATGATTATACCATGTATTGGGTCCGGCAGACTCCGGGGACGGGTCTGGAGTGGGTCGCTCTCATTACTTGGAATGGTGTCACGACAAGATATGCAGACTCTGTGCAGGGCCGATTTACCATCTCCAGAGACAACAGGAAAAACTCTCTGTCTCTGCAAATGAATAGCCTGAGACCTGGGGACAGCGGCTTATATTACTGTGCAAGAGATATCGGCCCCCTACGAGACAGTGACTACTATTACTACGGTGTGGGCGTCTGGGGCCTAGGGACCACGGTCACCGTCTCCTCA

[0145] The VL amino acid sequence is as shown in SEQ ID NO.6, its encoding nucleic acid is as shown in SEQ ID NO.7, and its LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO.8, 9, 10 respectively. VL amino acid sequence:

[0146] DIVMTQSPLSLPVTPGEPASISCRSS QSLLHINGYNY LHWYLQKPGQSPQLLIY FGS NRASGVSDRFSGSGSGTEFTLKISKVEPEDVGTYYC MQGLQTPLT FGGGTRVEIK

[0147] VL nucleic acid sequence:

[0148] GATATTGTGATGACCCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTACATATTAATGGATACAACTATTTGCATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTCGGTTCCAATCGGGCCTCCGGGGTCTCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGAGTTCACACTGAAAATTAGTAAAGTTGAGCCTGAGGATGTTGGGACCTATTATTGCATGCAAGGTCTACAAACTCCCCTCACTTTCGGCGGGGGGACGAGGGTGGAGATCAAA

[0149] PC0010

[0150] The VH amino acid sequence is as shown in SEQ ID NO.11, its encoding nucleic acid is as shown in SEQ ID NO.12, and its HCDR1, HCDR2, and HCDR3 are as shown in SEQ ID NO.13, 14, and 15 respectively. VH amino acid sequence:

[0151] QVQLQESGPGLVRPSETLSLMCTVS GASISNTKYY WGWIRQPPGKRLEWVGS LYFSGTT YYNPSLQSRLTMSVDTSKNQFSLNLRSVTAADTAVYYC ARRPFVMSRGVRSDP WGQGILVSVST

[0152] VH nucleic acid sequence:

[0153] CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAGGCCTTCGGAGACCCTGTCCCTCATGTGCACTGTCTCTGGTGCCTCCATCAGCAATACAAAATACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGCGACTGGAGTGGGTTGGAAGTCTCTACTTTAGTGGGACCACCTACTACAACCCGTCCCTCCAGAGTCGACTCACCATGTCCGTAGACACGTCGAAGAACCAGTTCTCCCTCAACCTGAGGTCTGTGACCGCCGCAGACACGGCTGTCTACTATTGTGCGCGACGCCCTTTTGTTATGAGTCGGGGAGTGAGGTCCGACCCCTGGGGCCAGGGAATCCTGGTCTCCGTCTCCACA

[0154] The VL amino acid sequence is shown in SEQ ID NO.16, its encoding nucleic acid is shown in SEQ ID NO.17, and its LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs. 18, 19, and 20 respectively. VL amino acid sequence:

[0155] DIVMTQTPLSLPVTPGEPASISCTSS QSLLQSNGYTY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRLEAEDVGVYYC MQALQTPFT FGPGTRVDIK

[0156] VL nucleic acid sequence:

[0157] GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCACGTCTAGTCAGAGCCTCCTGCAAAGTAATGGATACACCTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAACTCCTGATCTATTTGGGTTCCAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGTAGACTGGAGGCCGAGGATGTTGGAGTTTATTACTGCATGCAAGCTCTACAAACTCCGTTCACTTTCGGCCCTGGGACCAGAGTGGACATCAAA

[0158] PC0012

[0159] The VH amino acid sequence is shown in SEQ ID NO.21, the nucleic acid encoding it is shown in SEQ ID NO.22, and its HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO.23, 24, and 25, respectively.

[0160] VH amino acid sequence:

[0161] EVQLVESGSELKKPGASVKLSCKTS GYSFTTYA ISWVRQAPGQGLEWLGR INTFTGNP TYAQGFTGRFVFSLDTSVTTAYLEISSLKAEDTAVYFC ARGASHLSGLDS WGQGGLVSVSS

[0162] VH nucleic acid sequence:

[0163] GAGGTGCAGCTGGTGGAGTCTGGGTCTGAGTTGAAGAAGCCGGGGGCCTCTGTGAAGCTTTCCTGCAAGACCTCTGGATACTCCTTCACTACTTATGCTATCAGTTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGCTGGGAAGGATCAACACCTTCACTGGAAACCCAACCTATGCCCAGGGCTTCACAGGACGGTTTGTCTTCTCCTTGGACACCTCTGTCACCACGGCATATCTGGAGATCAGCAGCCTAAAGGCTGAGGACACCGCCGTCTATTTCTGTGCGAGGGGGGCGTCCCACCTAAGCGGCTTAGACTCCTGGGGCCAGGGAGGCCTGGTCAGCGTCTCCTCA

[0164] The VL amino acid sequence is shown in SEQ ID NO.26, its encoding nucleic acid is shown in SEQ ID NO.27, and its LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO.28, 29, and 30 respectively.

[0165] VH amino acid sequence:

[0166] DIQLTQSPSSLSASVGDRVTITCRAS HNIGNW LAWYQQKPGQAPNLLIF KAS NLEYGVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQSETYPWT FGQGTRVEVK

[0167] VL nucleic acid sequence:

[0168] GACATCCAGTTGACCCAGTCTCCTTCCTCCCTGTCTGCATCTGTTGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCACAACATTGGTAACTGGTTGGCCTGGTATCAGCAGAAACCAGGGCAAGCCCCTAACCTCCTCATCTTTAAGGCGTCTAATTTAGAATATGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCGACTTATTACTGCCAACAGTCTGAGACTTATCCGTGGACGTTCGGCCAAGGGACCAGGGTGGAAGTCAAA

[0169] PC0014

[0170] The VH amino acid sequence is as shown in SEQ ID NO.31, its encoding nucleic acid is as shown in SEQ ID NO.32, and its HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO.33, 34, 35 respectively.

[0171] VH amino acid sequence:

[0172] QVQLQESGAGLLKPSETLSLTCAIY GGSFGNNY WNWIRQPPGEGLEWIGE INHRGST NSNPSLKSRVTMSVDTSKNQFSLKLTSVTAADAAVYFC ARREQLLLPDVFDI WGLGTRVAVSS

[0173] VH nucleic acid sequence:

[0174] CAGGTGCAGCTGCAGGAGTCGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTATCTATGGTGGGTCCTTCGGTAATAACTACTGGAACTGGATCCGCCAGCCCCCAGGGGAGGGGCTGGAATGGATTGGCGAAATCAATCATCGTGGAAGCACCAACTCCAACCCGTCCCTCAAGAGTCGAGTCACCATGTCGGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGACCTCTGTGACCGCCGCGGACGCGGCTGTCTATTTCTGTGCGAGACGAGAGCAGCTCCTATTGCCTGATGTCTTTGATATCTGGGGCCTCGGGACAAGGGTCGCCGTCTCTTCC

[0175] The VL amino acid sequence is as shown in SEQ ID NO.36, its encoding nucleic acid is as shown in SEQ ID NO.37, and its LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO.38, 39, 40 respectively.

[0176] VL amino acid sequence:

[0177] DIQLTQSPSSLSASVGDSVTITCRAS QRMSSY LNWYQQKPGKAPNLLIY AAS SLHSGVPSRFSGSGSGTDFTLTIASLQPEDFATYYC QQSYSAPYT FGQGTKLEIK

[0178] VL nucleic acid sequence:

[0179] GACATCCAGTTGACCCAGTCTCCGTCCTCCCTGTCTGCATCTGTAGGAGACAGTGTCACCATCACTTGCCGGGCAAGTCAGAGAATGAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATCTATGCTGCATCCAGTTTGCATAGTGGAGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCGCCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTGCCCCGTACACTTTTGGCCAGGGGACCAAACTGGAGATCAAA

[0180] PC0031

[0181] The VH amino acid sequence is as shown in SEQ ID NO.41, the nucleic acid encoding it is as shown in SEQ ID NO.42, and its HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO.43, 44, 45 respectively.

[0182] VH amino acid sequence:

[0183] EVQLVESGGGVVQPGRSLRLSCAAS GFSFTKYA MHWVRQAPGKGLEWVAV IRSDGINK YYGDSVKGRFTISRDNSKSTVDLQMLSLRGEDTAVYYC AKGEGYTDYSTMYYYNGMDV WGQGTTVRVSS

[0184] VH nucleic acid sequence:

[0185] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCTTGTGCAGCGTCTGGATTTAGTTTCACAAAATATGCAATGCACTGGGTCCGCCAGGCCCCAGGCAAGGGGCTGGAATGGGTGGCAGTTATTCGGAGTGATGGAATTAATAAATATTATGGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAAAGCACAGTGGACCTGCAAATGCTCAGCCTGAGAGGCGAAGACACGGCTGTGTATTACTGTGCGAAAGGGGAGGGCTACACTGACTACTCCACCATGTACTATTACAATGGAATGGACGTCTGGGGCCAGGGGACCACGGTCAGAGTCTCCTCA

[0186] The VL amino acid sequence is shown in SEQ ID NO.46, its encoding nucleic acid is shown in SEQ ID NO.47, and its LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO.48, 49, and 50 respectively.

[0187] VL amino acid sequence:

[0188] QAVLTQPPSLSVSPGQTARISCSAR ELPNQY SHWYQQRPGQAPVLLIF KDT ERPPGIPERFSGSSSGTTVTLTISRIQPDDEADYYC QSSDNDGTHWV FGGGTHLTVRS

[0189] VL nucleic acid sequence:

[0190] CAGGCTGTGCTGACTCAGCCACCCTCGCTGTCAGTGTCCCCAGGCCAGACGGCCAGGATCTCCTGCTCTGCACGTGAATTGCCAAACCAATATTCTCATTGGTACCAGCAGAGGCCAGGCCAGGCCCCTGTATTGTTGATTTTCAAAGACACTGAGAGGCCCCCAGGCATCCCCGAGCGATTCTCTGGCTCCAGCTCAGGAACAACAGTCACGTTGACCATCTCTAGAATCCAACCAGACGACGAGGCTGACTATTATTGTCAATCATCAGACAACGATGGTACCCACTGGGTCTTCGGCGGGGGGACGCACTTAACCGTCCGCAGT

[0191] PC0034

[0192] The VH amino acid sequence is shown in SEQ ID NO.51, its encoding nucleic acid is shown in SEQ ID NO.52, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.53, 54, 55 respectively.

[0193] VH amino acid sequence:

[0194] QVQLVQSGGGLVQPGGSLRLSCAAS GFTFSSYA MIWVRQAPGKGLEWVSG ISGRASPT YYADSVKGRFTISRDNSKSTLYLQMNSLRAEDTAVYYC VKDYSGSDYDILPGITALDF WGRGTLVTVSS

[0195] VH nucleic acid sequence:

[0196] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGGTTGGTGCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCGGCCTCTGGATTCACGTTTAGCAGCTATGCCATGATCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTAGTGGTCGTGCAAGTCCCACATACTACGCAGACTCCGTAAAGGGCCGGTTTACCATCTCCAGAGACAATTCCAAGAGCACCCTGTATTTGCAAATGAACAGCCTGAGAGCTGAGGATACGGCCGTTTATTATTGTGTGAAAGATTATAGCGGCTCGGATTACGATATTTTGCCTGGAATCACCGCCCTTGACTTCTGGGGCCGGGGAACCCTGGTCACCGTGTCCTCA

[0197] The VL amino acid sequence is shown in SEQ ID NO.56, its encoding nucleic acid is shown in SEQ ID NO.57, and its LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO.58, 59, and 60 respectively.

[0198] VL amino acid sequence:

[0199] QSALTQPPSVSAAPGETARITCGGK NIGSKS VHWYQQKPGQAPVLVIH YDT DRPSGIPERFSGSNSGNTATLTISTVSAGDEADYYC QVWDSGSDHVV FGGGTKLTVL

[0200] VL nucleic acid sequence:

[0201] CAGTCTGCCCTGACTCAGCCACCCTCAGTGTCAGCGGCCCCAGGAGAGACGGCCAGGATTACCTGTGGGGGAAAGAACATTGGAAGTAAAAGTGTTCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTACTGGTCATCCATTATGATACCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCCGGCTCCAACTCTGGGAACACGGCCACCTTGACCATCAGCACGGTCTCAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATTCAGGTAGTGATCATGTGGTTTTCGGCGGAGGGACCAAGCTGACCGTCCTA

[0202] PC0035

[0203] The VH amino acid sequence is as shown in SEQ ID NO.61, its encoding nucleic acid is as shown in SEQ ID NO.62, and its HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO.63, 64, 65 respectively.

[0204] VH amino acid sequence:

[0205] QVQLVQSGSELMKPGASVKVSCKAS GYTFSYYA INWVRQVPGQGLEWMGW INTNTGKP SYARGLTGRFVFSLDTSVNTAFLQISSLLPDDSAIYYC ARGNLVRSLRGATGRNWIDP WGLGTLVTVSS

[0206] VH nucleic acid sequence:

[0207] CAGGTGCAGCTGGTGCAGTCTGGGTCTGAGTTGATGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCCTCTGGATACACTTTTAGTTATTATGCTATAAATTGGGTGCGACAGGTCCCTGGACAAGGACTTGAGTGGATGGGATGGATCAACACCAACACTGGGAAACCAAGTTATGCCCGGGGCCTCACAGGACGATTTGTCTTCTCCTTGGACACGTCTGTCAACACGGCTTTTCTGCAGATCAGTAGCCTATTGCCTGACGACTCTGCCATTTATTACTGTGCGCGGGGTAATTTGGTTCGTTCGCTTCGGGGAGCCACGGGGCGCAACTGGATCGACCCCTGGGGCCTGGGAACTCTGGTCACCGTCTCCTCA

[0208] The VL amino acid sequence is shown in SEQ ID NO.66, its encoding nucleic acid is shown in SEQ ID NO.67, and its LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO.68, 69, and 70 respectively.

[0209] VL amino acid sequence:

[0210] QSALTQPASVSGSPGESITVSCTGS TSDVGGYNY VSWYQQHPGKAPKLLIY DVS HRPAGVSSRFSASKSGNTASLTISWLQADDEGDYYC SSYTSSNSYV FGTGTSVTVL

[0211] VL nucleic acid sequence:

[0212] CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGAGAGTCGATCACCGTCTCCTGCACTGGAAGCACCAGTGACGTTGGTGGATACAACTATGTCTCCTGGTACCAGCAACACCCAGGCAAAGCCCCCAAACTCTTGATCTATGATGTCAGTCATCGGCCCGCAGGAGTTTCTAGTCGCTTCTCTGCGTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTTGGCTCCAGGCTGACGACGAGGGTGATTATTACTGCAGCTCATATACAAGCAGCAATTCCTATGTCTTCGGCACTGGGACTTCGGTCACCGTCCTG

[0213] PC0037

[0214] The VH amino acid sequence is shown in SEQ ID NO.71, the nucleic acid encoding it is shown in SEQ ID NO.72, and its HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO.73, 74, and 75 respectively.

[0215] VH amino acid sequence:

[0216] QVQLVQSGAEVKKPGESLKISCKGY GYNFATNW IGWVRQVPGKGLEWMGI IFPADSDT RYSPSFQGQVTISADKSTATAYLQWRGLKASDTAVYYC AKQSIPGWRWLDS WGQGALVTVSS

[0217] VH nucleic acid sequence:

[0218] CAGGTGCAGCTGGTGCAGTCTGGGGCAGAGGTGAAAAAGCCCGGGGAGTCCCTGAAGATCTCTTGTAAGGGTTATGGATACAACTTTGCCACGAACTGGATCGGCTGGGTGCGCCAGGTGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATATTTCCTGCTGACTCTGACACCAGATATAGTCCGTCCTTCCAAGGCCAGGTCACCATATCAGCCGACAAGTCAACCGCCACCGCCTACCTCCAGTGGCGTGGCCTGAAGGCCTCGGACACCGCCGTGTATTATTGTGCGA AACAGTCAATACCTGGATGGAGGTGGCTTGACTCATGGGGCCAGGGGGCCCTGGTCACCGTCTCCTCA

[0219] The VL amino acid sequence is as shown in SEQ ID NO.76, its encoding nucleic acid is as shown in SEQ ID NO.77, and its LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO.78, 79, 80 respectively.

[0220] VL amino acid sequence:

[0221] SYELTQPPSVSVSAGQTARITCSGD ALPKQH AHWYHQKPGQAPVLVMY KET ERPSGIPERFSGSSSGTTVTLTISAVRAEDEGDYYC QSEDSSATYLI FGGGTTLTVV

[0222] VL nucleic acid sequence:

[0223] TCTTATGAGCTGACTCAGCCACCCTCGGTGTCAGTGTCCGCAGGACAGACGGCCAGGATCACCTGCTCTGGAGATGCATTGCCAAAGCAACATGCTCATTGGTATCATCAGAAGCCAGGCCAGGCCCCTGTGTTGGTGATGTATAAAGAGACTGAGAGGCCGTCAGGGATACCTGAGCGATTCTCTGGCTCCAGTTCAGGGACAACAGTCACGTTGACAATCAGCGCAGTCCGGGCAGAGGACGAGGGCGACTATTACTGTCAATCAGAAGACAGCAGCGCCACTTATCTGATTTTTGGCGGAGGGACCACGCTGACCGTCGTA

[0224] The above 8 antibodies are all of IgG1 subtype. The amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO.81, and the encoding nucleic acid is as shown in SEQ ID NO.82.

[0225] Amino acid sequence of the heavy chain constant region:

[0226] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0227] Nucleic acid sequence of the heavy chain constant region:

[0228] GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAA

[0229] Among the above 8 antibodies, the light chains of PC0004, PC0010, PC0012, and PC0014 are κ light chains. The amino acid sequence of the κ light chain constant region is as shown in SEQ ID NO.89, and the nucleic acid encoding it is as shown in SEQ ID NO.90.

[0230] Amino acid sequence of the κ light chain constant region

[0231] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0232] Nucleic acid sequence of the κ light chain constant region

[0233] CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0234] Among the above 8 antibodies, the light chains of PC0031, PC0034, PC0035, and PC0037 are λ light chains. The amino acid sequence of the λ light chain constant region is as shown in SEQ ID NO.91, and the nucleic acid encoding it is as shown in SEQ ID NO.92.

[0235] The amino acid sequence of the λ light chain constant region is as follows:

[0236] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0237] The nucleotide sequence of the λ light chain constant region is as follows:

[0238] GGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA

[0239] Reactivity of antibodies in Example 3 that bind to CMV trimer and pentamer complexes

[0240] To determine the protein components in the CMV pentamer complex recognized by the isolated pentamer-reactive antibodies, in this example, recombinant CMV trimer and pentamer complexes ( Figure 1 A) were produced and purified and verified by known CMV gH / gL-reactive and pentamer-specific neutralizing antibodies ( Figure 3 ). Using 8I21 (gene synthesized, sequence from PDB database 5VOC) and 9I6 (gene synthesized, sequence from PDB database 5VOD) as CMV pentamer-specific antibodies and MSL-109 antibody (gene synthesized, sequence from PDB database 4LRI) as a control, the binding of 8 purified antibodies to the recombinant CMV trimer and pentamer complexes was tested in ELISA. The results showed that according to the reactivity to CMV trimer and pentamer, the 8 tested antibodies could be divided into 2 groups. The antibodies PC0004, PC0010, PC0012, PC0014, PC0035, and PC0037 in Group 1 could bind both pentamer and trimer simultaneously with the control antibody MSL-109, but the binding of PC0004 to the CMV trimer was relatively weak ( Figure 3 ). MSL-109 binds to the gH / gL subunit in the CMV trimer and pentamer complexes. PC0031 and PC0034 in Group 2 could only bind to the CMV pentamer with the control antibodies 8I21 and 9I6 ( Figure 3 ). Since gH / gL is present in both the CMV trimer and pentamer complexes, these results suggest that the antibodies in Group 1 may recognize gH / gL in the complex, while the two antibodies in Group 2 may recognize pUL128 / 130 / 131A (Figure 3 ) The fact that two groups of antibodies recognize different subunits or different antigenic epitopes reflects that the two groups of antibodies have different antiviral activities. This is of great clinical significance for developing an antibody that completely targets the pentamer-cell receptor binding region and completely blocks the binding of the virus to the cell.

[0241] Example 4 Neutralization Activity of Antibodies That Specifically Bind to CMV Pentamers

[0242] The neutralizing ability of all 8 purified antibodies against CMV was tested in human embryonic lung fibroblasts (MRC-5, purchased from ATCC, catalog number: CCL-171). It was found that 3 antibodies, PC0012, PC0014, and PC0035, among the 6 antibodies in Group 1, together with the control antibody MSL-109, neutralized the HCMV laboratory standard Towne strain, with EC50s of 0.648 - 0.938 μg / mL, and also neutralized the HCMV wild-type strain BE13 / 2012, with EC50s of 0.299 - 1.480 μg / mL ( Figure 4 A and 4B). In the fibroblast-based neutralization assay, the two pentamer-specific antibodies, PC0031 and PC0034, in Group 2 did not neutralize the CMV Towne strain ( Figure 4 A), nor did they neutralize the clinical isolate BE13 / 2012 (data not shown), which is the same as the known pentamer-specific neutralizing antibodies 8I21 and 9I6 (39). However, among the 2 pentamer-specific antibodies, only PC0034 was shown to neutralize 2 tested HCMV wild strains in the ARPE-19 epithelial cell-based neutralization assay, including VR1814 (GenBank: GU179289.1) (40), NR (GenBank: KX544831.1) (40), and the recombinant HCMV strain AD169 FIX (chimerically expressing the complete pentamer membrane protein on the standard strain AD169 (GenBank: FJ527563.1)) (40), with EC50s of 0.068, 0.070, and 0.079 μg / ml, respectively ( Figure 4 C).

[0243] Example 5 Binding Ability of Antibodies and Mechanism of Neutralizing CMV

[0244] (1) Binding and Cross-Reactivity of Neutralizing Antibodies

[0245] The binding affinities of these two groups of HCMV neutralizing antibodies to purified gH / gL / gO trimers and gH / gL / UL128 / UL130 / UL131A pentamers were determined by SPR and compared with the known neutralizing antibody MSL-109. The three gH / gL-binding neutralizing antibodies PC0012, PC0014, and PC0035 had high affinities for binding to the pentamer, with KD values of 2.85×10 -10 M, 3.23×10 -10 M, and 1.83×10 -10 M( Figure 5 A), and their KD values for binding to the trimer were 9.38×10 -11 M, 1.00×10 -9 M, and 1.01×10 -10 M( Figure 5 B). The binding affinities of antibodies PC0012, PC0014, and PC0035 to the pentamer were approximately 4 - 7 times higher than that of antibody MSL-109( Figure 5 A and Table 2), and their affinities for the trimer were approximately 20 - 230 times higher than that of antibody MSL-109( Figure 5 B and Table 2).

[0246] Table 2. Affinities of antibodies PC0012, PC0014 and PC0035 for binding to HCMV pentamers and trimers

[0247]

[0248] To gain insight into the relationship of the antigenic epitopes recognized by antibodies PC0012, PC0014, and PC0035 compared with the known neutralizing antibody MSL-109, the cross-competitive blocking binding of these antibodies to CMV pentamers was determined by SPR and ELISA( Figure 6 ). It was found that the antibodies in Group 1 did not compete with the antibodies in Group 2 for binding, and vice versa in the ELISA assay( Figure 6 ). Among the three newly isolated gH / gL-binding neutralizing antibodies in Group 1, PC0012 blocked the binding of antibodies PC0014 and PC0035 to CMV pentamers, while PC0014 only partially blocked it, and PC0035 did not block the binding of PC0012 to CMV pentamers at all. PC0014 and PC0035 did not block each other's binding to CMV pentamers( Figure 6 ). PC0012 and PC0035 completely blocked the binding of MSL-109 to CMV pentamers, while MSL-109 did not block the binding of PC0012 to CMV pentamers but could block the binding of PC0035 to CMV pentamers. PC0014 and MSL-109 did not block each other's binding to CMV pentamers(Figure 6 )。

[0249] Previous studies have shown that in virus mutants resistant to MSL-109, the W168C / R, P171H / S, and D446N mutations in the gH subunit result in virus tolerance to the antibody MSL-109, and at the same time, the antibody MSL-109 does not bind to the gH / gL with the W168C / R mutation (41). To evaluate whether the binding of antibodies PC0012, PC0014, and PC0035 to the pentamer is affected by mutations at these three sites on the gH subunit, pentamer mutants targeting these three sites were recombinantly expressed. The experimental results showed that antibodies PC0012, PC0014, and PC0035 strongly bound to these three pentamer mutants( Figure 7 A), while the antibody MSL-109 did not bind to these mutants or bound very weakly( Figure 7 A). Therefore, antibodies PC0012, PC0014, and PC0035 recognize antigenic epitopes in similar regions, and their epitopes are different from those of the antibody MSL-109, but may partially overlap or be close.

[0250] (2) Mechanism of neutralization of CMV by antibodies that bind to gH / gL

[0251] It has been reported that neutralizing antibodies that bind to gH / gL and gB play a neutralizing role after the virus adheres to host cells (35, 41). To clarify how antibodies PC0012, PC0014, and PC0035 affect virus-infected cells, first, complexes of antibodies PC0012, PC0014, and PC0035 with the virus were incubated with MRC-5 cells pre-cooled at 4°C. It was found that neither antibodies PC0012, PC0014, and PC0035 nor the antibody MSL-109 and the irrelevant antibody TRN006 (Patent: CN103910796B) reduced the amount of CMV virus on the cell membrane surface, while almost no viral DNA copies were detected in the positive control heparin( Figure 7 B). Subsequently, it was studied whether antibodies PC0012, PC0014, and PC0035 could interfere with the infection of cells by the virus attached to the cell membrane surface. CMV virus was pre-inoculated into MRC-5 cells at 4°C and incubated for 30 minutes, and then antibodies PC0012, PC0014, and PC0035 were added. It was found that antibodies PC0012, PC0014, and PC0035 as well as the antibody MSL-109 could block CMV from infecting cells( Figure 7 C). Therefore, antibodies PC0012, PC0014, and PC0035 as well as the antibody MSL-109 do not block the adhesion of the virus to cells, but block the invasion process after virus adhesion.

[0252] HCMV also spreads between cells by mediating syncytium formation (42). The inventors evaluated the effects of antibodies PC0012, PC0014, and PC0035 on the intercellular spread of HCMV. Five days after virus inoculation, the infection status of HCMV was judged by detecting the expression of CMV virus-specific CMV IE1 / IE2 protein. Larger and more numerous brown-stained satellite replication centers were seen in virus-infected cells without added antibody (only virus) or treated with the negative control antibody TRN006, while smaller-sized and fewer brown-stained satellite virus replication centers with uniform dispersion were observed in infected cells treated with antibodies PC0012, PC0014, PC0035, or MSL-109( Figure 7 D). These results indicate that antibodies PC0012, PC0014, and PC0035, as well as MSL-109, can inhibit the intercellular spread of HCMV in fibroblasts.

[0253] Example 6 Antibody PC0034 Blocks the Binding of HCMV Pentamer to Epithelial and Endothelial Cells

[0254] The CMV neutralizing antibody 9I6 has been clearly defined as recognizing the pentamer-specific site and blocking the binding of CMV pentamer to the cell surface receptor Nrp2 (43). The affinity of antibody PC0034 for binding to the CMV pentamer is 1.05×10 -10 M, while the affinity of antibody 9I6 for binding to the pentamer is slightly lower, with a KD of 4.84×10 -10 M, which is lower than that of PC0034( Figure 8 A). SPR analysis shows that antibodies PC0034 and 9I6 can completely block each other's binding to the CMV pentamer( Figure 8 B). The recombinant expressed HCMV pentamer protein can bind to epithelial cells (APRE-19, purchased from ATCC, catalog number: CRL-2302) and endothelial cells (HUVEC, purchased from ATCC, catalog number: CRL-1730), and there is a dose-response relationship( Figure 8 C). After antibody PC0034 binds to the HCMV pentamer protein, antibody PC0034 can inhibit the binding of the pentamer to epithelial and endothelial cells( Figure 8 D). Therefore, these data indicate that antibody PC0034 exerts antiviral activity by blocking the binding of the pentamer to the cell surface receptor.

[0255] Example 7 Study on the Epitope Recognized by Antibody PC0034

[0256] Figure 9The binding results of antibody PC0034 to the HCMV pentamer mutants are shown. The affinity changes of antibody PC0034 for these 4 pentamer mutants were evaluated by SPR, where UL131A_E23A decreased by 9.96-fold, UL131A_K27A decreased by 33.93-fold, UL128_K47A decreased by 9.33-fold, and UL128_T94A decreased by 81.06-fold( Figure 9 A to 9D and Table 3). The affinity of antibody PC0034 for the pentamer is closely related to 2 sites (UL128_T94 and UL131A_K27) on the pentamer, but the affinity changes of antibody 9I6 for the pentamer mutants at these 2 sites are not obvious. Therefore, the binding epitope of antibody PC0034 is different from that of 9I6. Meanwhile, in the experiment of pentamer protein binding to cells, it was found that the binding of pentamer mutants (UL128_T94A and UL131A_K27A) to epithelial cells was significantly reduced( Figure 9 F and 9G). These results indicate that the binding epitope of antibody PC0034 on the pentamer overlaps with the binding epitope of the pentamer to the cell surface receptor.

[0257] Table 3. Affinity of antibodies binding to HCMV pentamer and pentamer mutants

[0258]

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Claims

1. An antibody against human cytomegalovirus or an antigen-binding fragment thereof, wherein the CDR combinations of the heavy-chain and light-chain variable regions are as follows: The heavy-chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO. 33 - 35, and the light-chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO. 38 - 40.

2. The antibody or antigen-binding fragment thereof according to claim 1, which has the following combination of heavy-chain variable region and light-chain variable region: The heavy-chain variable region comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO. 31, and the light-chain variable region comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO.

36.

3. The antibody or antigen-binding fragment thereof according to claim 1, which has the following combination of heavy-chain variable region and light-chain variable region: The heavy-chain variable region has the amino acid sequence shown in SEQ ID NO. 31, and the light-chain variable region has the amino acid sequence shown in SEQ ID NO.

36.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 - 3, wherein the antibody is an IgG1, IgG2, IgG3, or IgG4 type antibody.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein the antibody is an IgG1 type antibody.

6. The antibody or antigen-binding fragment thereof according to claim 4, wherein the constant region of the heavy chain comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO. 81; and / or The constant region of the light chain comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO. 89 or 91.

7. The antibody or antigen-binding fragment thereof according to claim 4, wherein the constant region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 81; and / or the constant region of the light chain has the amino acid sequence shown in SEQ ID NO. 89 or 91.

8. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 - 7.

9. The nucleic acid according to claim 8, wherein the nucleic acid has a nucleic acid sequence encoding the heavy-chain variable region of the antibody of SEQ ID NO. 32 or any variant thereof, and a nucleic acid sequence encoding the light-chain variable region of the antibody of SEQ ID NO. 37 or any variant thereof.

10. A vector comprising the nucleic acid according to claim 8 or 9.

11. A host cell comprising the nucleic acid according to claim 8 or 9 or the vector according to claim 10.

12. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-7, the nucleic acid according to claim 8 or 9, the vector according to claim 10, and / or the cell according to claim 11.

13. An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-7 and a label.

14. A method for generating the antibody or antigen-binding fragment thereof according to any one of claims 1-7, the method comprising culturing a host cell comprising the nucleic acid according to claim 8 or 9 or the vector according to claim 10.

15. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-7, the nucleic acid according to claim 8 or 9, the vector according to claim 10, the host cell according to claim 11, or the immunoconjugate according to claim 13 in the preparation of a pharmaceutical composition or kit for detecting, treating, preventing, and / or alleviating HCMV infection or HCMV-related diseases.

16. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-7, the nucleic acid according to claim 8 or 9, the vector according to claim 10, the host cell according to claim 11, the pharmaceutical composition according to claim 12, or the immunoconjugate according to claim 13 in the preparation of a drug for enhancing, strengthening, or stimulating the resistance of a human individual infected with HCMV.

17. A method for neutralizing HCMV in a sample for non-diagnostic and non-therapeutic purposes, the method comprising contacting the antibody or antigen-binding fragment thereof according to any one of claims 1-7 with the sample and testing the ability of the antibody or antigen-binding fragment thereof to bind to neutralize HCMV.

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