BDCA2-targeting antibody or antigen-binding fragment thereof, and use thereof

Abstract

Provided are a BDCA2-targeting antibody or antigen-binding fragment thereof, and a use thereof, relating to the technical field of biology. The antibody or antigen-binding fragment thereof comprises: a heavy chain complementarity determining region (HCDR), the HCDR including at least one selected from SEQ ID NOs: 1-12, or an amino acid sequence having at least 80% homology therewith. The provided antibody or antigen-binding fragment thereof can bind to human and monkey BDCA2, effectively blocking the release of IFN-α by pDCs, and has advantages such as high specificity, affinity and solubility. A drug prepared on the basis of the antibody or antigen-binding fragment thereof can be used for effectively treating diseases associated with pDC and IFN-α abnormalities, such as autoimmune diseases.

Description

Antibodies targeting bdca2 or antigen-binding fragments thereof and uses thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and in particular relates to antibodies targeting BDCA2 or antigen-binding fragments thereof and uses thereof. BACKGROUND

[0002] Blood dendritic cell antigen 2 (BDCA-2), also known as CLEC4C or CD303, is a type II C-type lectin that is uniquely expressed on plasmacytoid dendritic cells (pDC). pDC plays an important role in the immune system. When pDC is abnormally activated in the peripheral blood or at the site of a lesion, it produces a large amount of interferon IFN-α and other inflammatory cytokines and chemokines, which are contributing factors in the pathogenesis of some autoimmune diseases, such as systemic lupus erythematosus (SLE), cutaneous lupus erythematosus (CLE), scleroderma (SSc), polymyositis and dermatomyositis, psoriasis, Sjogren's syndrome, rheumatoid arthritis, Graves' disease, and Hashimoto's disease, etc.

[0003] Studies have shown that the signaling of BDCA-2 affects the production of IFN-α by pDC. Since the production of IFN-α by pDC is considered to be a major pathophysiological factor in autoimmune diseases, BDCA-2 has become a potential target for blocking the production of IFN-α in autoimmune diseases. Drugs targeting BDCA-2 can achieve the purpose of treating autoimmune diseases by reducing the production of IFN-α and other inflammatory cytokines.

[0004] Currently, there are not many drugs targeting BDCA-2 in the world. The most advanced one is the BDCA2 monoclonal antibody BIIB059 (Litifilimab) developed by Biogen, which binds to BDCA-2 to reduce the production of inflammatory cytokines such as IFN-α, thereby achieving the purpose of treating lupus. Among them, the treatment of SLE and CLE has been advanced to clinical phase III. In addition, Capella Bioscience is also developing BDCA2 monoclonal antibody 3E5 (CBS004), which is in the preclinical stage. This project has been authorized to AnaptysBio company in 2023. At present, there is still a lack of drugs targeting BDCA-2 on the market. SUMMARY

[0005] The present application aims to solve at least one of the above technical problems. To this end, one object of the present application is to provide a BDCA2 antibody with high affinity, high specificity and human-macaque species cross.

[0006] Specifically, the present application provides the following technical solutions:

[0007] In a first aspect, the present application provides an antibody or an antigen binding fragment thereof. According to embodiments of the present application, the antibody or the antigen binding fragment thereof comprises a heavy chain complementarity determining region (HCDR) comprising an amino acid sequence selected from at least one of SEQ ID NOs: 1-12 or having at least 80% homology thereto. In some examples of the present application, the antibody or the antigen binding fragment thereof is capable of binding to human and monkey BDCA2 and effectively blocking pDCs from releasing IFN-a, with advantages of high specificity, affinity and solubility. A medicament prepared based on the antibody or the antigen binding fragment thereof can be used to effectively treat diseases associated with abnormal pDCs and IFN-a, such as autoimmune diseases.

[0008] In a second aspect, the present application provides a nucleic acid molecule. According to embodiments of the present application, the nucleic acid molecule encodes the antibody or the antigen binding fragment thereof of the first aspect of the present application. In some examples of the present application, the antibody or the antigen binding fragment thereof encoded by the nucleic acid molecule can bind to human and monkey BDCA2 with high affinity and specificity. A medicament prepared based on the antibody or the antigen binding fragment thereof can be used to effectively treat diseases associated with abnormal pDCs and IFN-a, such as autoimmune diseases.

[0009] In a third aspect, the present application provides an expression vector. According to embodiments of the present application, the expression vector carries the nucleic acid molecule of the second aspect of the present application. In some examples of the present application, the expression vector can efficiently express the antibody or the antigen binding fragment thereof in a suitable host cell, and the obtained antibody or the antigen binding fragment thereof can bind to human and monkey BDCA2 with high affinity and specificity. A medicament prepared based on the antibody or the antigen binding fragment thereof can be used to effectively treat diseases associated with abnormal pDCs and IFN-a, such as autoimmune diseases.

[0010] In a fourth aspect, the present application provides a method for preparing the antibody or the antigen binding fragment thereof of the first aspect. According to embodiments of the present application, the method comprises introducing the expression vector of the third aspect of the present application into a cell; and culturing the cell under conditions suitable for protein expression and secretion, so as to obtain the antibody or the antigen binding fragment thereof. In some examples of the present application, the antibody or the antigen binding fragment thereof obtained by the method can bind to human and monkey BDCA2 with high affinity and specificity. A medicament prepared based on the antibody or the antigen binding fragment thereof can be used to effectively treat diseases associated with abnormal pDCs and IFN-a, such as autoimmune diseases.

[0011] In a fifth aspect, the present application provides a recombinant cell. According to embodiments of the present application, the recombinant cell carries the antibody or antigen binding fragment thereof of the first aspect, the nucleic acid molecule of the second aspect, or the expression vector of the third aspect. The recombinant cell is obtained by transfecting or transforming the expression vector. In some embodiments of the present application, the recombinant cell can efficiently express the antibody or antigen binding fragment thereof described above, which can specifically bind to human and monkey BDCA2 with high affinity. The drug prepared based on the foregoing antibody or antigen binding fragment thereof can be used to effectively treat diseases associated with pDC and IFN-a abnormalities, such as autoimmune diseases.

[0012] In a sixth aspect, the present application provides an immunoconjugate. According to embodiments of the present application, the immunoconjugate comprises the antibody or antigen binding fragment thereof of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, or the recombinant cell of the fifth aspect. As described above, the antibody or antigen binding fragment can effectively bind to the BDCA2 protein. Therefore, the immunoconjugate comprising the antibody or antigen binding fragment can also bind to the BDCA2 protein of a human or monkey, and has a good effect of preventing and / or treating BDCA2-mediated autoimmune diseases, such as systemic lupus erythematosus (SLE), cutaneous lupus erythematosus (CLE), scleroderma (SSc), polymyositis and dermatomyositis, psoriasis, Sjogren's syndrome, rheumatoid arthritis, Graves' disease, and Hashimoto's disease, etc.

[0013] In a seventh aspect, the present application provides a composition. According to embodiments of the present application, the composition comprises the antibody or antigen binding fragment thereof of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fifth aspect, or the immunoconjugate of the sixth aspect. As described above, the antibody or antigen binding fragment thereof can effectively bind to human and monkey BDCA2, and therefore, the composition comprising the antibody or antigen binding fragment thereof, such as a food composition, a pharmaceutical composition, etc., can also effectively treat or prevent autoimmune diseases, such as systemic lupus erythematosus (SLE), cutaneous lupus erythematosus (CLE), scleroderma (SSc), polymyositis and dermatomyositis, psoriasis, Sjogren's syndrome, rheumatoid arthritis, Graves' disease, and Hashimoto's disease, etc.

[0014] In an eighth aspect of the present application, the present application provides use of the antibody or antigen binding fragment thereof of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fifth aspect, the immunoconjugate of the sixth aspect, or the composition of the seventh aspect in the manufacture of a medicament for treating or preventing a disease associated with BDCA2. As described above, the antibody or antigen binding fragment thereof can effectively bind to human and monkey BDCA2, and thus the medicament comprising the antibody or antigen binding fragment thereof and the like has a significant effect on treating or preventing autoimmune diseases (e.g., systemic lupus erythematosus (SLE), cutaneous lupus erythematosus (CLE), scleroderma (SSc), polymyositis and dermatomyositis, psoriasis, Sjogren's syndrome, rheumatoid arthritis, Graves' disease, and Hashimoto's disease, etc.) mediated by BDCA2.

[0015] In a ninth aspect of the present application, the present application provides a medicament. According to an embodiment of the present application, the medicament comprises: the antibody or antigen binding fragment thereof of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fifth aspect, the immunoconjugate of the sixth aspect, or the composition of the seventh aspect, and the medicament is used for treating or preventing a disease associated with BDCA2. As described above, the antibody or antigen binding fragment thereof can effectively bind to human and monkey BDCA2. Thus, the medicament comprising the antibody or antigen binding fragment thereof and the like has a significant effect on treating or preventing autoimmune diseases (e.g., systemic lupus erythematosus (SLE), cutaneous lupus erythematosus (CLE), scleroderma (SSc), polymyositis and dermatomyositis, psoriasis, Sjogren's syndrome, rheumatoid arthritis, Graves' disease, and Hashimoto's disease, etc.) mediated by BDCA2.

[0016] In a tenth aspect of the present application, the present application provides use of the antibody or antigen binding fragment thereof of the first aspect in the manufacture of a kit for detecting BDCA2. As described above, the antibody or antigen binding fragment thereof can effectively bind to human and monkey BDCA2, and thus the kit comprising the antibody or antigen binding fragment thereof can be used to effectively detect BDCA2. In some examples of the present application, the aforementioned kit can be used for scientific research, such as qualitative or quantitative detection of BDCA2 protein in a biological sample.

[0017] In an eleventh aspect, the present application provides a kit. According to embodiments of the present application, the kit comprises the antibody or antigen binding fragment thereof of the first aspect. As previously described, the antibody or antigen binding fragment thereof is capable of effectively binding to human and monkey BDCA2, and thus the kit comprising the antibody or antigen binding fragment thereof can be used to effectively detect BDCA2. In some examples of the present application, the kit can be used for scientific research, such as qualitatively or quantitatively detecting BDCA2 protein in a biological sample, or can be used to determine the state of an individual, such as determining whether the BDCA2 level of the individual is higher or lower than the normal level after obtaining the BDCA2 level of the individual.

[0018] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0019] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings, wherein:

[0020] FIG. 1 is a schematic diagram of the results of detecting the affinity of the BDCA2 monoclonal antibody provided by embodiments of the present application to human BDCA2 protein;

[0021] FIG. 2 is a schematic diagram of the results of detecting the affinity of the BDCA2 monoclonal antibody provided by embodiments of the present application to monkey BDCA2 protein;

[0022] FIG. 3 is a schematic diagram of the results of detecting the affinity of the BDCA2 monoclonal antibody provided by embodiments of the present application to human BDCA2 overexpressing cells;

[0023] FIG. 4 is a schematic diagram of the results of detecting the affinity of the BDCA2 monoclonal antibody provided by embodiments of the present application to monkey BDCA2 overexpressing cells;

[0024] FIG. 5 is a schematic diagram of the results of detecting the specificity of the BDCA2 monoclonal antibody provided by embodiments of the present application;

[0025] FIG. 6 is a schematic diagram of the results of detecting the blocking activity of the BDCA2 monoclonal antibody provided by embodiments of the present application;

[0026] FIG. 7A is a schematic diagram of the results of detecting the monoclonal antibody-induced internalization of BDCA2 at different temperatures provided by embodiments of the present application;

[0027] FIG. 7B is a schematic diagram of the results of detecting the effect of the monoclonal antibody on the expression of cell surface BDCA2 at different temperatures by flow cytometry provided by embodiments of the present application. DETAILED DESCRIPTION

[0028] Embodiments of the present application are described in detail below with reference to the attached drawing figures, wherein the embodiments described are examples of implementations and do not limit the application. Embodiments described below are examples of implementations and do not limit the application.

[0029] In the present application, the terms "first", "second" are used only for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, the features defined with "first", "second" can explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "a plurality of" is at least two, such as two, three, etc., unless otherwise explicitly and specifically limited.

[0030] In the present application, the term "antibody" refers to an immunoglobulin, which is a four polypeptide chain structure connected by inter-chain disulfide bonds, consisting of two identical heavy chains and two identical light chains. The antigenicity of immunoglobulin heavy chain constant region is different due to different amino acid composition and arrangement order. Immunoglobulin can be divided into five types, namely IgM, IgD, IgG, IgA, and IgE. The same type of immunoglobulin can also be divided into different subtypes according to the amino acid composition, such as IgG1, IgG2, IgG3, IgG4. Immunoglobulin light chain is divided into κ chain or λ chain according to the constant region.

[0031] In the present application, the antibody heavy chain variable region can further comprise a heavy chain constant region, which comprises human or monkey IgG1, IgG2, IgG3, IgG4 or variants thereof.

[0032] In the present application, the antibody light chain variable region can further comprise a light chain constant region, which comprises human or monkey κ, λ chain or variants thereof.

[0033] The sequence of about 110 amino acids near the N-terminal of antibody heavy chain and light chain varies greatly, which is the variable region (Fv region); the remaining amino acid sequence near the C-terminal is relatively stable, which is the constant region. The variable region includes 3 hypervariable regions (HVR) and 4 relatively conserved framework regions (FR). The 3 hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 3 CDR regions and 4 FR regions, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminal to the carboxyl terminal. The 3 CDR regions of light chain are LCDR1, LCDR2, and LCDR3; the 3 CDR regions of heavy chain are HCDR1, HCDR2, and HCDR3.

[0034] In the present application, the antibodies can be from rabbit, murine, ovine, caprine, primate (including human and monkey) sources or mutants thereof and chimeric antibodies of antibodies of different origins.

[0035] In the present application, the term "antigen binding fragment" refers to Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments, ScFv fragments that bind to human BDCA2; which comprise one or more CDR regions of the antibodies described in the present application selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 24. Fv fragments contain the variable region of the heavy chain and the variable region of the light chain, but no constant region, and are the smallest antibody fragments that have the entire antigen binding site. Generally, Fv antibodies further comprise a polypeptide linker between the VH and VL domains, and are capable of forming the structure required for antigen binding. The two antibody variable regions can also be joined by a different linker to form a single polypeptide chain known as a single chain antibody or single chain Fv.

[0036] In the present application, the amino acid sequences of the listed CDRs are shown according to the IMGT definition rule. However, it is well known to those skilled in the art that the CDRs of antibodies can be defined by various methods in the art, such as the Kabat rule, the Chothia rule, etc. Those skilled in the art should understand that, unless otherwise specified, the terms "CDR" and "complementary determining region" of a given antibody or region thereof (e.g. variable region) should be understood to encompass the complementary determining regions defined by any of the above known schemes described in the present application. Although the scope of protection claimed in the present application is based on the sequences shown according to the IMGT definition rule, the amino acid sequences corresponding to the CDRs defined according to other rules should also be included in the scope of protection of the present application.

[0037] In the present application, the term "amino acid" is represented by a single letter or a three letter code, and has the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gin (glutamine); E: Glu (glutamic acid); G: Gly (glycine); H: His (histidine); I: Ile (isoleucine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Pro (proline); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).

[0038] For nucleotides, the terms "homology," "identity," or "similarity" are used to describe or compare the degree of nucleotide similarity between two or more nucleotide sequences. The percent "sequence homology" between a first sequence and a second sequence can be calculated by dividing [the number of nucleotides in the first sequence that are identical to the nucleotides at the corresponding positions] by [the total number of nucleotides in the second sequence] minus [the number of insertions, deletions, or additions of nucleotides in the second sequence relative to the first nucleotide sequence] and then multiplying by [100%]. Each deletion, insertion, substitution, or addition of a nucleotide in the second nucleotide sequence relative to the first nucleotide sequence is considered a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using known computer algorithms for sequence alignment, such as NCBI Blast v2.0, using standard settings. Some other techniques, computer algorithms, and settings for determining the degree of sequence identity are described, for example, in WO 04 / 037999, EP 0 967 284, EP 1 085 089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185, and GB 2357768-A.

[0039] For polypeptides, the terms "(substantial) homology," "identity," or "similarity" are used to describe or compare the degree of amino acid similarity between two or more polypeptides or specified sequences thereof when optimally aligned and compared (with appropriate insertions or deletions of nucleotides). The percent homology between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), when the sequences are optimally aligned and compared. Optimal alignment of sequences for comparison can be achieved using various algorithms known in the art, such as those described in the following non-limiting examples.

[0040] In the present application, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids can be substituted, added and / or deleted from the sequences of the present application by one of skill in the art without materially affecting the activity of the antibody (retaining at least 95% activity) to obtain variants of the sequences of the antibody or functional fragments thereof. They are all considered to be included in the scope of protection of the present application. Amino acids with similar properties are substituted in the variable region. The variant sequences described in the present application can have at least 80% identity (or homology) with the reference sequences, which means at least 80%, which can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity with each reference sequence. The sequence identity described in the present application can be measured using sequence analysis software. For example, using the computer program BLAST, especially BLASTP or TBLASTN with default parameters. The amino acid sequences referred to in the present application are all shown in the order of N-terminal to C-terminal.

[0041] In the present application, the term "vector" generally refers to a nucleic acid molecule that is capable of inserting into a suitable host and self-replicating, which transfers the inserted nucleic acid molecule to the host cell and / or between host cells. The vector can include a vector mainly for inserting DNA or RNA into a cell, a vector mainly for replicating DNA or RNA, and a vector mainly for the transcription and / or translation of expression of DNA or RNA. The vector also includes a vector having a plurality of the above-mentioned functions. The vector can be a polynucleotide that is capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the vector can produce the desired expression product by culturing a suitable host cell containing the vector.

[0042] In the present application, the term "composition" generally refers to a unit dosage form, and can be prepared by any one of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the carrier that constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both.

[0043] In the present application, the term "pharmaceutically acceptable" ingredient is a substance suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reaction), i.e., having a reasonable benefit / risk ratio.

[0044] In the present application, the term "pharmaceutically acceptable excipient" can include any solvent, solid excipient, diluent or other liquid excipient, and the like, which are suitable for the particular intended dosage form. Except insofar as any conventional excipient is incompatible with the compounds of the present application, such as by producing any adverse biological effect or otherwise interacting in a deleterious manner with any other component(s) of a pharmaceutically acceptable composition, their use is contemplated to be within the scope of this application.

[0045] In the present application, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means. The antibodies or antigen binding fragments, recombinant proteins, multispecific antibodies, conjugates or pharmaceutical compositions of the present application can be administered by any common route, so long as it reaches the intended tissue. Various modes of administration are contemplated, including intraperitoneal, intravenous, intramuscular, subcutaneous, and the like, although the present application is not limited to these exemplified modes of administration. Preferably, the compositions of the present application are administered intravenously or subcutaneously.

[0046] In the present application, the term "treatment" refers to obtaining a desired pharmacological and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure of a disease and / or adverse effect attributable to the disease. "Treatment" as used herein covers the treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or condition from occurring in an individual which can be predisposed to the disease but has not yet developed or diagnosed with the disease; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing the partial or complete regression of the disease. "Treatment" as used herein covers any use of a drug or compound to treat, cure, alleviate, improve, reduce the symptoms of, or inhibit a disease in an individual, including but not limited to administering a drug comprising a compound described herein to an individual in need thereof.

[0047] In the present application, the term "effective amount" or "effective dose" refers to an amount that is functional or active and acceptable to a human and / or an animal.

[0048] In the present application, the amino acid sequences are shown in Table 1.

[0049] The present application provides an antibody or antigen binding fragment thereof targeting BDCA2, an expression vector, a preparation method, a recombinant cell, an immunoconjugate, a composition, a pharmaceutical use, a medicament, a preparation kit use, a kit, a disease treatment method, and a disease treatment use, which will be described in detail respectively.

[0050] Antibody or antigen binding fragment thereof

[0051] In an aspect of the present application, the present application provides an antibody or an antigen binding fragment thereof, comprising: a heavy chain complementarity determining region (HCDR), the HCDR comprising an amino acid sequence selected from at least one of SEQ ID NOs: 1-12 or having at least 80% homology thereto. In some examples of the present application, the antibody or the antigen binding fragment thereof having the HCDR amino acid sequence is capable of binding to human and monkey BDCA2 protein with high affinity and specificity.

[0052] In some examples of the present application, the HCDR comprises: a HCDR1, a HCDR2 and a HCDR3, wherein the HCDR1 comprises an amino acid sequence as shown in SEQ ID NOs: 1-4 or having at least 80% homology thereto, the HCDR2 comprises an amino acid sequence as shown in SEQ ID NOs: 5-8 or having at least 80% homology thereto, and the HCDR3 comprises an amino acid sequence selected from as shown in SEQ ID NOs: 9-12 or having at least 80% homology thereto. The antibody or the antigen binding fragment thereof targeting BDCA2 based on the HCDR sequence has high binding affinity and specificity to BDCA2.

[0053] In some preferred examples of the present application, the HCDR comprises: a HCDR1 comprising an amino acid sequence as shown in SEQ ID NOs: 1-4, respectively; a HCDR2 comprising an amino acid sequence as shown in SEQ ID NOs: 5-8, respectively; and a HCDR3 comprising an amino acid sequence as shown in SEQ ID NOs: 9-12, respectively. It has been verified by experiments that the antibody or the antigen binding fragment thereof targeting BDCA2 having the amino acid sequence as shown in SEQ ID NOs: 1-12 has high binding affinity and specificity to BDCA2.

[0054] In some examples of the present application, the anti-BDCA2 antibody further comprises: a light chain complementarity determining region (LCDR), the LCDR comprising: a LCDR1, a LCDR2 and a LCDR3, wherein the LCDR1 comprises an amino acid sequence as shown in SEQ ID NOs: 13-16 or having at least 80% homology thereto, the LCDR2 comprises an amino acid sequence as shown in SEQ ID NOs: 17-20 or having at least 80% homology thereto, and the LCDR3 comprises an amino acid sequence as shown in SEQ ID NOs: 21-24 or having at least 80% homology thereto. The antibody or the antigen binding fragment thereof targeting BDCA2 based on the LCDR sequence has high binding affinity and specificity to BDCA2.

[0055] In some preferred examples of the present application, the aforementioned LCDRs comprise: LCDR1 of the amino acid sequence as set forth in SEQ ID NOs: 13-16, respectively; LCDR2 of the amino acid sequence as set forth in SEQ ID NOs: 17-20, respectively; and LCDR3 of the amino acid sequence as set forth in SEQ ID NOs: 21-24, respectively. It has been experimentally verified that the targeting BDCA2 antibody or antigen-binding fragment thereof having the amino acid sequence as set forth in SEQ ID NOs: 13-24 has higher binding affinity and specificity to BDCA2.

[0056] It should be noted that one or more amino acid residues in the aforementioned targeting BDCA2 antibody HCDRs and / or LCDRs can be replaced by other amino acid residues from the same side chain family, and the retained function of the altered antibody can be tested using the functional assays described herein. Preferably, the conservative modification does not exceed 1 or 2 in number.

[0057] In some examples of the present application, the aforementioned antibody or antigen-binding fragment thereof further comprises: a heavy chain framework region.

[0058] In some examples of the present application, the aforementioned antibody or antigen-binding fragment thereof further comprises: a light chain framework region.

[0059] In some examples of the present application, at least a portion of the aforementioned heavy chain framework region and the aforementioned light chain framework region are independently derived from at least one of a rabbit-derived antibody, a mouse-derived antibody, a sheep-derived antibody, a goat-derived antibody, a primate-derived antibody, or a mutant thereof.

[0060] In some preferred examples of the present application, at least a portion of the aforementioned light chain framework region is independently derived from at least one of a primate-derived kappa chain and / or lambda chain, or a mutant thereof.

[0061] In some preferred examples of the present application, at least a portion of the aforementioned heavy chain framework region is independently derived from at least one of a primate-derived IgG1, IgG2, and IgG3, or a mutant thereof.

[0062] In some examples of the present application, the aforementioned primate-derived includes human-derived or monkey-derived.

[0063] In some preferred examples of the present application, the heavy chain variable region of the aforementioned antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-28 or having at least 80% homology with SEQ ID NOs: 25-28. In some more preferred examples of the present application, the amino acid sequence of the heavy chain variable region of the aforementioned antibody or antigen-binding fragment thereof is as set forth in SEQ ID NOs: 25-28.

[0064] In some preferred examples of the present application, the light chain variable region of the aforementioned antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NOs: 29-32 or has at least 80% homology with SEQ ID NOs: 29-32. In some more preferred examples of the present application, the amino acid sequence of the light chain variable region of the aforementioned antibody or antigen-binding fragment thereof is as set forth in SEQ ID NOs: 29-32.

[0065] The aforementioned antibody or antigen-binding fragment thereof based on the aforementioned heavy chain variable region and light chain variable region sequence has high binding affinity and specificity to BDCA2.

[0066] In some examples of the present application, the aforementioned antibody or antigen-binding fragment thereof further comprises a heavy chain constant region.

[0067] In some examples of the present application, the aforementioned antibody or antigen-binding fragment thereof further comprises a light chain constant region.

[0068] In some examples of the present application, at least one of the aforementioned heavy chain constant region and the aforementioned light chain constant region is independently derived from at least one of a rabbit-derived antibody, a mouse-derived antibody, a sheep-derived antibody, a goat-derived antibody, a primate-derived antibody, or a mutant thereof. In some preferred examples of the present application, at least one of the aforementioned heavy chain constant region and the aforementioned light chain constant region is derived from a primate-derived antibody.

[0069] In some examples of the present application, the heavy chain of the aforementioned antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-36 or has at least 80% homology with SEQ ID NOs: 33-36. In some preferred examples of the present application, the amino acid sequence of the heavy chain of the aforementioned antibody or antigen-binding fragment thereof is as set forth in SEQ ID NOs: 33-36.

[0070] In some examples of the present application, the light chain of the aforementioned antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-40 or has at least 80% homology with SEQ ID NOs: 37-40. In some preferred examples of the present application, the amino acid sequence of the light chain of the aforementioned antibody or antigen-binding fragment thereof is as set forth in SEQ ID NOs: 37-40.

[0071] In some examples of the present application, the aforementioned antibody comprises at least one selected from the group consisting of a polyclonal antibody and a monoclonal antibody; or the aforementioned antigen-binding fragment comprises at least one selected from the group consisting of a F(ab’)2 fragment, a Fab’ fragment, a Fab fragment, a Fv fragment, a scFv fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein, and a minimal recognition unit. In some preferred examples of the present application, the aforementioned antibody is a monoclonal antibody.

[0072] The antibody or antigen-binding fragment thereof having the above sequence is more hydrophilic and can effectively induce the internalization of BDCA2 on the surface of pDC cells.

[0073] Nucleic acid molecule

[0074] In another aspect of the present application, a nucleic acid molecule is provided, which encodes the antibody or antigen-binding fragment thereof as described above. Based on the nucleic acid molecule, the antibody or antigen-binding fragment thereof can be prepared in vitro in large quantities.

[0075] In some examples of the present application, the nucleic acid molecule is DNA.

[0076] It should be noted that, for the nucleic acid molecule mentioned in the specification and claims of the present application, it is understood by those skilled in the art that either one of the complementary double strands or both are actually included. For the convenience, in the specification and claims, although only one strand is given in most cases, the other complementary strand is also disclosed. In addition, the nucleic acid sequence in the present application includes DNA form or RNA form, and the disclosure of one means the disclosure of the other.

[0077] Expression vector

[0078] In another aspect of the present application, an expression vector is provided, which carries the nucleic acid molecule as described above. Based on the expression vector, the antibody or antigen-binding fragment thereof can be prepared in vitro in large quantities.

[0079] It should be noted that, when the nucleic acid molecule is connected to the vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the vector, as long as the control elements can control the translation and expression of the nucleic acid molecule. Of course, the control elements can be directly from the vector itself, or can be exogenous, i.e., not from the vector itself. Of course, the nucleic acid molecule can be operably connected to the control elements.

[0080] According to an embodiment of the present application, the vector can refer to a cloning vector or an expression vector, which can be obtained by operably connecting the nucleic acid to a commercially available vector such as a plasmid or a viral vector. The vector in the present application is not particularly limited, and commonly used plasmids can be used, such as pSeTag2, PEE14, pMH3, etc.

[0081] In the present application, the term "operably linked" refers to the linkage of an exogenous gene to a vector, so that the control elements within the vector, such as the amino acid sequences for transcription control, such as and the amino acid sequences for translation control, such as and the like, can exert their intended functions of regulating the transcription and translation of the exogenous gene. The commonly used vectors can be, for example, viral vectors, plasmids, bacteriophages, and the like. After the expression vector according to some embodiments of the present application is introduced into a suitable recipient cell, the expression of the aforementioned nucleic acid molecules can be effectively realized under the mediation of the regulatory system, and thus the aforementioned proteins encoded by the nucleic acid molecules can be obtained in large quantities in vitro.

[0082] In some examples of the present application, the aforementioned vector is a eukaryotic vector or a prokaryotic vector.

[0083] In some examples of the present application, the aforementioned vector comprises at least one selected from the group consisting of a plasmid vector, an adenovirus vector, a lentivirus vector, and an adeno-associated virus vector.

[0084] Method for preparing an antibody or antigen-binding fragment thereof

[0085] In another aspect of the present application, a method for preparing the aforementioned antibody or antigen-binding fragment thereof is provided, comprising: introducing the aforementioned expression vector into a cell; and culturing the aforementioned cell under conditions suitable for protein expression and secretion, so as to obtain the aforementioned antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof can be prepared in large quantities in vitro by the method.

[0086] In some examples of the present application, the aforementioned cell is a eukaryotic cell.

[0087] In some specific examples of the present application, the aforementioned eukaryotic cell is a mammalian cell. When the cell is a mammalian cell, the expression efficiency of the aforementioned antibody or antigen-binding fragment thereof is higher.

[0088] It should be noted that the aforementioned eukaryotic cell does not include an animal reproductive cell, a fertilized egg, or an embryonic stem cell.

[0089] Recombinant cell

[0090] In another aspect of the present application, a recombinant cell carrying the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, or the aforementioned expression vector is provided. In some examples of the present application, the aforementioned recombinant cell is obtained by transfecting or transforming the aforementioned expression vector, and the aforementioned recombinant cell can efficiently express the aforementioned antibody or antigen-binding fragment under suitable conditions.

[0091] In some examples of the present application, the aforementioned recombinant cell is a prokaryotic cell, a eukaryotic cell, or a bacteriophage. It should be noted that the aforementioned eukaryotic cell does not include an animal reproductive cell, a fertilized egg, or an embryonic stem cell.

[0092] In some examples of the present application, the aforementioned prokaryotic cell is E. coli, B. subtilis, Streptomyces, or P. mirabilis.

[0093] In some examples of the present application, the aforementioned eukaryotic cell is a fungus, an insect cell, a plant cell, or a mammalian cell.

[0094] In some examples of the present application, the aforementioned fungus is P. pastoris, S. cerevisiae, S. pombe, or T. reesei.

[0095] In some examples of the present application, the aforementioned insect cell is a M. sexta cell; in some examples of the present application, the aforementioned plant cell is a tobacco plant cell; in some examples of the present application, the aforementioned mammalian cell is a BHK cell, a CHO cell, a COS cell, a myeloma cell, or a human embryonic kidney 293 cell; and the animal reproductive cell, a fertilized egg, or an embryonic stem cell is excluded.

[0096] In some examples of the present application, the aforementioned recombinant cell is a mammalian cell. When the cell is a mammalian cell, the expression efficiency of the aforementioned antibody or antigen-binding fragment thereof is higher.

[0097] In some examples of the present application, the aforementioned recombinant cell is a BHK cell, a CHO cell, a COS cell, or an NSO cell.

[0098] It should be noted that the aforementioned "suitable conditions" in the present application refer to conditions suitable for the expression of the bispecific antibody of the present application. Those skilled in the art can easily understand that the conditions suitable for the expression of the bispecific antibody include, but are not limited to, a suitable transformation or transfection method, a suitable transformation or transfection condition, a healthy state of the host cell, a suitable density of the host cell, a suitable cell culture environment, and a suitable cell culture time. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the bispecific antibody according to the specific environment of the laboratory.

[0099] Immunoconjugate

[0100] In another aspect of the present application, an immunoconjugate is provided, comprising the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell. The aforementioned immunoconjugate can be used for treating diseases related to BDCA2, such as autoimmune diseases.

[0101] In some examples of the present application, the aforementioned immunoconjugate can further comprise a therapeutic agent. The aforementioned therapeutic agent includes a radioisotope and a cytotoxic agent, etc.

[0102] Composition

[0103] In another aspect of the present application, the present application provides a composition comprising: the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, or the aforementioned immunoconjugate. The aforementioned composition can be used for treating a disease associated with BDCA2, such as an autoimmune disease.

[0104] It should be noted that the aforementioned composition comprises components separated in time and / or space, as long as they can act together to achieve the purpose of the present application. For example, the components contained in the aforementioned composition can be administered to the subject as a whole, or administered to the subject separately. When the components contained in the aforementioned composition are administered to the subject separately, each component can be administered to the subject simultaneously or sequentially.

[0105] Pharmaceutical use

[0106] In yet another aspect of the present application, the present application provides use of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, the aforementioned immunoconjugate, or the aforementioned composition in the manufacture of a medicament for treating or preventing a disease associated with BDCA2. The medicament prepared based on the aforementioned antibody or antigen-binding fragment thereof can specifically bind to BDCA2, inhibit the signal transduction of BDCA2, reduce the production of INF-α, and thus treat an autoimmune disease.

[0107] In some examples of the present application, the aforementioned disease associated with BDCA2 includes an autoimmune disease.

[0108] In some examples of the present application, the aforementioned autoimmune disease includes at least one of systemic lupus erythematosus, cutaneous lupus erythematosus, scleroderma, polymyositis and dermatomyositis, psoriasis, Sjogren's syndrome, rheumatoid arthritis, Graves' disease, and Hashimoto's disease.

[0109] Medicament

[0110] In yet another aspect of the present application, the present application provides a medicament comprising: the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, the aforementioned immunoconjugate, or the aforementioned composition, for treating or preventing a disease associated with BDCA2. The aforementioned medicament can specifically bind to BDCA2, inhibit the signal transduction of BDCA2, reduce the production of INF-α, and thus treat an autoimmune disease.

[0111] In some examples of the present application, the aforementioned disease associated with BDCA2 includes an autoimmune disease.

[0112] In some examples of the present application, the aforementioned autoimmune disease includes at least one of systemic lupus erythematosus, cutaneous lupus erythematosus, scleroderma, polymyositis and dermatomyositis, psoriasis, Sjogren's syndrome, rheumatoid arthritis, Graves' disease, and Hashimoto's disease.

[0113] In some examples of the present application, the aforementioned pharmaceutically acceptable excipient further includes one or more excipient, diluent, stabilizer, or carrier.

[0114] In some examples of the present application, the aforementioned excipient includes one or more pharmaceutically acceptable excipient, diluent, stabilizer, or carrier.

[0115] In some examples of the present application, the aforementioned pharmaceutical composition is an injection.

[0116] It is to be noted that the aforementioned pharmaceutical composition includes a combination separated in time and / or space as long as it can act together to achieve the purpose of the present application. For example, the components contained in the aforementioned pharmaceutical composition can be administered to the subject as a whole, or administered to the subject separately. When the components contained in the aforementioned pharmaceutical composition are administered to the subject separately, each component can be administered to the subject simultaneously or sequentially.

[0117] The pharmaceutical of the present application contains a safe and effective amount of the active ingredient (bispecific antibody) of the present application and a pharmaceutically acceptable excipient. Such excipients include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should be matched with the administration mode, and the dosage form of the pharmaceutical of the present application is injection, oral preparation (tablet, capsule, oral liquid), transdermal preparation, sustained-release preparation. For example, it is prepared by conventional methods with normal saline or aqueous solution containing glucose and other auxiliary agents. The aforementioned pharmaceutical is preferably manufactured under sterile conditions.

[0118] The effective amount of the aforementioned active ingredient of the present application can vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art according to various factors (for example, by clinical trials). The aforementioned factors include, but are not limited to, the pharmacokinetic parameters of the aforementioned active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, several separate doses can be given daily, or the dose can be proportionally reduced, according to the exigencies of the therapeutic situation.

[0119] The aforementioned pharmaceutically acceptable excipient of the present application includes, but is not limited to, water, saline, liposome, lipid, protein, protein-antibody conjugate, peptide substance, cellulose, nanogel, or combinations thereof. The selection of the carrier should be matched with the administration mode, which is well known to a person of ordinary skill in the art.

[0120] Use in preparing a kit

[0121] In yet another aspect of the present application, the present application provides use of the aforementioned antibody or antigen-binding fragment thereof in preparing a kit, wherein the aforementioned kit is used for detecting BDCA2.

[0122] As aforementioned, the antibody or antigen-binding fragment thereof of the embodiments of the present application can specifically bind to BDCA2, thus the aforementioned antibody or antigen-binding fragment thereof can be used for detecting BDCA2. Further, it can be used for preparing a BDCA2 related kit and for scientific research, such as qualitatively or quantitatively detecting BDCA2 protein molecules in a biological sample. More specifically, it can be used for a kit related to immunoblotting, immunoprecipitation and the like, which involves the use of the specific binding property of BDCA2 and the antibody. These kits can comprise any one or more of the following: antagonists, bispecific antibodies of the present application or pharmaceutical reference materials; protein purification columns; immunoglobulin affinity purification buffers; assay diluents for cells. The bispecific antibodies of the present application can be used in different types of diagnostic tests, for example, various diseases or the presence of drugs, toxins or other proteins and the like can be detected in vitro or in vivo. For example, the serum or blood of a subject can be detected to test BDCA2 mediated related diseases.

[0123] Kit

[0124] In yet another aspect of the present application, the present application provides a kit comprising the aforementioned antibody or antigen-binding fragment thereof of the first aspect. The aforementioned kit can be used for portable detection of BDCA2.

[0125] Method for treating a disease

[0126] In yet another aspect of the present application, the present application provides a method for preventing and / or treating a disease mediated by BDCA2. According to the embodiments of the present application, the aforementioned method comprises: administering a pharmaceutically acceptable amount of the antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, recombinant cell, immunoconjugate, composition or drug to a subject. Based on the aforementioned method, autoimmune diseases mediated by BDCA2 can be effectively treated.

[0127] It should be noted that the terms "subject", "individual" and "patient" are used interchangeably herein and refer to a mammal being evaluated for treatment and / or being treated. In one embodiment, the mammal is a human. The terms "subject", "individual" and "patient" include, but are not limited to, individuals having cancer, individuals having autoimmune diseases, individuals having pathogen infections, and the like. The subject can be a human, but also includes other mammals, particularly mammals useful as laboratory models of human disease, e.g., mice, rats, and the like.

[0128] The effective amount of the foregoing antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, immunoconjugate, composition or medicament of the present application can vary according to factors such as the mode of administration and the severity of the disease to be treated. The selection of an optimal effective amount can be made by one of ordinary skill in the art (e.g., by clinical trial). The foregoing factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient(s) such as the bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated; the age, body weight, and immune status of the patient, the route of administration, etc. For example, the dosage can be administered several times a day, or proportionally reduced, according to the exigencies of the therapeutic situation.

[0129] In some examples of the present application, the foregoing BDCA2-mediated related disease includes an autoimmune disease.

[0130] In some examples of the present application, the foregoing autoimmune disease includes at least one of systemic lupus erythematosus, cutaneous lupus erythematosus, scleroderma, polymyositis and dermatomyositis, psoriasis, Sjogren's syndrome, rheumatoid arthritis, Graves' disease, and Hashimoto's disease.

[0131] Therapeutic uses

[0132] In yet another aspect of the present application, the present application provides the use of the foregoing antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, recombinant cell, immunoconjugate, composition or medicament in the treatment or prevention of a BDCA2-mediated disease. In some examples of the present application, the administration of an effective amount of the antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, immunoconjugate, composition or medicament to a subject is effective in treating a BDCA2-mediated disease.

[0133] In some examples of the present application, the foregoing BDCA2-mediated related disease includes an autoimmune disease.

[0134] In some examples of the present application, the foregoing autoimmune disease includes at least one of systemic lupus erythematosus, cutaneous lupus erythematosus, scleroderma, polymyositis and dermatomyositis, psoriasis, Sjogren's syndrome, rheumatoid arthritis, Graves' disease, and Hashimoto's disease.

[0135] Table 1. BDCA2 antibody sequences

[0136] The following will be specifically described in the examples. The technical or conditions not specified in the examples are carried out according to the technical or conditions described in the literature in the art or according to the product manual. The reagents or instruments not specified by the manufacturer are all conventional products that can be obtained by purchase. Among them,

[0137] The amino acid sequences of the heavy chain and light chain of the HR04 antibody are shown in SEQ ID NO: 33 and SEQ ID NO: 37, respectively;

[0138] The amino acid sequences of the heavy chain and light chain of the HR09 antibody are shown in SEQ ID NO: 34 and SEQ ID NO: 38, respectively;

[0139] The amino acid sequences of the heavy chain and light chain of the HR13 antibody are shown in SEQ ID NO: 35 and SEQ ID NO: 39, respectively;

[0140] The amino acid sequences of the heavy chain and light chain of the HR17 antibody are shown in SEQ ID NO: 36 and SEQ ID NO: 40, respectively.

[0141] Example 1 Screening of BDCA2 monoclonal antibodies

[0142] Balb / c mice were immunized with soluble recombinant human BDCA2 protein (R&D, Cat. No. BCA-HM102) for multiple rounds. The antigen protein was mixed with the immunoadjuvant to form an immunogen reagent, which was then injected subcutaneously through the groin or injected intraperitoneally. In the first round of immunization, each mouse received an immunization with 50 μg of human BDCA2 protein mixed with complete Freund's adjuvant (Sigma, Cat. No. F5881) at a volume ratio of 1:1. In each subsequent round of booster immunization, each mouse received an immunization with 50 μg of human BDCA2 protein mixed with Freund's incomplete adjuvant (Sigma, Cat. No. F5506) at a volume ratio of 1:1. The interval between each round of booster immunization was at least two weeks, and there were usually no more than five rounds of booster immunization, and the antibody titers of the mouse serum were detected after the 3rd and 4th immunizations. Three days before cell fusion, the last booster immunization was performed at a dose of 25 μg of human BDCA2 protein per mouse.

[0143] When the antibody titers specific to BDCA2 in the mouse serum reached a certain level, the spleen cells of the mice were taken out and fused with a myeloma cell line to obtain hybridoma cells; after multiple rounds of screening and cloning of the hybridoma cells, several hybridoma cells expressing anti-BDCA2 monoclonal antibody molecules were isolated. The amino acid sequences encoding the variable domains of the antibody molecules were obtained by using conventional hybridoma sequencing methods. The SEQ ID NO numbers corresponding to the variable region sequences and CDR sequences of the monoclonal antibodies obtained by hybridoma sequencing are shown in Table 1.

[0144] Example 2 Recombinant expression of BDCA2 monoclonal antibody

[0145] The hybridoma antibody variable region sequence (see Table 1) was connected to the human IgG1 Fc sequence (SEQ ID NO: 33), synthesized into the expression plasmid pcDNA3.4, and after transformation into E. coli, the plasmid was extracted. After filtering the expression plasmid through a 0.22 μm filter, it was mixed with CHO cells and an electrotransformation solution, added to a 1 ml electroporation tube, and after electroporation, the cells in the electroporation tube were subpackaged into pre-prepared 20 ml culture solution shake flasks, and incubated for 40 min. After the incubation was completed, the shake flasks were placed in a 37°C, 270 rpm, 8% CO2 culture, and after 24 h, the feed / butyric acid sodium / dual antibody was added, and after 3-6 days of continuous culture, the cell supernatant was collected. The cell supernatant was added to a Protein A affinity chromatography column, the chromatography column was washed with 20 mL of PBS buffer, and then 5 mL of sodium acetate buffer (pH 3.4) was added to elute the chromatography column, and the eluate was collected in a dialysis bag and dialyzed into PBS buffer to obtain the target product BDCA2 recombinant monoclonal antibody.

[0146] Example 3 Affinity detection of BDCA2 monoclonal antibody

[0147] Test Example 1: Affinity detection by ELISA method

[0148] Human BDCA2 protein (Kaiyuan, item number BCA-HM102), and monkey BDCA2 protein (Kaiyuan, item number BCA-CM102) were diluted to prepare a 1 μg / mL coating solution, and 100 μL / well was added to the enzyme-labeled plate, and coated at 2-8°C for 12 hours or more. The residual solution was discarded, 2% BSA-PBS was added to each well at 200 μL, and after 2 hours of 37°C blocking, 300 μL of PBST was added to each well and washed 3 times. The sample to be tested (HR04, HR09, HR13, HR17, BIIB059, 3E5) was diluted to 20 nM with PBS, and then diluted 3 times to 12 gradients, and 100 μL / well was added to the enzyme-labeled plate. Incubate at 37°C for 1 hour, wash 3 times with 300 μL of PBST per well, and then add Goat anti Human Fc-HRP (abcam, item number ab97225) diluted 10000 times, 100 μL / well. Incubate at 37°C for 1 hour, wash 3 times with 300 μL of PBST per well, and then tap dry. Add TMB color developing solution, 100 μL per well. After 5 minutes of reaction at room temperature, add 2M H2SO4 to terminate the reaction, 50 μL / well. Place the enzyme-labeled plate after stopping the reaction on the enzyme-labeled instrument, read the absorbance OD450 value at 450 nm wavelength, and analyze the data using GraphPad Prism 8.0 software. Through four-parameter nonlinear fitting, the binding activity fitting was performed to obtain the binding curve and EC50 value parameters.

[0149] The smaller the EC50 value, the stronger the binding activity. As shown in Table 2 and Figure 1, HR04, HR09, HR13, and HR17 have obvious binding activity to human BDCA2 protein, and the binding activity is equivalent to that of BIIB059 and 3E5.

[0150] Table 2. Affinity of BDCA2 monoclonal antibodies to human BDCA2 protein

[0151] As shown in Table 3 and Figure 2, HR04, HR09, HR13, and HR17 have obvious binding activity to monkey BDCA2 protein, and the binding activity is better than that of 3E5 and equivalent to that of BIIB059.

[0152] Table 3. Affinity of BDCA2 monoclonal antibodies to monkey BDCA2 protein

[0153] Test Example 2: FACS method for affinity detection

[0154] To study the affinity of BDCA2 monoclonal antibodies to human / monkey BDCA2 on the cell membrane surface, jurkat cell strains overexpressing human BDCA2 (Jiamin Biotech, item number GM-C20052) and jurkat cell strains overexpressing monkey BDCA2 (Jiamin Biotech, GM-C20051) were used for cell level binding experiments. Briefly, the cell density was adjusted to 1x10 6 cells / mL with PBS. 100 μL of cells per well were inoculated in a 96-well U-bottom plate. The samples to be tested (HR04, HR09, HR13, HR17, BIIB059, and 3E5) were diluted to 400 nM with PBS, and then diluted 3 times to obtain 12 gradients, 100 μL per well was added to the U-bottom plate. The cells were incubated at 4°C in the dark for 1 hour. Then, 200 μL per well of pre-cooled PBS was added to rinse the cells twice, centrifuged at 500g at 4°C for 5 minutes, and the supernatant was discarded. Then, 100 μL per well of fluorescent secondary antibody (abcam, item number ab6785) was added, and the cells were incubated at 4°C in the dark for 30 minutes. The cells were washed twice with 200 μL per well of pre-cooled PBS, centrifuged at 500g at 4°C for 5 minutes, and the supernatant was discarded. Finally, 200 μL per well of pre-cooled PBS was used to resuspend the cells. The BD FACS Symphony A1 was used to read the fluorescence signal value. The data were processed and plotted using the software GraphPad Prism 8.0, and the binding curve and EC50 value were obtained by four-parameter nonlinear fitting.

[0155] The smaller the EC50 value, the stronger the binding activity. As shown in Table 4 and FIG. 3, HR04, HR09, HR13, and HR17 have obvious binding activity to human BDCA2 overexpressed cells, and the binding activity is better than that of 3E5 and comparable to that of BIIB059.

[0156] Table 4. Affinity of BDCA2 monoclonal antibodies to human BDCA2 overexpressed cells

[0157] As shown in Table 5 and FIG. 4, HR04, HR09, HR13, and HR17 have obvious binding activity to monkey BDCA2 overexpressed cells, and the binding activity is better than that of 3E5 and comparable to that of BIIB059.

[0158] Table 5. Affinity of BDCA2 monoclonal antibodies to monkey BDCA2 overexpressed cells

[0159] Example 4: Blocking activity study of BDCA2 monoclonal antibodies

[0160] CpG-A ODN (working concentration 5 μg / mL) and gradient concentration mixture of monoclonal antibodies (starting concentration 20 μg / mL, 6-fold dilution, 9 concentrations) were prepared with 1640+10% FBS medium, 50 uL per well was added to a 96-well plate. PBMC was resuscitated and counted to adjust the cell density to 5×10 6 / mL, 100 uL of cells per well was added to the 96-well plate containing the mixed solution, i.e. 5×10 5 uL per well. Incubate at 37°C, 5% CO2 for 16-18h. Centrifuge to take the supernatant, and use ELISA method (Human IFN-Alpha Multi-Subtype ELISA Kit (TCM), pbl assay science, 41105-1) to quantitatively detect the secretion level of IFN-α. According to the standard curve of standard concentration and OD450 value, the sample concentration is calculated by the standard curve equation. The data processing and graph analysis are performed by using software GraphPad Prism 8.0, and the binding curve and IC50 value are obtained by four-parameter nonlinear fitting.

[0161] The smaller the IC50 value, the stronger the blocking activity. As shown in Table 6 and FIG. 5, HR04, HR09, HR13, HR17, BIIB059, and 3E5 can effectively block the secretion of IFN-α mediated by BDCA2. Among them, the ability of HR04, HR09, HR13, and HR17 to block the secretion of IFN-α is better than that of 3E5 and comparable to that of BIIB059.

[0162] Table 6. Blocking of IFN-α secretion in PBMC by BDCA2 monoclonal antibodies

[0163] Example 5: Specificity study of BDCA2 monoclonal antibodies

[0164] Human BDCA2 protein (Aatree, Cat: BCA-HM102), CLEC4A protein (Aatree, Cat: CLE-HM24A), human CLEC4D protein (Aatree, Cat: 11485-H07H), human CLEC4E protein (Abnova, Cat: C588), human CLEC4N protein (Aatree, Cat: 10250-H01H) were diluted to 1 μg / mL coating solution, 100 μL / well was added to the enzyme-labeled plate, and coated at 2-8 ℃ for 12 hours or more. Discard the residual liquid in the plate, add 2% BSA-PBS, 200 μL per well, and incubate at 37 ℃ for 2 hours. Then wash 3 times with 300 μL of PBST per well. The test samples (HR04, HR09, HR13, HR17, BIIB059, 3E5) were diluted with PBS to 60 nM, and then diluted 3 times to 11 gradients, 100 μL / well was added to the enzyme-labeled plate. Incubate at 37 ℃ for 1 hour, wash 3 times with 300 μL of PBST per well, and then add Goat anti Human Fc-HRP (abcam, ab97225) diluted 10000 times, 100 μL / well. Incubate at 37 ℃ for 1 hour, wash 3 times with 300 μL of PBST per well, and then dry. Add TMB developing solution, 100 μL per well. React for 5 minutes at room temperature, then stop the reaction with 2M H2SO4, 50 μL / well. Place the enzyme-labeled plate in the enzyme-labeled instrument, and read the absorbance OD450 value at 450 nm wavelength. Use software GraphPad Prism 9.0 for data processing and graph analysis, and obtain the binding curve and EC50 value parameters by four-parameter nonlinear fitting.

[0165] The smaller the EC50 value, the stronger the binding activity and the weaker the specificity. The results are shown in Table 7 and FIG. 6. HR04, HR09, HR13, HR17, BIIB059, and 3E5 all bind to BDCA2, but not to CLEC4A, CLEC4D, CLEC4E, and CLEC4N in the same group, and have good specificity.

[0166] Table 7. Specificity of BDCA2 monoclonal antibodies

[0167] Example 6: Hydrophobicity study of BDCA2 monoclonal antibodies

[0168] The sample to be tested (HR04, HR09, HR13, HR17, BIIB059) was diluted with mobile phase A (1.8 M ammonium sulfate + 0.1 M sodium dihydrogen phosphate solution, pH 6.5) to a final ammonium sulfate concentration of 1.0 M in the test sample solution, with mobile phase A (1.8 M ammonium sulfate + 0.1 M sodium dihydrogen phosphate solution, pH 6.5), mobile phase B (0.1 M sodium dihydrogen phosphate solution, pH 6.5) as the mobile phase, a chromatographic column (manufacturer: Saipien Technology, model: Protemix HIC Butyl-NP54.6*10mm, 5μm), wavelength 214nm, flow rate 1.0mL / min, injection amount 5μg, column temperature 25℃, sample chamber temperature 8℃. The gradient elution method was 0-5min, 44% B; 5-15min, 44% B→100% B; 15-20min, 100% B; 20.1-25min, 44% B. Elution was performed, and by comparing the retention time of the target protein, the hydrophilicity of the target protein was determined.

[0169] The shorter the elution time, the stronger the hydrophilicity, and the better the solubility of the protein with strong hydrophilicity. The results are shown in Table 8. The elution time of HR04, HR09, HR13, and HR17 is less than that of BIIB059, so it is inferred that the hydrophilicity of HR04, HR09, HR13, and HR17 is better than that of BIIB059.

[0170] Table 8. Hydrophilicity and hydrophobicity of BDCA2 monoclonal antibody

[0171] Example 7: Monoclonal antibody-induced BDCA2 internalization

[0172] Freshly collected PBMC cells were obtained, and pDC cell separation was performed according to the pDC kit (Stem cell) instructions. After centrifugation at 400 g for 5 min, the cell density was adjusted to 3E+05 / mL. 100 uL was aliquoted into 10 1.5 ml EP tubes, i.e. 100 uL / tube, 3E4 / tube. 100 uL of the antibody to be tested at a concentration of 20 ug / mL was added to each tube, mixed, and the antibody concentration was 10 ug / mL. The control group was immediately placed on ice (4℃ group), and the detection group was placed at 37℃, and incubated for 1.5 h. 1 mL of pre-cooled FACS buffer was added, centrifuged at 4℃ and 400 g for 3 min, and then reverse centrifuged for 10 s to ensure that the cells were at the bottom of the tube. The supernatant was removed, and the step was repeated. The detection antibody (a labeled antibody that has been verified to have no blocking effect) was diluted with pre-cooled FACS buffer at 1:2000, added to the cells, 100 uL per group, mixed gently, and stained on ice for 30 min in the dark. The antibody was washed off, and the fixation buffer was diluted with pre-cooled FACS buffer at 1:1, added to the cells, and fixed on ice for 20-60 min before flow detection.

[0173] If the monoclonal antibody induces the internalization of BDCA2, the detection antibody cannot label BDCA2, and if there is no internalization, it can be labeled and detected by fluorescence. As shown in FIGS. 7A and 7B, after incubation of pDC with antibodies BIIB059, HR04, HR09, HR13 and HR17 at 37℃ for 1.5 h, compared with incubation on ice, the BDCA2 on the pDC at 37℃ was significantly internalized, and the isotype control IgG1 did not induce internalization.

[0174] In summary, the anti-BDCA2 antibody prepared in the present application has better affinity than the positive control 3E5, better inhibition of IFN-α than the positive control 3E5, and better hydrophilicity than the positive control BIIB059.

[0175] The above describes the preferred embodiments of the present application, but the present application is not limited thereto. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as disclosed in the present application and fall within the protection scope of the present application.

[0176] In the description of the specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are contained in at least one embodiment or example of the present application. In the specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the specification and the features of the different embodiments or examples without contradiction.

[0177] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and the person skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. An antibody or antigen-binding fragment thereof, characterized in that, comprises at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1-12 or an amino acid sequence having at least 80% homology thereto. The HCDRs comprise: an HCDR1, an HCDR2, and an HCDR3, wherein:

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, The HCDR1 comprises an amino acid sequence as set forth in or having at least 80% homology to SEQ ID NOs: 1-4, The HCDR2 comprises an amino acid sequence as set forth in or having at least 80% homology to SEQ ID NOs: 5-8, and The HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-12 or an amino acid sequence having at least 80% homology thereto; Preferably, the HCDRs comprise: an HCDR1 of an amino acid sequence as set forth in SEQ ID NOs: 1-4, respectively; Preferably, the HCDRs comprise: an HCDR2 of an amino acid sequence as set forth in SEQ ID NOs: 5-8, respectively; Preferably, the HCDRs comprise: an HCDR3 of an amino acid sequence as set forth in SEQ ID NOs: 9-12, respectively. Further comprising:

3. The antibody or antigen-binding fragment thereof of claim 1, wherein, light chain complementarity determining regions (LCDRs) comprising: an LCDR1, an LCDR2, and an LCDR3, wherein: The LCDR1 comprises an amino acid sequence as set forth in or having at least 80% homology to SEQ ID NOs: 13-16, The LCDR2 comprises an amino acid sequence as set forth in or having at least 80% homology to SEQ ID NOs: 17-20, and The LCDR3 comprises an amino acid sequence as set forth in or having at least 80% homology to SEQ ID NOs: 21-24; Preferably, the LCDRs comprise: an LCDR1 of an amino acid sequence as set forth in SEQ ID NOs: 13-16, respectively; Preferably, the LCDRs comprise: an LCDR2 of an amino acid sequence as set forth in SEQ ID NOs: 17-20, respectively; Preferably, the LCDRs comprise: an LCDR3 of an amino acid sequence as set forth in SEQ ID NOs: 21-24, respectively. The antibody or antigen-binding fragment thereof further comprises: a heavy chain framework region; 4. The antibody or antigen-binding fragment thereof of claim 2 or 3, wherein, Optionally, the antibody or antigen-binding fragment thereof further comprises: a light chain framework region; Optionally, at least a portion of the heavy chain framework region and the light chain framework region are independently derived from at least one of a rabbit-derived antibody, a murine-derived antibody, an ovine-derived antibody, a caprine-derived antibody, a primate-derived antibody, or a mutant thereof. ​ Preferably, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-28 or having at least 80% homology with SEQ ID NOs: 25-28; preferably, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is as set forth in SEQ ID NOs: 25-28; Preferably, the light chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NOs: 29-32 or having at least 80% homology with SEQ ID NOs: 29-32; preferably, the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof is as set forth in SEQ ID NOs: 29-32.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein, The antibody or antigen-binding fragment thereof further comprises a heavy chain constant region; Optionally, the antibody or antigen-binding fragment thereof further comprises a light chain constant region; Optionally, at least one of the heavy chain constant region and the light chain constant region is independently derived from a rabbit-derived antibody, a murine-derived antibody, a sheep-derived antibody, a goat-derived antibody, a primate-derived antibody, or a mutant thereof; preferably, at least one of the heavy chain constant region and the light chain constant region is derived from a primate-derived antibody; Preferably, the heavy chain of the antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-36 or having at least 80% homology with SEQ ID NOs: 33-36; preferably, the amino acid sequence of the heavy chain of the antibody or antigen-binding fragment thereof is as set forth in SEQ ID NOs: 33-36; Preferably, the light chain of the antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-40 or having at least 80% homology with SEQ ID NOs: 37-40; preferably, the amino acid sequence of the light chain of the antibody or antigen-binding fragment thereof is as set forth in SEQ ID NOs: 37-40.

6. The antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody comprises at least one selected from the group consisting of a polyclonal antibody and a monoclonal antibody; or The antigen-binding fragment comprises at least one selected from the group consisting of a F(ab’)2 fragment, a Fab’ fragment, a Fab fragment, a Fv fragment, a scFv fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein, and a minimal recognition unit; Preferably, the antibody is a monoclonal antibody.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1-6; Optionally, the nucleic acid molecule is DNA.

8. An expression vector, characterized by, Carrying the nucleic acid molecule according to claim 7.

9. A method of producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that, Comprising: Introducing the expression vector according to claim 8 into a cell; Culturing the cell under conditions suitable for protein expression and secretion so as to obtain the antibody or antigen-binding fragment thereof; Optionally, the cell is a eukaryotic cell.

10. A recombinant cell, characterized in that, The recombinant cell carries the nucleic acid molecule according to claim 7 or the expression vector according to claim 8.

11. An immunoconjugate, characterized in that, Comprising: The antibody or antigen-binding fragment thereof of any one of claims 1-6, the nucleic acid molecule of claim 7, the expression vector of claim 8, or the recombinant cell of claim 10; Optionally, the immunoconjugate further comprises a therapeutic agent.

12. A composition characterized in that, Comprising: The antibody or antigen-binding fragment thereof of any one of claims 1-6, the nucleic acid molecule of claim 7, the expression vector of claim 8, the recombinant cell of claim 10, or the immunoconjugate of claim 11.

13. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-6, the nucleic acid molecule of claim 7, the expression vector of claim 8, the recombinant cell of claim 10, the immunoconjugate of claim 11, or the composition of claim 12 in the manufacture of a medicament for the treatment or prevention of a BDCA2-mediated related disease.

14. Use according to claim 13, characterized in that, The BDCA2-mediated related disease comprises an autoimmune disease; Optionally, the autoimmune disease comprises at least one of systemic lupus erythematosus, cutaneous lupus erythematosus, scleroderma, polymyositis and dermatomyositis, psoriasis, Sjogren's syndrome, rheumatoid arthritis, Graves' disease, and Hashimoto's disease.

15. A medicament, characterized by comprising: Comprising: The antibody or antigen-binding fragment thereof of any one of claims 1-6, the nucleic acid molecule of claim 7, the expression vector of claim 8, the recombinant cell of claim 10, the immunoconjugate of claim 11, or the composition of claim 12, for use in the treatment or prevention of a BDCA2-mediated related disease.

16. The medicament according to claim 15, characterized in that, The BDCA2-mediated related disease comprises an autoimmune disease; Optionally, the autoimmune disease comprises at least one of systemic lupus erythematosus, cutaneous lupus erythematosus, scleroderma, polymyositis and dermatomyositis, psoriasis, Sjogren's syndrome, rheumatoid arthritis, Graves' disease, and Hashimoto's disease. Optionally, further comprising a pharmaceutically acceptable excipient; Optionally, the excipient comprises: one or more pharmaceutically acceptable excipients, diluents, stabilizers, or carriers; Optionally, the medicament is an injection.

17. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-6 in the manufacture of a kit for detecting BDCA2.

18. A kit comprising, The kit comprises: The antibody or antigen-binding fragment thereof of any one of claims 1-6.

19. The kit of claim 18, wherein The kit is for detecting BDCA2.

20. A method of preventing and / or treating a BDCA2-mediated disease, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-19. Comprising: administering to a subject a pharmaceutically acceptable amount of the antibody or antigen-binding fragment thereof of any one of claims 1-6, the nucleic acid molecule of claim 7, the expression vector of claim 8, the recombinant cell of claim 10, the immunoconjugate of claim 11, the composition of claim 12, or the medicament of any one of claims 15-16.

21. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 6, the nucleic acid molecule of claim 7, the expression vector of claim 8, the recombinant cell of claim 10, the immunoconjugate of claim 11, the composition of claim 12, or the medicament of any one of claims 15 to 16, in the treatment or prevention of a BDCA2-mediated disease.

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