Cd180 antibodies and uses thereof
By developing monoclonal or humanized antibodies against human CD180, the problem of lacking antibodies that specifically bind to human CD180 in existing technologies has been solved, enabling effective diagnosis and treatment of CD180-mediated diseases.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
- Filing Date
- 2025-01-24
- Publication Date
- 2026-07-24
Smart Images
Figure CN122444871A_ABST
Abstract
Description
Technical Field
[0001] This disclosure relates to the field of biomedicine, and specifically, to an antibody against human CD180 and its applications. Background Technology
[0002] CD180 is a type I single-pass transmembrane protein composed of 661 amino acids with a molecular weight of 74 kDa. CD180 is expressed on the cell surface as a 2:2 heterodimer with MD1, and its expression on the cell membrane depends on binding to MD1. CD180 belongs to the Toll-like receptor (TLR) family, shares high homology with TLR4, and participates in various physiological functions. CD180 is known to be expressed on B lymphocytes, dendritic cells, monocytes, and macrophages, participating in signaling activation, regulating TLR immune responses, and mediating B cell innate immune responses to lipopolysaccharide (LPS). Simultaneously, CD180 participates in the pathological processes of various diseases, including tumors, autoimmune diseases, and inflammation. High expression of CD180 in B-lymphocyte malignancies has been reported, including chronic lymphocytic leukemia, mantle cell lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, marginal zone lymphoma, and follicular lymphoma. In various autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and Kawasaki disease, the expression level of CD180 on B cells changes, suggesting that CD180 may play an important role in the pathological process of autoimmune diseases. Furthermore, studies related to cardiovascular diseases suggest that CD180 may exert a protective effect against ischemic cardiac injury by inhibiting TLR4-mediated inflammatory responses.
[0003] Immunotherapy based on antibody technology has developed rapidly in fields such as cancer treatment. However, there are currently no antibody drugs targeting CD180 that have been applied clinically, so there is a need to further develop molecules that specifically bind to human CD180 to treat CD180-mediated diseases. Summary of the Invention
[0004] Technical problems to be solved:
[0005] One aspect of this disclosure is to provide an antibody against human CD180, addressing the problem of the lack of an ideal molecule that specifically binds to human CD180 in the prior art.
[0006] Technical solution:
[0007] An isolated antibody or antigen-binding moiety that specifically binds to human CD180 protein, the antibody or antigen-binding moiety comprising:
[0008] (a) The complementarity-determining region (CDRH) of the heavy chain variable region: selected from at least one of the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 7, SEQ ID No. 8 or SEQ ID No. 9; and / or
[0009] (b) Complementarity-determining region (CDRL) of the light chain variable region: selected from at least one of the amino acid sequences shown in SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 10, SEQ ID No. 11, and SEQ ID No. 12.
[0010] In some embodiments, the antibody or antigen-binding portion comprises:
[0011] (c) the heavy chain variable region CDRH1 as shown in SEQ ID No. 1, the heavy chain variable region CDRH2 as shown in SEQ ID No. 2, and the heavy chain variable region CDRH3 as shown in SEQ ID No. 3; and / or
[0012] Light chain variable region CDRL1 as shown in SEQ ID No. 4, light chain variable region CDRL2 as shown in SEQ ID No. 5, and light chain variable region CDRL3 as shown in SEQ ID No. 6; or
[0013] (d) Heavy chain variable region CDRH1 as shown in SEQ ID No. 7, heavy chain variable region CDRH2 as shown in SEQ ID No. 8, and heavy chain variable region CDRH3 as shown in SEQ ID No. 9; and / or
[0014] Light chain variable region CDRL1 as shown in SEQ ID No. 10, light chain variable region CDRL2 as shown in SEQ ID No. 11, and light chain variable region CDRL3 as shown in SEQ ID No. 12.
[0015] In one embodiment, the antibody or antigen-binding portion comprises:
[0016] (g) the heavy chain variable region CDRH1 as shown in SEQ ID No. 1, the heavy chain variable region CDRH2 as shown in SEQ ID No. 2, and the heavy chain variable region CDRH3 as shown in SEQ ID No. 3; and
[0017] Light chain variable region CDRL1 as shown in SEQ ID No. 4, light chain variable region CDRL2 as shown in SEQ ID No. 5, and light chain variable region CDRL3 as shown in SEQ ID No. 6; or
[0018] (h) the heavy chain variable region CDRH1 as shown in SEQ ID No. 7, the heavy chain variable region CDRH2 as shown in SEQ ID No. 8, and the heavy chain variable region CDRH3 as shown in SEQ ID No. 9; and
[0019] Light chain variable region CDRL1 as shown in SEQ ID No. 10, light chain variable region CDRL2 as shown in SEQ ID No. 11, and light chain variable region CDRL3 as shown in SEQ ID No. 12.
[0020] In this disclosure, the antibody or antigen-binding moiety (g) is labeled as A24C9, and the antibody or antigen-binding moiety (h) is labeled as C12F11.
[0021] In some embodiments, the antibody or antigen-binding portion comprises:
[0022] (e) the heavy chain variable region as shown in SEQ ID No. 13 and an amino acid sequence having more than 90% identity with said heavy chain variable region; and / or
[0023] The light chain variable region as shown in SEQ ID No. 14 and the amino acid sequence having more than 90% identity with the light chain variable region; or
[0024] (f) the heavy chain variable region as shown in SEQ ID No. 15 and an amino acid sequence having more than 90% identity with said heavy chain variable region; and / or
[0025] The light chain variable region shown in SEQ ID No. 16 and the amino acid sequence having more than 90% identity with the light chain variable region.
[0026] In one embodiment, the antibody or antigen-binding portion comprises:
[0027] (i) the heavy chain variable region as shown in SEQ ID No. 13 and an amino acid sequence having more than 90% identity with said heavy chain variable region; and
[0028] The light chain variable region as shown in SEQ ID No. 14 and the amino acid sequence having more than 90% identity with the light chain variable region; or
[0029] (j) the heavy chain variable region as shown in SEQ ID No. 15 and an amino acid sequence having more than 90% identity with said heavy chain variable region; and
[0030] The light chain variable region shown in SEQ ID No. 16 and the amino acid sequence having more than 90% identity with the light chain variable region.
[0031] In some embodiments, the antibody may be a mammalian-derived antibody, such as that from mice, rabbits, sheep, horses, monkeys, pigs, camels, sharks, chickens, etc. In other embodiments, the antibody may be a chimeric antibody, a humanized antibody, or a fully human antibody.
[0032] In some embodiments, the antibody may be IgG, IgA, IgM, IgD, or IgE. Preferably, in some embodiments, the antibody may be IgG. Further, in some embodiments, the antibody may be one or more selected from IgG1, IgG2, IgG3, or IgG4. Preferably, the antibody may be IgG1.
[0033] In some embodiments, the antibody is a monoclonal antibody.
[0034] In some embodiments, the antibody or antigen-binding portion is modified, including N-glycosylation, O-glycosylation, phosphorylation, methylation, acetylation, or labeling.
[0035] In some embodiments, the antibody includes an Fc moiety. Preferably, in some embodiments, the Fc moiety of the antibody is modified or altered to enhance its ADCC, CDC, or ADCP activity.
[0036] In some embodiments, the antigen-binding moiety is Fab, Fab', F(ab')2, Fd, FCL, dAb, or a single-chain antibody scFv. Preferably, in some embodiments, the antigen-binding moiety is a single-chain antibody scFv.
[0037] Another aspect of this disclosure is to provide a multivalent antibody comprising the antibody or antigen-binding portion described above.
[0038] The multivalent antibody can be, for example, bivalent, trivalent, tetravalent, hexavalent, nonavalent, etc. The multivalent antibody can be prepared using suitable methods in the prior art. Preferably, in some embodiments, the multivalent antibody is a bispecific antibody or a trispecific antibody.
[0039] Another aspect of this disclosure is to provide a multispecific antibody that selectively binds at least to human CD180, the multispecific antibody comprising the antibody or antigen-binding portion described above; the multispecific antibody is a monovalent antibody or a multivalent antibody.
[0040] Another aspect of this disclosure is to provide an isolated polynucleotide encoding the aforementioned antibody or antigen-binding moiety, or encoding the aforementioned multivalent antibody. Further, in some embodiments, the polynucleotide encodes an amino acid sequence as shown in SEQ ID No. 1-16.
[0041] Another aspect of this disclosure is to provide a vector comprising the aforementioned polynucleotide.
[0042] Another aspect of this disclosure is to provide a cell comprising the aforementioned antibody or antigen-binding moiety, the aforementioned multivalent antibody, the aforementioned polynucleotide, or the aforementioned carrier. In some embodiments, the cell may be any suitable host cell used as a tool for producing the target protein. For example, SP2 / 0, YB2 / 0, IR983F, human myeloma Namalwa, PERC6 or CHO cell lines, insect cells, or Escherichia coli cells.
[0043] Another aspect of this disclosure is to provide a method for generating an anti-human CD180 antibody or antigen-binding moiety, wherein the above-mentioned cells are subjected to protein expression to obtain the anti-human CD180 antibody or antigen-binding moiety.
[0044] Another aspect of this disclosure is to provide a pharmaceutical composition comprising the aforementioned antibody or antigen-binding moiety, the aforementioned multivalent antibody, the aforementioned polynucleotide, the aforementioned carrier or the aforementioned cell, and a pharmaceutically acceptable carrier. In some embodiments, to achieve better therapeutic effects, the pharmaceutical composition may further comprise other therapeutic agents.
[0045] Another aspect of this disclosure is to provide an immunoconjugate comprising:
[0046] a) The antibody or antigen-binding moiety described above, or the multivalent antibody described above; and
[0047] b) Therapeutic agents or detectable markers; and
[0048] c) The connecting body between parts a) and b) above;
[0049] The therapeutic agents include drugs, enzymes, toxins, cytokines, or radionuclides.
[0050] Another aspect of this disclosure is the use of the above-mentioned antibody or antigen-binding moiety, the above-mentioned multivalent antibody, the above-mentioned polynucleotide, the above-mentioned carrier, the above-mentioned cell, the above-mentioned pharmaceutical composition or the above-mentioned immune conjugate in the preparation of a medicament for treating the following diseases: tumors, autoimmune diseases, graft-versus-host disease or infections.
[0051] Furthermore, in some embodiments, the tumor is selected from breast cancer, neurotumor, melanoma, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, prostate cancer, ovarian cancer, cervical cancer, glioblastoma, esophageal cancer, bladder cancer, renal cell carcinoma, endometrial cancer, skin cancer, testicular cancer, thyroid cancer, lymphoma, leukemia, and multiple myeloma;
[0052] Furthermore, in some embodiments, the tumor is selected from acute myeloid leukemia, chronic lymphocytic leukemia, and B-cell lymphoma.
[0053] Another aspect of this disclosure is the use of the aforementioned antibody or antigen-binding moiety, the aforementioned multivalent antibody, the aforementioned polynucleotide, the aforementioned carrier, the aforementioned cell, the aforementioned pharmaceutical composition, or the aforementioned immunoconjugate in the preparation of a medicament for treating diseases mediated by TLR4.
[0054] Another aspect of this disclosure is the use of the aforementioned antibody or antigen-binding moiety or the aforementioned multivalent antibody in the preparation of products for detecting the presence or level of human CD180 molecules in a sample.
[0055] Beneficial effects:
[0056] The anti-human CD180 antibodies A24C9 and C12F11 disclosed herein exhibit strong affinity for human CD180 protein, with Kd values of 0.4748 nM and 78.19 nM, respectively. They also demonstrate high binding specificity, specifically recognizing human CD180-positive AML cell lines (OCI-AML2, MV4-11, THP1) and human CD180-positive B-cell lymphoma cell lines (Daudi, BJAB, Nalm6). Furthermore, they show no cross-reactivity with various human CD180-negative cell lines (K562, NB4, Kasumi1, TF1, NK92, Jurkat, SupT1, and MM1S). These antibodies can be used to detect human CD180 protein expression and can be applied alone or in combination with other methods for immunotherapy, demonstrating diagnostic and therapeutic value in tumors, autoimmune diseases, graft-versus-host disease, and inflammatory diseases. Attached Figure Description
[0057] Figure 1 This is a graph showing the detection results of antibody A24C9 and antibody C12F11 subtypes in the embodiments of this disclosure;
[0058] Figure 2 The images shown are SDS-PAGE images of the pure antibodies A24C9 and C12F11 in the embodiments of this disclosure. Lane M is the marker, lane 1 is the ascites supernatant, and lane 2 is the purified product.
[0059] Figure 3This is a graph showing the flow cytometry results for determining the affinity constants of antibodies A24C9 and C12F11 in this embodiment of the present disclosure.
[0060] Figure 4 This diagram shows the flow cytometry results of the binding of antibodies A24C9 and C12F11 to hCD180-positive AML and B lymphoma cell lines in embodiments of this disclosure. Negative: negative isotype control; positive: positive control, commercial antibody MHR73-11.
[0061] Figure 5 This is a flow cytometry result of the non-specific binding of antibodies A24C9 and C12F11 to hCD180 negative cell lines in the embodiments of this disclosure. In the figure, negative: isotype control; positive: positive control commercial antibody MHR73-11.
[0062] Sequence Description
[0063]
[0064] Detailed Implementation
[0065] This invention discloses an antibody against human CD180 and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. Furthermore, those skilled in the art can clearly modify or appropriately alter and combine the content described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.
[0066] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising of," etc., shall be understood to include the stated elements or components without excluding other elements or other components. The term "a," "an," and "the" includes plural indicators. The term "a plurality of" means two or more. The terms "such as," "for example," etc., are intended to refer to exemplary embodiments and are not intended to limit the scope of this disclosure.
[0067] In this disclosure, when a range of values is provided, it should be understood that, unless the context otherwise explicitly indicates otherwise, the range includes endpoints and each intermediate value between the upper and lower limits of the range, as well as any other specified value or intermediate value within the specified range and any value within a smaller range between specified values.
[0068] In this disclosure, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.
[0069] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Definitions of common molecular biology terms can be found in Lewin's *GENES*, Twelfth Edition, Jocelyn E. Krebs, Elliott S. Goldstein, Stephen T. Kilpatrick, Jones & Bartlett Learning. Definitions of common biochemistry terms can be found in Lehninger's *Principles of Biochemistry*, Eighth Edition, David L. Nelson, Michael M. Cox, WH Freeman. Definitions of common cell biology terms can be found in *Molecular Biology of the Cell*, Sixth Edition, Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter, Garland Science. Definitions of common genetics terms can be found in *Genetics: Analysis of Genes and Genomes*, Eighth Edition, Daniel L. Hartl, Maryellen Ruvolo, Jones & Bartlett Learning.
[0070] Unless otherwise specified, the experimental techniques used in this paper employ standard techniques from immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which can be found in standard books such as *Molecular Cloning: A Laboratory Manual* and *Cell Biology: A Laboratory Handbook*.
[0071] definition:
[0072] The term "separated" in this disclosure refers to a substance or entity that has been removed from its natural environment or the environment in which it existed prior to separation and is separate from other components. For example, a separated protein substantially does not originate from cellular material or other proteins derived from the cell or tissue from which it originates. The separation ratio can be, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. Separated substances may have different levels of purity relative to the substances before their separation.
[0073] The term "antibody" in this disclosure refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair having one "light" (L) chain and one "heavy" (H) chain). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both the light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant regions of an antibody mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antibody-binding sites. The term "antibody" is not limited to any particular method of antibody production. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be different isotypes of antibody, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibody. In some embodiments, the antibody can be IgG, IgA, IgM, IgD, or IgE. Preferably, in some embodiments, the antibody type can be IgG. Further, in some embodiments, the antibody can be one or more selected from IgG1, IgG2, IgG3, or IgG4. Preferably, the antibody can be IgG1.
[0074] Antibody preparation:
[0075] In some embodiments, the antibodies are generated using mammalian cells. For example, monoclonal antibodies are generated in mammalian cells using hybridoma technology. The monoclonal antibodies can be prepared using the hybridoma preparation method reported by Kohler et al. in Nature 256:495 (1975). Mice or other suitable host animals are first immunized with an immunogen (with adjuvants added if necessary).
[0076] Immunogens or adjuvants are typically administered via subcutaneous multi-site injection or intraperitoneal injection. Adjuvants can include Freund's adjuvant (complete or incomplete) or MPL-TDM, etc. After immunization, animals produce lymphocytes that secrete antibodies specifically binding to the immunogen. Target lymphocytes are collected and fused with myeloma cells using a suitable fusion agent (such as PEG4000) to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996).
[0077] The hybridoma cells prepared above are seeded into a suitable culture medium for growth, the medium containing one or more substances that can inhibit the growth of unfused maternal myeloma cells. For example, for maternal myeloma cells lacking hypoxanthine-guanine phosphotransferase (HGPRT or HPRT), the addition of substances such as hypoxanthine, aminopterin, and thymine (HAT medium) to the culture medium can inhibit the growth of HGPRT-deficient cells.
[0078] Preferred myeloma cells should possess high fusion rates, stable antibody secretion capabilities, and sensitivity to HAT culture medium. Among these, murine myeloma cells are preferred, such as the MOP-21 and MC-11 mouse tumor-derived lines (THE Salk Institute Cell Distribution Center, San Diego, Calif.USA), and SP-2 / 0 or X63-Ag8-653 cell lines (American Type Cell Collection, Rockville, Md.USA). Additionally, human monoclonal antibodies can be prepared using human myeloma and human-mouse heterologous myeloma cell lines (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63, Marcel Dekker, Inc., New York, 1987).
[0079] The culture medium used for hybridoma cell growth is used to detect the production of monoclonal antibodies against specific antigens. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined using methods such as immunoprecipitation or in vitro binding assays, including radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). For example, the affinity of monoclonal antibodies can be determined using the Scatchard assay described by Munson et al. in Anal. Biochem. 107:220 (1980).
[0080] After determining the specificity, affinity, and reactivity of the antibodies produced by hybridomas, the target cell line can be subcloned using the limiting dilution method described in Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996. Suitable culture media include DMEM or RPMI-1640. Additionally, hybridoma cells can also grow in animals in the form of ascites tumors.
[0081] Traditional immunoglobulin purification methods, such as protein A agarose gel chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography, can be used to separate monoclonal antibodies secreted by subclonal cells from cell culture medium, ascites fluid, or serum, thereby obtaining the monoclonal antibodies.
[0082] In other embodiments, antibodies against human CD180 can also be generated by known recombinant methods, such as selecting a recombinant antibody library in a phage or similar vector, see, for example, the description in Smith GP. Filamentous fusionphage: novel expression vectors that display cloned antigens on the virionsurface. Science. 1985; 228:1315–17.
[0083] Antibody modification and alteration:
[0084] In some embodiments, the isolated antibody may be a humanized antibody. Humanization of the antibody may improve its affinity or other characteristics. A description and method for humanized antibodies can be found in Riechmann, L., Clark, M., Waldmann, H., & Winter, G. (1988). Reshaping human antibodies for therapy. Nature, 332(6162), 323–327.
[0085] In some embodiments, the antibody Fc (crystallizable region fragment, Fc) is modified to enhance its effector functions triggered by binding to Fc receptors or complement. These functions may include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). The modifications may include: 1) modifying glycosylation, for example, modifying the aspartic acid at position 297 (N297) in the Fc region with N-acetylglucosamine. Mutations of N297 to alanine (A), glutamine (Q), or glycine (G) all inhibit antibody glycosylation, thereby reducing Fc-mediated effector functions. Antibody deglycosylation reduces its ability to induce ADCC or CDC activity; sialic acid modification reduces its binding affinity to FcγRIIIa, thus leading to a decrease in CDC and ADCC activity.
[0086] In addition to the functions mentioned above, glycosylation modification of antibodies can also affect their conformation and stability. For example, the glycans in glycosylation can maintain the antibody's conformation, preventing aggregation or unfolding. For instance, the sugars on the a1-3 arm do not contact the antibody surface but are embedded deep within the space formed by the Fc segments of the two heavy chains. The mannose on the a1-3 arms of the two glycans interacts with each other, which is crucial for maintaining the antibody's conformation. Without the presence of glycans, the CH2 domain of the Fc segment would be slightly enlarged, leading to earlier elution times in size exclusion chromatography and increased sensitivity and aggregation in thermally accelerated stability experiments. Simultaneously, glycosylation modification can also affect the binding of antibodies to receptors on cell membranes, forming complexes that play an important role in signal transduction. This regulation of signal transduction is crucial for physiological processes such as cell proliferation, differentiation, and apoptosis.
[0087] The above modifications may also include: 2) point mutations, for example, LALA mutations (L234A / L235A) can lead to changes in the antibody's affinity for FcγR (eliminating binding to low-affinity FcγR and reducing binding to FcγRI), thereby significantly reducing its ADCC and CDC activities. Additionally, combining trans-subtype antibodies can also modulate the antibody's effector function.
[0088] In some embodiments, the point mutation results in the substitution of some conserved amino acids, thereby obtaining a “conservative amino acid substitution variant”. The change results in some amino acids being substituted by others with similar chemical properties and / or functions. Providing conserved substitutions of amino acids with similar chemical properties and / or functions is well known in the art. Typical examples of mutually conserved substitutions include, for example, (1) alanine (A), glycine (G); (2) aspartic acid (D), glutamic acid (E); (3) asparagine (N), glutamine (Q); (4) arginine (R), lysine (K); (5) isoleucine (I), leucine (L), methionine (M), valine (V); (6) phenylalanine (F), tyrosine (Y), tryptophan (W); (7) serine (S), threonine (T); (8) cysteine (C), methionine (M).
[0089] The above modifications can also include: 3) Phosphorylation modification. Phosphorylation modification refers to the process of adding phosphate groups to amino acids of proteins within cells. Phosphorylation antibodies can specifically recognize specific phosphorylation sites, thereby detecting the increase or decrease in the phosphorylation level of proteins when cells are stimulated. These antibodies play an important role in life science research fields such as cell signaling, apoptosis, and cancer. 4) Methylation modification: Methylation modification is an important dynamic modification and biological phenomenon catalyzed by methyltransferases acting on specific residues of proteins. Methylation antibodies can specifically recognize specific methylated amino acid sites, used to distinguish between methylated and unmethylated forms of proteins. They have wide applications in research fields such as epigenetics, cancer, Alzheimer's disease, and aging. 5) Acetylation modification. Acetylation is one of the most common types of acylation modification. Acetylation antibodies can specifically recognize the acetylated form of target proteins and specific acetylated amino acid sites, detecting the activity level of the protein. These antibodies are widely used in research on cell cycle regulation, signal transduction, neurodegenerative diseases, metabolic diseases, and the occurrence and development of cancer.
[0090] The above modifications can also include: 6) Labeling. Antibodies can be cross-linked with different chemical reagents to attach to substances such as enzymes, fluorescent dyes, biotin, or colloidal gold, thereby altering their detection or analytical performance. For example, enzyme labeling: Antibodies can be cross-linked to enzymes such as horseradish peroxidase (HRP) and alkaline phosphatase. This is commonly used in immunohistochemistry, ELISA, and other experiments, where a color reaction is produced through enzyme catalysis, thus detecting the presence of the antibody. For example, HRP-labeled antibodies can produce a color precipitate after binding to an antigen by adding a substrate, facilitating observation and quantification. Fluorescent dye labeling: Antibodies can also be bound to fluorescent dyes (such as FTC, PE, APC, etc.) for use in flow cytometry, fluorescence microscopy, and other detection methods. Fluorescently labeled antibodies can locate specific antigens in cells or tissue sections, and the intensity of the fluorescence signal can be used to determine the antigen expression level. Biotin labeling: Biotin is a small molecule compound that can bind to antibodies without affecting their antigen-binding ability. Biotin-labeled antibodies can amplify and detect signals by binding to avidin (such as streptavidin). It is commonly used in multiplex immunolabeling assays to detect multiple antigens simultaneously.
[0091] In this disclosure, modifications and alterations to the antibody typically occur in the Fc region and the framework regions (FRs) of the antibody variable region, but not in the complementarity-determining regions (CDRs) of the antibody variable region. The framework regions of the antibody variable region have relatively conserved amino acid sequences, providing stable support for the hypervariable structure and participating in maintaining the three-dimensional conformation of the antigen-binding groove. Therefore, modifications and alterations to these regions do not affect the antibody's binding capacity. In this disclosure, the term "having more than 90% identity" means that, under the aforementioned modifications or alterations, it has approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% identity with the sequence shown in SEQ ID No. 1-16.
[0092] Separated antigen-binding portion:
[0093] The term "antigen-binding moiety" in this disclosure refers to a polypeptide fragment containing a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as an "antigen-binding fragment." See Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)). Antigen-binding fragments of antibodies can be generated through recombinant DNA technology or through enzymatic or chemical fragmentation of intact antibodies. In some cases, antigen-binding fragments include Fab, Fab′, F(ab′)2, Fd, Fv, etc.
[0094] The term "Fab fragment" refers to an antibody fragment composed of VL, VH, CL, and CH1 domains; the term "F(ab′)2 fragment" refers to an antibody fragment containing two Fab fragments connected by a disulfide bridge on the hinge region; the term "Fd fragment" refers to an antibody fragment composed of VH and CH1 domains; and the term "Fv fragment" refers to an antibody fragment composed of the VL and VH domains of a single arm of the antibody.
[0095] In some embodiments, the antigen-binding moiety is prepared using protease digestion, such as papain or pepsin. In other embodiments, the antigen-binding moiety is prepared using chemical reagent treatment. In still other embodiments, the antigen-binding moiety is prepared using genetic engineering methods. That is, a fragment containing all or part of the gene sequence of the antigen-binding moiety is ligated into a suitable vector and expressed. Examples of the expression vector include bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.
[0096] Multivalent and multispecific antibodies:
[0097] In some embodiments, the multivalent antibody comprises at least two antibody or antigen-binding moieties as described in this disclosure, capable of competitively binding to human CD180 molecules and producing effects different from those of monovalent antibodies. For example, by increasing the number of antigen-binding sites, a tighter antigen-antibody complex can be formed, thereby enhancing binding affinity and stability. This enhanced binding affinity helps improve the affinity of anti-human CD180 antibodies for antigens and enhances the interaction between antigens and cell surface receptors or other molecules. The multivalent antibody can be obtained, for example, by protein fusion, the addition of linkers, covalent bonds, or non-covalent bonds.
[0098] In some embodiments, the multispecific antibody, such as a bispecific antibody or a trispecific antibody, includes the antibody or antigen-binding portion described in this disclosure, which, in addition to competitively binding to human CD180 molecules, can also bind to at least one other different site or target molecule. Non-limiting examples include CD33, CD123, CLEC12A, CD47, FLT-3, PDGFR, VEGFR, KIT, IDH1, IDH2, SMO, BCL-2, ALT, c-KIT, CD70, CD45, PD-1 / PD-L1, CTLA-4, TIM-3, TLR-2, CD3, CD4, IL-2, CD20, BAFF, TLR-4, TLR-7, TLR-8, TNF-α, IL-6, IL-6R, IL-17A, IL-17RA, IL-12, IL-23, IL-4, α4 integrin, cell adhesion molecules, complement factor D, JAK1, JAK2, JAK3, TYK2, IL-5, URAT1, TSLP, MASP-2, CSF1R, and ROCK2. These multispecific antibodies can be linked together directly or via linkers. The multispecific antibody can be expressed through recombination. To achieve better therapeutic effects, the multispecific antibody can be monovalent or multivalent.
[0099] The aforementioned multivalent and multispecific antibodies can be prepared using conventional techniques in the art. For the preparation of engineered antibodies, please refer to, for example, Hantao Lou, Xuetao Cao, Antibody variable region engineering for improving cancer immunotherapy, Cancer Communications. 2022; 42:804–827.
[0100] The term "polynucleotide" in this disclosure may also be used interchangeably with "nucleic acid," referring to a chain of nucleotides of any length, including DNA or RNA. It may include any known nucleotide analogs or modified nucleotides or bases.
[0101] The term "vehicle" as used in this disclosure refers to a polynucleotide molecule capable of delivering and / or expressing one or more target genes. Examples of vectors include viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as generating cells.
[0102] Pharmaceutical composition:
[0103] The term "pharmaceutical composition" as used in this disclosure refers to a composition that, in addition to containing the antibody, the antigen-binding moiety, the multivalent antibody, the polynucleotide, the carrier, the cell, or the multispecific antibody described in this disclosure, also contains at least one other substance. In some embodiments, this other substance may be, for example, a pharmaceutically acceptable carrier (a substance that does not affect the function of the mesenchymal stem cells and has no effect on the patient's physical condition, such as physiological saline, cell culture medium, glucose, water for injection, glycerol, ethanol, and combinations thereof), excipients, stabilizers, surfactants, preservatives, isotonic agents, etc. It can also be other therapeutic agents, such as chemotherapy drugs: melphalan, doxorubicin, cyclophosphamide, vincristine, etc.; glucocorticoids: prednisone, dexamethasone, betamethasone, etc.; immunomodulatory drugs: thalidomide, lenalidomide, pomalidomide, etc.; and biologics targeting the following exemplary targets: CD33, CD123, CLEC12A, CD47, FLT-3, PDGFR, VEGFR, KIT, IDH1, IDH2, SMO, BCL-2, ALT, c-KIT, CD70, CD45, P D-1 / PD-L1, CTLA-4, TIM-3, TLR-2, CD3, CD4, IL-2, CD20, BAFF, TLR-4, TLR-7, TLR-8, TNF-α, IL-6, IL-6R, IL-17A, IL-17RA, IL-12, IL-23, IL-4, α4 integrin, cell adhesion molecule, complement factor D, JAK1, JAK2, JAK3, TYK2, IL-5, URAT1, TSLP, MASP-2, CSF1R, ROCK2. The above biologics can be antibodies, small molecule inhibitors, or agonists targeting this target.
[0104] In some embodiments, the above pharmaceutical compositions can be prepared into any suitable formulation. For example, pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, gels, films, tubes, solutions, or suspensions. The above pharmaceutical compositions can be administered by any suitable route of administration, such as intralesional, intravenous, topical, rectal, parenteral, topical, inhalation or subcutaneous, submuscular, intrathecal, transabdominal, oral, and intracerebral administration.
[0105] Immunoconjugates:
[0106] In some embodiments, the immunoconjugates provided in this disclosure may be in any suitable form, such as antibody-drug conjugates (ADCs), radionuclide drug conjugates (RDCs), antibody fusion proteins, etc.
[0107] The aforementioned antibody-drug conjugates comprise the antibody, antigen-binding moiety, multivalent or multispecific antibody, linker, and payload described in this disclosure. Known linkers include, for example, N-succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC), hydrazone linkers, Val-Cit dipeptide, tetrapeptide Gly-Gly-Phe-Gly, glucuronic acid-containing linkers, β-galactosidase-containing linkers, etc. Known payloads include, for example, calendula extract, maytansin derivatives, tubulysins, cryptomycins (CR), pyrrolo[2,1-c][1,4]benzodiazepines (PBD), ducamycin, camptothecin (CPT), chachiin, apoptosis inducers, thailanstatin A, amatoxins, nicotinamide phosphoribosyltransferase, carbamycin, etc.
[0108] Indications:
[0109] In some embodiments, the drugs prepared from the antibodies or antigen-binding moieties, multivalent antibodies, polynucleotides, carriers, cells, pharmaceutical compositions, or immunoconjugates described in this disclosure can be used to treat the following diseases: tumors, autoimmune diseases, graft-versus-host disease, or infections.
[0110] Exemplary examples of tumors include breast cancer, neurotumor, melanoma, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, stomach cancer, kidney cancer, bladder cancer, prostate cancer, ovarian cancer, cervical cancer, glioblastoma, esophageal cancer, bladder cancer, renal cell carcinoma, endometrial cancer, skin cancer, testicular cancer, thyroid cancer, acute myeloid leukemia, chronic lymphocytic leukemia, and B-cell lymphoma.
[0111] Exemplary examples of autoimmune diseases include, for example, autoimmune hematologic disorders (including, for example, hemolytic anemia, aplastic anemia, simple erythrocytic anemia, and idiopathic thrombocytopenic purpura), systemic lupus erythematosus, polychondritis, scleroderma, Wegener granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, Steven-Johnson syndrome, idiopathic diarrhea, autoimmune inflammatory bowel disease (including, for example, ulcerative colitis and Crohn's disease), endocrine ophthalmopathy, Graves' disease, sarcoidosis, multiple sclerosis, primary biliary cirrhosis, type I diabetes, uveitis, keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitial pulmonary fibrosis, psoriatic arthritis, and glomerulonephritis.
[0112] In some embodiments, drugs prepared from antibodies or antigen-binding moieties, multivalent antibodies, polynucleotides, carriers, cells, pharmaceutical compositions, or immunoconjugates described in this disclosure can be used to treat TLR4-mediated diseases. Exemplary examples include, for instance, psoriasis, atopic dermatitis, asthma, COPD, adult respiratory diseases, arthritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, septic shock, endotoxic shock, Gram-negative sepsis, toxic shock syndrome, stroke, cardiac and renal reperfusion injury, glomerulonephritis, thrombosis, Alzheimer's disease, malaria, acute respiratory distress syndrome, delayed-type hypersensitivity reactions, atherosclerosis, cerebral and cardiac ischemia, osteoarthritis, angiogenesis, osteoporosis, gingivitis, respiratory viruses, herpesviruses, hepatitis viruses, HIV, Kaposi's sarcoma-associated virus, meningitis, cystic fibrosis, preterm birth, cough, pruritus, sprains, strains, contusions, psoriatic arthritis, herpes, encephalitis, central nervous system vasculitis, traumatic brain injury, central nervous system tumors, subarachnoid hemorrhage, and postoperative conditions. Trauma, interstitial pneumonia, hypersensitivity reactions, crystal arthritis, acute and chronic pancreatitis, acute alcoholic hepatitis, necrotizing enterocolitis, chronic sinusitis, vasculogenic eye disease, ocular inflammation, retinopathy of prematurity, diabetic retinopathy, polymyositis, vasculitis, acne, gastric and duodenal ulcers, celiac disease, esophagitis, glossitis, airflow obstruction, airway hyperresponsiveness, bronchiectasis, bronchiolitis, obliterative bronchiolitis, chronic bronchitis, cor pulmonale, cough, dyspnea, emphysema, hypercapnia, hyperinflation, hypoxemia, inflammation caused by hyperoxia, hypoxia, surgical lung reduction, pulmonary fibrosis, pulmonary hypertension, right ventricular hypertrophy, peritonitis associated with continuous ambulatory peritoneal dialysis, granulocytic erythrocyte disease, sarcoidosis, small airway disease, wheezing, common cold, gout, alcoholic liver disease, burn treatment, periodontitis.
[0113] Example:
[0114] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.
[0115] Example 1: Screening of monoclonal antibodies for mouse hybridomas
[0116] Balb / c mice were immunized intraperitoneally using CD180-positive cells as antigen cells. Booster immunizations were administered at weeks 2 and 4 after the initial immunization. On day 8 post-booster immunization, 20 μl of tail blood was collected from mice and added to 180 μl of PBS. After standing at room temperature for 1 hour, the mixture was centrifuged at 3000 rpm for 10 minutes. The tail blood supernatant was collected and diluted with PBS to different concentrations: 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800. CD180-overexpressing cell lines were prepared, and the cell density was adjusted to 1 x 103. 7 / ml, 100ul of serum at different dilutions was added to 100μl of cells. CD180-negative cell line K562 was used as a negative control. Cells were incubated at 4℃ for 30min, washed twice with PBS, and then PE-labeled anti-mouse F(ab)2 secondary antibody was added at a ratio of 1:500. Cells were incubated at room temperature in the dark for 30min, washed twice with PBS, and resuspended in 200μl of PBS. Flow cytometry was used to detect the percentage of antibody binding to different cells and the fluorescence intensity. An effective titer was defined as an average fluorescence intensity of positive cells that was more than twice that of the negative control group. Fusion was only performed when the effective titer was higher than 6400. Three days before fusion, CD180-positive cells were injected intravenously into immunized mice that met the titer requirement for a pulse immunization. Spleen cells from successfully immunized mice were fused with myeloma SP2 / 0 cells at a ratio of 10:1. During fusion, 50% PEG was added to the mixed spleen and myeloma cell clusters (with supernatant discarded) within 1 minute in a 37°C water bath. The mixture was then shaken at 37°C for 1.5 minutes, followed by the addition of 10 ml of serum-free 1640 medium within 5 minutes. The cells were centrifuged at 800 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in semi-solid medium containing HAT. The mixture was thoroughly mixed and transferred to 10 cm Petri dishes (10 ml / plate). The clones were cultured at 37°C in a 5% CO2 incubator for 7–10 days. When the clones were large enough, single clones were picked and seeded into 96-well plates with 200 μl of complete medium per well for further culture for 3–4 days. When the clones in the 96-well plate are large enough under a microscope, 100 μl of the supernatant from the corresponding well is taken and co-incubated with the detection cells (CD180 positive cells) using the same method as for detection titers. A control is set up using K562 negative cells. Wells with average fluorescence intensity higher than those of negative cells are designated as positive wells for further clonal culture. The selected positive hybridoma clones are transferred from the 96-well plate to a 24-well plate and cultured for 3–5 days. The culture supernatant is then analyzed again. Positive clones are then further processed for subclonal culture, and the remaining cells are cryopreserved. Hybridoma cells were collected from 24-well plates, counted, and the cell density was adjusted to 10 cells / ml. The cells were then seeded into 96-well plates at 200 μl per well and incubated at 37°C with 5% CO2 for approximately 10 days. Clonal formation was observed. Wells containing only a single clone were selected, and 100 μl of the culture supernatant was collected. The detection method was the same as before. Positive clones were selected and expanded to 24-well plates. After another supernatant detection, positive clones were selected for a second round of subclonal culture. Three rounds of subclonal culture were performed. When all wells showed positive results, a stable hybridoma cell line was obtained. The positive hybridoma culture supernatant was selected, and antibody subtype detection strips (Roche, #11493027001) were used according to the instructions to detect the antibody subtype. In this example, the two monoclonal antibodies were numbered A24C9 and C12F11, both of which were murine IgG1 subtypes, and both had the κ chain light chain (results are shown in Figure 1). Figure 1 (As shown).
[0117] Example 2: Ascites preparation and purification
[0118] Hybridoma cells were washed and resuspended in sterile PBS solution at 5 x 10⁻⁶ ppm. 6 500 μl of cells per mouse were intraperitoneally injected into Balb / c mice pre-sensitized with liquid paraffin. Ascites fluid was collected 7-10 days later and centrifuged at 3000 rpm for 10 min at room temperature. The supernatant was collected. The antibody was crudely purified with 33% saturated ammonium sulfate. The method was as follows: one part ascites fluid was added to one part PBS, and one part saturated ammonium sulfate was added dropwise while stirring. The mixture was incubated overnight at 4°C, centrifuged at 10000 rpm for 10 min to remove the supernatant, and the precipitate was dissolved with a small amount of PBS. The precipitate was dialyzed against PBS at 4°C for 24 h to remove salts, with three medium changes during this period. The crudely purified antibody was further purified according to the purification manual provided by GE using an AKTA protein purification system through a 1 ml Protein G pre-packed column. The obtained purified antibody was used for subsequent antibody detection and functional experiments. The protein concentration of the purified antibody was determined by BCA quantification. 10 μg of protein was subjected to SDS-PAGE electrophoresis and stained with Coomassie Brilliant Blue. The pure antibody showed a band of all antibodies at 150 kDa. After reduction with mercaptoethanol, light and heavy chain bands were observed at 25 kDa and 50 kDa, respectively (results are shown in the figure). Figure 2 (As shown).
[0119] Example 3: Monoclonal Antibody Titer Detection
[0120] The purified antibodies were quantified using BCA assay. The quantified A24C9 and C12F11 antibodies were then dispensed at final concentrations of 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.2 nM, 1.6 nM, 0.8 nM, 0.4 nM, 0.2 nM, 0.1 nM, 0.05 nM, and 0.025 nM, respectively, and then mixed with 2 x 10⁻⁶ antibodies. 5 Daudi (CD180 positive cell line) was co-incubated at room temperature for 30 min, washed twice with PBS, and then 1 μl / well of PE-labeled anti-mouse F(ab)2 secondary antibody was added. The cells were incubated at room temperature in the dark for 30 min, washed twice with PBS, and resuspended in 200 μl of PBS. Fluorescence intensity was measured by flow cytometry, and the mean value was calculated. The Kd value of the antibody was calculated using GraphPad Prism. The Kd value of A24C9 was 0.4748 nM; the Kd value of C12F11 was 78.19 nM (results are shown in the figure). Figure 3 (As shown).
[0121] Example 4: Cloning of variable region genes by RT-PCR
[0122] Total RNA was extracted from A24C9 and C12F11 hybridoma cell lines using Trizol reagent, and cDNA libraries were synthesized using reverse transcriptase with the RNA as a template. The variable region gene fragments of the anti-human CD180 antibody heavy chain (VH) and light chain (VL) were amplified by RT-PCR. Primer sequences are shown in Table 1.
[0123] Table 1
[0124]
[0125] The PCR reaction mixture (50 μl) was prepared as follows:
[0126] cDNA: 2 μl; upstream primer (10 μM): 2 μl; downstream primer (10 μM): 2 μl; 2X pfu DNA polymerase: 25 μl; ddH2O: bring to 50 μl. Reaction conditions: 95℃ pre-denaturation for 5 min; repeat the following cycles 35 times: 95℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min; finally, 72℃ extension for 10 min. VH and VL fragments were separated and recovered by agarose gel electrophoresis. The recovered VH and VL fragments were ligated to the pMD19-T (simple) vector (Takara) using T4 ligase (Takara). The ligation system was as follows: 70 ng each of VH PCR product and VL PCR product; 1 μl of pMD19-T (simple) vector; 5 μl of Solution I ligation reaction solution; ddH2O to 10 μl. Ligation was performed at 16℃ for 30 min, and the ligation product was transformed into E. coli JM109 competent bacteria. After overnight incubation at 37℃, a single colony was selected for sequencing. The heavy and light chain variable region sequences of A24C9 and C12F11 were successfully cloned, consistent with typical antibody variable region sequence characteristics. Finally, complete VH and VL sequences of two antibodies were obtained.
[0127] Example 5: Flow cytometry detection of the specific binding of antibodies A24C9 and C12F11 to hCD180 positive cell lines
[0128] FACS detection of the binding of antibodies A24C9 and C12F11 to hCD180-positive cell lines OCI-AML2, MV4-11, THP1, Daudi, BJAB, and Nalm6 on the cell membrane surface: Antibodies A24C9 and C12F11 bound to hCD180 molecules on the cell membrane surface of 2x10⁻¹¹ cells respectively. 5OCI-AML2, MV4-11, THP1, Daudi, BJAB, and Nalm6 cells were incubated with a commercial CD180 antibody (Biolegend #312906MHR73-11) as a positive control. Cells were incubated at room temperature for 30 min, washed twice with PBS, resuspended in 100 μl of PBS, and incubated at room temperature in the dark for 30 min. Cells were washed twice with PBS, resuspended in 200 μl of PBS, and analyzed by flow cytometry. Results are shown below. Figure 4 AML cell lines (OCI-AML2, MV4-11, THP1) and B lymphoma cell lines (Daudi, BJAB, Nalm6) all highly express hCD180. Figure 4 As can be seen, the antibodies A24C9 and C12F11 prepared in this embodiment can effectively bind to the six positive cell lines.
[0129] Example 6: Flow cytometry detection of nonspecific cross-reactivity between antibodies A24C9, C12F11 and hCD180 negative cells
[0130] Pure antibodies A24C9 and C12F11 were reacted with 2x10⁻¹¹ samples respectively. 5 hCD180 negative cell lines K562, NB4, Kasumi1, TF1, NK92, Jurkat, SupT1, and MM1S were co-incubated, with a commercial CD180 antibody (Biolegend #312906MHR73-11) as a positive control. Cells were incubated at room temperature for 30 min, washed twice with PBS, resuspended in 100 μl, and incubated for 30 min at room temperature in the dark with 1 μl of PE-labeled anti-mouse F(ab)2 secondary antibody. Cells were washed twice with PBS, resuspended in 200 μl of PBS, and analyzed by flow cytometry. Results are shown below. Figure 5 Antibodies A24C9 and C12F11 showed no cross-reactivity with eight different hCD180-negative cell lines, indicating good binding specificity.
[0131] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A separated antibody or antigen-binding moiety, characterized in that, The antibody or antigen-binding moiety specifically binds to human CD180 protein, and the antibody or antigen-binding moiety comprises: (a) The complementarity-determining region (CDRH) of the heavy chain variable region: selected from at least one of the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 7, SEQ ID No. 8 or SEQ ID No. 9; and / or (b) Complementarity-determining region (CDRL) of the light chain variable region: selected from at least one of the amino acid sequences shown in SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 10, SEQ ID No. 11, and SEQ ID No.
12.
2. The isolated antibody or antigen-binding moiety according to claim 1, characterized in that, The antibody or antigen-binding portion comprises: (c) the heavy chain variable region CDRH1 as shown in SEQ ID No. 1, the heavy chain variable region CDRH2 as shown in SEQ ID No. 2, and the heavy chain variable region CDRH3 as shown in SEQ ID No. 3; and / or Light chain variable region CDRL1 as shown in SEQ ID No. 4, light chain variable region CDRL2 as shown in SEQ ID No. 5, and light chain variable region CDRL3 as shown in SEQ ID No. 6; or (d) Heavy chain variable region CDRH1 as shown in SEQ ID No. 7, heavy chain variable region CDRH2 as shown in SEQ ID No. 8, and heavy chain variable region CDRH3 as shown in SEQ ID No. 9; and / or Light chain variable region CDRL1 as shown in SEQ ID No. 10, light chain variable region CDRL2 as shown in SEQ ID No. 11, and light chain variable region CDRL3 as shown in SEQ ID No.
12.
3. The isolated antibody or antigen-binding moiety according to claim 1 or 2, characterized in that, The antibody or antigen-binding portion comprises: (e) the heavy chain variable region as shown in SEQ ID No. 13 and an amino acid sequence having more than 90% identity with said heavy chain variable region; and / or The light chain variable region as shown in SEQ ID No. 14 and the amino acid sequence having more than 90% identity with the light chain variable region; or (f) the heavy chain variable region as shown in SEQ ID No. 15 and an amino acid sequence having more than 90% identity with said heavy chain variable region; and / or The light chain variable region shown in SEQ ID No. 16 and the amino acid sequence having more than 90% identity with the light chain variable region.
4. The antibody or antigen-binding moiety according to any one of claims 1 to 3, characterized in that, The antibody is a chimeric antibody, a humanized antibody, or a fully human antibody.
5. The isolated antibody or antigen-binding moiety according to any one of claims 1 to 3, characterized in that, The antibody is selected from one or more of IgG1, IgG2, IgG3 or IgG4; Preferably, the antibody is IgG1.
6. The antibody or antigen-binding moiety according to any one of claims 1 to 3, characterized in that, The antibody or antigen-binding portion is modified. The modifications include N-glycosylation, O-glycosylation, phosphorylation, methylation, acetylation, or labeling.
7. The antibody or antigen-binding moiety according to any one of claims 1 to 3, characterized in that, The antigen-binding portion is Fab, Fab', F(ab')2, Fd, FCL, dAb, or a single-chain antibody scFv.
8. A multivalent antibody, characterized in that, The multivalent antibody comprises the antibody or antigen-binding moiety as described in any one of claims 1 to 6; Preferably, the multivalent antibody is a bispecific antibody or a trispecific antibody.
9. A multispecific antibody, characterized in that, The multispecific antibody selectively binds to at least human CD180, and the multispecific antibody comprises the antibody or antigen-binding moiety as described in any one of claims 1 to 6; the multispecific antibody is a monovalent antibody or a multivalent antibody.
10. An isolated polynucleotide, characterized in that, The polynucleotide encodes an antibody or antigen-binding moiety as described in any one of claims 1 to 7, or encodes a multivalent antibody as described in claim 8 or 9.
11. The polynucleotide according to claim 10, characterized in that, The polynucleotide encodes an amino acid sequence as shown in SEQ ID No. 1-16.
12. A carrier, characterized in that, The vector comprises the polynucleotide as described in claim 10 or 11.
13. A cell characterized in that, The cells include an antibody or antigen-binding portion as described in any one of claims 1 to 7, a multivalent antibody as described in claim 8 or 9, a polynucleotide as described in claim 10 or 11, or a vector as described in claim 12.
14. A method for generating anti-human CD180 antibodies or antigen-binding moieties, characterized in that, After expressing proteins in the cells as described in claim 13, the anti-human CD180 antibody or antigen-binding moiety is obtained.
15. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an antibody or antigen-binding moiety as described in any one of claims 1 to 7, a multivalent antibody as described in claim 8 or 9, a polynucleotide as described in claim 10 or 11, a carrier as described in claim 12 or a cell as described in claim 13, and a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition further comprises other therapeutic agents.
16. An immunoconjugate, characterized in that, The immunoconjugate includes: a) an antibody or antigen-binding moiety as described in any one of claims 1 to 7, or a multivalent antibody as described in claim 8 or 9; and b) Therapeutic agents or detectable markers; and c) The connecting body between parts a) and b) above; The therapeutic agents include drugs, enzymes, toxins, cytokines, or radionuclides.
17. The use of the antibody or antigen-binding portion of any one of claims 1 to 7, the multivalent antibody of claim 8 or 9, the polynucleotide of claim 10 or 11, the carrier of claim 12, the cell of claim 13, the pharmaceutical composition of claim 15, or the immunoconjugate of claim 16 in the preparation of a medicament for treating: tumors, autoimmune diseases, graft-versus-host disease, or infections. Preferably, the tumor is selected from breast cancer, neurotumor, melanoma, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, prostate cancer, ovarian cancer, cervical cancer, glioblastoma, esophageal cancer, bladder cancer, renal cell carcinoma, endometrial cancer, skin cancer, testicular cancer, thyroid cancer, lymphoma, leukemia, and multiple myeloma. More preferably, the tumor is selected from acute myeloid leukemia, chronic lymphocytic leukemia, and B-cell lymphoma.
18. The use of the antibody or antigen-binding moiety as described in any one of claims 1 to 7, the multivalent antibody as described in claim 8 or 9, the polynucleotide as described in claim 10 or 11, the carrier as described in claim 12, the cell as described in claim 13, the pharmaceutical composition as described in claim 15, or the immunoconjugate as described in claim 16 in the preparation of a medicament for treating TLR4-mediated diseases.
19. The use of the antibody or antigen-binding portion as described in any one of claims 1 to 7, or the multivalent antibody as described in claim 8 or 9, in the preparation of a product for detecting the presence or level of human CD180 molecules in a sample.