Antibody or antigen-binding fragment thereof, for binding to BDCA2, recombinant protein, nucleotide molecule, expression vector, host cell, method for preparing an antibody or antigen-binding fragment thereof, for binding to BDCA2, composition, application of antibody or antigen-binding fragment thereof, for binding to BDCA2 and construction of the cell.

Description

1 / 71 “ANTIBODY OR ANTIGEN-BINDING FRAGMENT THEREOF, FOR BINDING TO BDCA2, RECOMBINANT PROTEIN, NUCLEOTIDE MOLECULE, EXPRESSION VECTOR, HOST CELL, METHOD FOR PREPARING AN ANTIBODY OR ANTIGEN-BINDING FRAGMENT THEREOF, FOR BINDING TO BDCA2, COMPOSITION, APPLICATION OF AN ANTIBODY OR ANTIGEN-BINDING FRAGMENT THEREOF, FOR BINDING TO BDCA2 AND CAR CONSTRUCTION” TECHNICAL FIELD

[001] The present invention relates to the area of ​​antibodies. Specifically, it relates to an anti-BDCA2 antibody. BACKGROUND OF THE INVENTION

[002] Type I interferon (IFN-I) is an inflammatory mediator produced primarily by plasmacytoid dendritic cells (pDCs), a component of the innate immune system. The IFN-I gene is subject to tight regulation. Under normal circumstances, IFN-α formation is hardly detectable in healthy individuals. However, continuous production of type I IFN, accompanied by increased expression of IFN-α-regulated genes, is common among patients with systemic autoimmune diseases. IFN-α activates both the innate and adaptive immune systems. When individuals possess genetic predispositions that lead to robust IFN-α production and / or pronounced responses to IFN-α, tolerance is disrupted, resulting in the generation of antibodies against autoantigens. These antibodies form immune complexes with autoantigens, stimulating pDCs to synthesize IFN-α and activating B cells to promote autoantibody production.This creates a vicious cycle of sustained IFN-α generation and autoimmunity.

[003] Blood dendritic cell antigen 2 Petition 870250087810, dated 09 / 29 / 2025, page 16 / 101 2 / 71 (BDCA2), also designated CLEC4C or CD303, is specifically expressed on pDCs. Upon activation, BDCA2 triggers the SYK protein, leading to the activation of a complex comprising BLNK, BTK, and PLCy2. This complex mobilizes intracellular Ca2+, thereby inhibiting the release of inflammatory mediators, such as IFN-I and IL-6, mediated by the TLR7 and TLR9 signaling pathways.

[004] Drugs targeting BDCA2 can interfere with pDC function by promoting rapid internalization of BDCA2 on the pDC surface. This indirectly inhibits the release of IFN-I and pro-inflammatory cytokines. Furthermore, BDCA2 signaling is not affected by other IFN-I-induced stimuli, making BDCA2 a novel therapeutic target for autoimmune diseases. However, existing therapies targeting BDCA2 have numerous shortcomings, necessitating the development of new anti-BDCA2 antibodies in this field. Summary of the invention

[005] The present invention aims to provide an anti-BDCA2 antibody.

[006] In a first aspect of the present invention, an antibody or antigen-binding fragment thereof is provided that binds to BDCA2, characterized by comprising a variable region of the heavy chain and a variable region of the light chain. Said variable region of the heavy chain comprises the H-CDR1, H-CDR2 and H-CDR3 heavy chain complementarity-determining regions, wherein: The amino acid sequence of H-CDR1 is shown in SYX1IH (Seq ID No.: 30), and the amino acid sequence of H-CDR2 is shown in Petition 870250087810, dated 09 / 29 / 2025, page 17 / 101 3 / 71 TIYPGX2GDTSYNQKFKG (Seq ID No.: 31), the amino acid sequence of H-CDR3 is shown in MGDNEYFDY (Seq ID No.: 8), and the aforementioned variable region of the light chain comprises complementarity-determining regions of the L-CDR1 light chain, L-CDR2,L-CDR3; wherein, the amino acid sequence of L-CDR1 is shown in RASGNIHNYLA (Seq ID No: 9), the amino acid sequence of L-CDR2 is shown in DAETLAX3 (Seq ID No: 32), the amino acid sequence of L-CDR3 is shown in QHFWSTPYT (Seq ID No: 11); where X1 is I or L; X2 is selected from the group consisting of the amino acids: N, G, A, S, R; and / or X3 is selected from the group consisting of the amino acids: D, I, or E.

[007] In another example of preferential selection, X1, X2, X3 are selected from the group: X1 is I, X2 is N, X3 is D; X1 is L, X2 is N, X3 is I; X1 is L, X2 is N, X3 is E; X1 is L, X2 is G, X3 is I; X1 is L, X2 is G, X3 is E; X1 is L, X2 is R, X3 is I; X1 is L, X2 is R, X3 is E; X1 is I, X2 is G, X3 is I; X1 is I, X2 is G, X3 is E; X1 is I, X2 is R, X3 is I; or X1 is I, X2 is R, X3 is E.

[008] In another example of preferential selection, the aforementioned variable region of the heavy chain comprises regions Petition 870250087810, dated 09 / 29 / 2025, page 18 / 101 4 / 71 heavy chain complementarity determinants H-CDR1, H-CDR2 and H-CDR3, wherein the amino acid sequence of H-CDR1 is shown in SYIIH (Seq ID No: 6), the amino acid sequence of H-CDR2 is shown in TIYPGXTIYPGNGDTSYNQKFKG (Seq ID No: 7), the amino acid sequence of H-CDR3 is shown in MGDNEYFDY (Seq ID No: 8), and said variable region of the light chain comprises light chain complementarity determinants L-CDR1, L-CDR2, L-CDR3; wherein, the amino acid sequence of L-CDR1 is shown in RASGNIHNYLA (Seq ID No.: 9), the amino acid sequence of L-CDR2 is shown in DAETLAD (Seq ID No.: 10), the amino acid sequence of L-CDR3 is shown in QHFWSTPYT (Seq ID No.: 11); wherein, in the above amino acid sequences, any of the amino acid sequences may further comprise a derived sequence that optionally contains the addition, omission, modification and / or substitution of at least one amino acid, while maintaining the binding affinity to BDCA2.

[009] In another example of preferential selection, the ratio (F1 / F0) between the F1 affinity with which the said derived antibody binds to BDCA2 and the F0 affinity with which the corresponding non-derived antibody binds to BDCA2 is 0.5-4, more preferably 0.7-1.5 and, even more preferably, 0.8-1.2.

[0010] In another example of preferential selection, the aforementioned number of amino acids added, omitted, Petition 870250087810, dated 09 / 29 / 2025, p. 19 / 101 5 / 71 modification and / or replacement is 1-5 (e.g., 1-3, more preferably 1-2, more preferably 1).

[0011] In another example of preferential selection, the derived sequence comprises a variable region of the heavy chain and a variable region of the light chain, wherein the variable region of the heavy chain comprises regions that determine heavy chain complementarity with the following amino acid sequence: The amino acid sequence of H-CDR1 is shown in SYX1IH, where X1 is I or L; and / or the amino acid sequence of H-CDR2 is shown in TIYPGX2GDTSYNQKFKG, where X2 is selected from the group consisting of the amino acids: N, G, A, S, R (preferably G); and / or the aforementioned variable region of the light chain comprises complementarity-determining regions of the light chain with the following amino acid sequence: The amino acid sequence of L-CDR2 is shown in DAETLAX3, where X3 is selected from the group consisting of the amino acids: D, I, or E; where X1, X2, and X3 are each independently parallel to I, N, and D.

[0012] In another example of preferential selection, in the derived sequence, X1, X2 and X3 are selected from the group: X1 is L, X2 is N, X3 is I; X1 is L, X2 is N, X3 is E; X1 is L, X2 is G, X3 is I; X1 is L, X2 is G, X3 is E; X1 is L, X2 is R, X3 is I; X1 is L, X2 is R, X3 is E; Petition 870250087810, dated 09 / 29 / 2025, p. 20 / 101 6 / 71 X1 is I, X2 is G, X3 is I; X1 is I, X2 is G, X3 is E; X1 is I, X2 is R, X3 is I; or X1 is I, X2 is R, X3 is E.

[0013] In another example of preferential selection, the aforementioned variable region of the heavy chain within the aforementioned derived sequence comprises: an H-CDR1 with the amino acid sequence shown in SYLIH (Seq ID No.: 14), an H-CDR2 with the amino acid sequence shown in TIYPGGGDTSYNQKFKG (Seq ID No.: 15), an H-CDR3 with the amino acid sequence shown in MGDNEYFDY (Seq ID No.: 8), the said variable region of the light chain comprises an L-CDR1 with the amino acid sequence shown in RASGNIHNYLA (Seq ID No.: 9), an L-CDR2 with the amino acid sequence shown in DAETLAE (Seq ID No.: 20), an L-CDR3 with the amino acid sequence shown in QHFWSTPYT (Seq ID No.: 11).

[0014] In another example of preferential selection, the aforementioned variable region of the heavy chain within the aforementioned derived sequence comprises: an H-CDR1 with the amino acid sequence shown in SYIIH (Seq ID No.: 6), an H-CDR2 with the amino acid sequence shown in TIYPGGGDTSYNQKFKG (Seq ID No.: 15), an H-CDR3 with the amino acid sequence shown in MGDNEYFDY (Seq ID No.: 8), the aforementioned variable region of the light chain comprises Petition 870250087810, dated 09 / 29 / 2025, page 21 / 101 7 / 71 an L-CDR1 with the amino acid sequence shown in RASGNIHNYLA (Seq ID No: 9), an L-CDR2 with the amino acid sequence shown in DAETLAE (Seq ID No: 20), an L-CDR3 with the amino acid sequence shown in QHFWSTPYT (Seq ID No: 11).

[0015] In another example of preferential selection, the aforementioned variable region of the heavy chain within the aforementioned derived sequence comprises: an H-CDR1 with the amino acid sequence shown in SYLIH (Seq ID No.: 14), an H-CDR2 with the amino acid sequence shown in TIYPGGGDTSYNQKFKG (Seq ID No.: 7), an H-CDR3 with the amino acid sequence shown in MGDNEYFDY (Seq ID No.: 8), the aforementioned variable region of the light chain comprises an L-CDR1 with the amino acid sequence shown in RASGNIHNYLA (Seq ID No.: 9), an L-CDR2 with the amino acid sequence shown in DAETLAE (Seq ID No.: 20), an L-CDR3 with the amino acid sequence shown in QHFWSTPYT (Seq ID No.: 11).

[0016] In another example of preferential selection, the antibody in question is a murine antibody, a chimeric antibody, or a humanized antibody.

[0017] In another example of preferential selection, the variable region of the heavy chain of the antibody in question also comprises a region of human structure and / or the variable region of the light chain of the antibody in question also comprises a region of human structure. Petition 870250087810, dated 09 / 29 / 2025, page 22 / 101 8 / 71

[0018] In another example of preferential selection, the variable region of the heavy chain of the antibody in question also comprises a murine structure region and / or the variable region of the light chain of the antibody in question also comprises a murine structure region.

[0019] In another example of preferential selection, the aforementioned constant region of the heavy chain is derived from humans and / or the aforementioned constant region of the light chain is derived from humans.

[0020] In another example of preferential selection, the aforementioned constant region of the heavy chain is the IgG1 constant region of a human antibody and the aforementioned constant region of the light chain is the kappa constant region of a human antibody light chain.

[0021] In another example of preferential selection, the aforementioned constant region of the heavy chain comprises an Fc fragment.

[0022] In another example of preferential selection, the aforementioned Fc fragment is an Fc variant, wherein the aforementioned modified Fc variant comprises the following mutations: M428L / N434S; G236A / S239D / I332E; S239D / I332E / A330L; S239D / I332E / G236A; S298A; A330L; I332E; E333A; and / or K334A; wherein the numbering of residues in the Fc variant corresponds to the EU index in Kabat.

[0023] In another example of preferential selection, the antibody in question is selected from the group: antibodies of animal origin, chimeric antibodies, humanized antibodies, fully human antibodies, or combinations thereof.

[0024] In another example of preferential selection, the Petition 870250087810, dated 09 / 29 / 2025, page 23 / 101 9 / 71 The referred antibody is either a double-chain antibody or a single-chain antibody.

[0025] In another example of preferential selection, the antibody in question is either a full-length antibody protein or an antigen-binding fragment.

[0026] In another example of preferential selection, the antibody in question is a bispecific antibody or a multispecific antibody.

[0027] In another example of preferential selection, the antibody in question is in the form of a pharmacological conjugate.

[0028] In another example of preferential selection, the affinity of the aforementioned antibody for the BDCA2 antigen is ^1E-9 M; more preferably ^1E-10 M; even more preferably ^1E-11 M.

[0029] In another example of preferential selection, the aforementioned BDCA2 is human BDCA2; more preferentially, the extracellular domain of human BDCA2.

[0030] In another example of preferential selection, the sequence of the extracellular domain of the aforementioned human BDCA2 is shown in SEQ ID No.: 1.

[0031] In another example of preferential selection, the antigen-binding fragment in question comprises a Fab fragment, an F(ab')2 fragment, an Fv fragment, or an scFv fragment.

[0032] In another example of preferential selection, the amino acid sequence of the variable region of the heavy chain of said antibody that binds to BDCA2 or its antigen-binding fragment is that shown in SEQ ID No. 3, and the amino acid sequence of the variable region of the chain Petition 870250087810, dated 09 / 29 / 2025, page 24 / 101 10 / 71 light is shown in No. ID SEQ: 5.

[0033] In another example of preferential selection, the amino acid sequence of the variable region of the heavy chain of said antibody that binds to the BDCA2 or antigen-binding fragment thereof is that shown in SEQ ID No.: 12, and the amino acid sequence of the variable region of the light chain is that shown in SEQ ID No.: 13.

[0034] In another example of preferential selection, the amino acid sequence of the variable region of the heavy chain of said antibody that binds to the BDCA2 or antigen-binding fragment thereof is selected from SEQ ID No.: 21, 22, 23, 24 or 25, and the amino acid sequence of the variable region of the light chain is selected from SEQ ID No.: 26 or 27.

[0035] In another example of preferential selection, the amino acid sequence of said variable region of the heavy chain shares at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequences shown in No. ID SEQ 3, 12, 21, 22, 23, 24 or 25.

[0036] In another example of preferential selection, the amino acid sequence of said variable region of the light chain shares at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequences shown in No. ID SEQ 5, 13, 26 or 27.

[0037] In a second aspect of the present invention, a recombinant protein is provided, characterized in that the recombinant protein comprises: (i) the said antibody that binds to BDCA2 or fragment Petition 870250087810, dated 09 / 29 / 2025, p. 25 / 101 11 / 71 of antigen binding as described in the first aspect of the present invention; and (ii) optionally, a labeling sequence to facilitate expression and / or purification.

[0038] In another example of preferential selection, the said tagging sequence comprises a 6His tag.

[0039] In another example of preferential selection, the said recombinant protein (or peptide) comprises a fusion protein.

[0040] In another example of preferential selection, the recombinant protein in question is a monomer, dimer, or polymer.

[0041] In the third aspect of the present invention, a nucleotide molecule is provided, characterized in that the nucleotide molecule encodes said antibody that binds to BDCA2 or an antigen-binding fragment thereof as per the first aspect of the present invention.

[0042] In another example of preferential selection, the aforementioned nucleotide molecules encode a nucleotide sequence for the variable region of the heavy chain, as shown in No. ID SEQ: 2, and a nucleotide sequence for the variable region of the light chain, as shown in No. ID SEQ: 4.

[0043] In the fourth aspect of the present invention, an expression vector is provided, characterized in that the expression vector contains said nucleotide molecules, as per the third aspect of the present invention.

[0044] In the fifth aspect of the present invention, a host cell is provided, characterized in that said host cell contains said expression vector, as per Petition 870250087810, dated 09 / 29 / 2025, page 26 / 101 12 / 71 the fourth aspect of the present invention.

[0045] In the sixth aspect of the present invention, a method is provided for preparing said antibody that binds to BDCA2 or an antigen-binding fragment thereof, as described in the first aspect of the present invention, characterized in that said method comprises the following steps: (a) cultivate said host cell referred to in the fifth aspect of the present invention under expression conditions to express said antibody that binds to BDCA2 or antigen-binding fragment thereof; b) Separate and purify a) said antibody that binds to BDCA2 or its antigen-binding fragment.

[0046] In the seventh aspect of the present invention, a composition is provided, characterized in that said composition comprises said antibody that binds to BDCA2 or an antigen-binding fragment thereof as per the first aspect of the present invention, and a pharmacologically acceptable vector.

[0047] In the eighth aspect of the present invention, an application is provided for said antibody that binds to BDCA2 or an antigen-binding fragment thereof, as referred to in the first aspect of the present invention, or for the composition referred to in the seventh aspect of the present invention, in the preparation of a drug for the treatment of inflammatory or autoimmune diseases.

[0048] In another example of preferential selection, inflammatory or autoimmune diseases are selected from the group: systemic lupus erythematosus, cutaneous lupus erythematosus, discoid lupus, lupus nephritis, cutaneous lupus, Petition 870250087810, dated 09 / 29 / 2025, p. 27 / 101 13 / 71 rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type 1 diabetes, dermatomyositis and polymyositis

[0049] In the ninth aspect of the present invention, a CAR construct is provided, characterized in that the scFv fragment of the antigen-binding region of the monoclonal antibody of said CAR construct is a binding domain that specifically binds to BDCA2, and in which the variable region of the heavy chain of said scFv comprises: The regions determining complementarity of the heavy chain H-CDR1, H-CDR2, and H-CDR3, wherein the amino acid sequence of H-CDR1 is shown in SYIIH (Seq ID No.: 6), the amino acid sequence of H-CDR2 is shown in TIYPGNGDTSYNQKFKG (Seq ID No.: 7), the amino acid sequence of H-CDR3 is shown in MGDNEYFDY (Seq ID No.: 8), and the variable region of the light chain of said scFv comprises: regions determining complementarity of the light chain L-CDR1, L-CDR2, and L-CDR3, wherein the said amino acid sequence of L-CDR1 is shown in RASGNIHNYLA (Seq ID No.: 9), the amino acid sequence of L-CDR2 is shown in DAETLAD (Seq ID No.: 10), and the amino acid sequence of L-CDR3 is shown in QHFWSTPYT (Seq ID No.: 11). SEQ: 11); wherein any of the above amino acid sequences may optionally comprise at least one amino acid by addition, omission, modification and / or substitution, and may comprise a derived sequence capable of retaining the binding affinity to BDCA2.

[0050] In another example of preferential selection, the derived sequence comprises a variable region of the heavy chain and a variable region of the light chain, in Petition 870250087810, dated 09 / 29 / 2025, p. 28 / 101 14 / 71 that the aforementioned variable region of the heavy chain comprises regions that determine the complementarity of the heavy chain with the following amino acid sequence: The amino acid sequence of H-CDR1 is shown in SYX1IH, where X1 is I or L; and / or the amino acid sequence of H-CDR2 is shown in TIYPGX2GDTSYNQKFKG, where X2 is selected from the group consisting of the amino acids: N, G, A, S, R (preferably G); and / or the aforementioned variable region of the light chain comprises complementarity-determining regions of the light chain with the following amino acid sequence: The amino acid sequence of L-CDR2 is shown in DAETLAX3, where X3 is selected from the group consisting of the amino acids: D, I, or E; where X1, X2 and X3 are each independently parallel to I, N and D.

[0051] In the tenth aspect of the present invention, a recombinant immune cell is provided, characterized in that said immune cell expresses an exogenous CAR construct, as per the ninth aspect of the present invention.

[0052] In the eleventh aspect of the present invention, an antibody-drug conjugate is provided, characterized in that said antibody-drug conjugate contains: an antibody portion, said antibody portion comprising said antibody or its antigen-binding fragment, as per the first aspect of the present invention; and a coupling portion conjugated to said antibody portion, said coupling portion selected from Petition 870250087810, dated 09 / 29 / 2025, p. 29 / 101 15 / 71 group: a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0053] In the twelfth aspect of the present invention, a method is provided for the in vitro detection of the BDCA2 protein in a sample, characterized in that said method comprises the following steps: (1) placing said sample in vitro in contact with said antibody or its antigen-binding fragment, according to the first aspect of the present invention, or with said antibody-drug conjugate, according to the eleventh aspect of the present invention; (2) detect if an antibody complex is formed, where the formation of the complex indicates the presence of the BDCA2 protein in the sample.

[0054] In the thirteenth aspect of the present invention, a method is provided for preventing and / or treating diseases associated with BDCA2, said method comprising: administering to an individual in need said antibody that binds to BDCA2 or a BDCA2-binding antigen fragment thereof, as per the first aspect of the present invention, said composition as per the seventh aspect of the present invention, said recombinant immune cell as per the tenth aspect of the present invention, or said antibody-drug conjugate as per the eleventh aspect of the present invention, or a combination thereof.

[0055] In another example of preferential selection, said BDCA2-associated disease is selected from inflammatory or autoimmune diseases, including, but not limited to, systemic lupus erythematosus, lupus erythematosus Petition 870250087810, dated 09 / 29 / 2025, p. 30 / 101 16 / 71 cutaneous, discoid lupus, lupus nephritis, cutaneous lupus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type 1 diabetes, dermatomyositis and polymyositis, or combinations thereof.

[0056] It should be understood that, within the scope of the present invention, the technical features described above and those specifically described below the present invention (for example, in the implementation example) can be combined to constitute new or preferred technical solutions. Due to space limitations, these are not exhaustively enumerated in this document. DESCRIPTION OF THE DRAWINGS

[0057] Figures 1A-1B illustrate the binding capacity of the murine antibody to human BDCA2-Fc.

[0058] Figures 2A-2B illustrate the binding affinity assay of murine antibodies to the stably transfected 293FT-BDCA2 cell line.

[0059] Figure 3 illustrates the binding capacity of the antibody that binds to human-murine BDCA2.

[0060] Figure 4 illustrates the inhibitory effect of the antibody that binds to human-murine BDCA2 on CpG-A-induced IFN-α secretion from PBMCs.

[0061] Figure 5 illustrates ELISA screening of different mutation sites affecting antibody 230 affinity.

[0062] Figure 6 illustrates ELISA affinity detection between mutant strains of anti-BDCA2 antibodies and the BDCA2-his antigen protein.

[0063] Figure 7 illustrates the use of PBMCs to detect the inhibitory effect of candidate antibodies on the secretion of Petition 870250087810, dated 09 / 29 / 2025, p. 31 / 101 17 / 71 IFN-α induced by CpG-A in PBMCs.

[0064] Figure 8 illustrates the inhibitory effect of candidate antibodies on CpG-A-induced IgM secretion in PBMCs.

[0065] Figure 9 illustrates the inhibitory effect of candidate antibodies on IFN-α secretion in mice. DETAILED DESCRIPTION OF THE INVENTION

[0066] Through extensive and thorough research and exhaustive screening, the inventors of the present invention unexpectedly discovered a series of murine antibodies that bind to the target antigen BDCA2-Fc with excellent binding activity. Further selection of candidate antibodies, through humanization and CDR engineering, yielded humanized antibodies with affinity and expression levels comparable to or superior to those of the parental antibody. The antibody of the present invention effectively inhibits the secretion of IFNα and IgM by CpG-A stimulated PBMCs, exhibiting significantly higher activity compared to the positive control antibody Litifilimab. The present invention is concluded on this basis. TERMINOLOGY

[0067] Unless otherwise indicated, all technical and scientific terms used herein shall have the same meanings as are commonly understood by experts in the technical field to which this invention relates.

[0068] The term approximately may refer to a specific value or composition determined by a person skilled in the art, or to a value or composition within an acceptable range of error, which will depend in part on how the value or composition is measured or determined. Petition 870250087810, dated 09 / 29 / 2025, p. 32 / 101 18 / 71

[0069] As used in this document, the terms contain or “include (including)” can be open, semi-closed or closed. In other words, these terms also include consisting essentially of or consisting of. Antibody

[0070] As used in this document, the term antibody or immunoglobulin denotes a heterotetrameric glycoprotein of approximately 150,000 Daltons that has identical structural characteristics, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain through a covalent disulfide bond, with the number of disulfide bonds varying between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intra-chain disulfide bonds. One end of each heavy chain harbors a variable region (VH), followed by multiple constant regions.One end of each light chain contains a variable region (VL), while the other end harbors a constant region; the constant region of the light chain faces the first constant region of the heavy chain, and the variable region of the light chain faces the variable region of the heavy chain. Specialized amino acid residues form an interface between the variable regions of the light and heavy chains.

[0071] As used in this document, the term variable denotes that certain parts of the variable regions within antibodies exhibit sequence diversity, which determines antibody binding and specificity. Petition 870250087810, dated 09 / 29 / 2025, page 33 / 101 19 / 71 specific with respect to their specific antigens. However, this variability is not uniformly distributed throughout all variable regions of the antibody. It is concentrated in three segments in the variable regions of the light and heavy chains, called complementarity-determining regions (CDRs) or hypervariability regions. The most conserved parts of the variable regions are called structural regions (FRs). The variable regions of the natural heavy and light chains contain four FRs each, which adopt a broadly folded conformation linked by three CDRs that form connecting loops and, in some cases, may adopt partially folded structures. The CDRs within each chain are held together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)).The constant regions do not participate directly in antibody-antigen binding, but exhibit distinct effector functions, such as involvement in antibody-dependent cellular cytotoxicity.

[0072] The light chains of vertebrate antibodies (immunoglobulins) can be classified into two distinct categories (referred to as Kα and βλ) based on the amino acid sequence of the constant region of the heavy chain. Immunoglobulins can be categorized into different classes according to the amino acid sequence of the constant region of the heavy chain. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which are subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The constant regions Petition 870250087810, dated 09 / 29 / 2025, page 34 / 101 The 20 / 71 heavy chain segments corresponding to the different classes of immunoglobulins are designated κα, γδ, αε, βγ, and κμ, respectively. The subunit structure and three-dimensional conformation of immunoglobulins from different classes are well known to specialists in the field.

[0073] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain and the variable region of the light chain, called constant regions (CDRs). These regions are interspersed with four frames (FRs), whose amino acid sequences are relatively conserved and do not directly participate in the binding reactions. These CDRs create loop structures, with β-turns formed by the intermediate FRs, which spatially approximate them. The CDRs in the heavy chain and the corresponding CDRs in the light chain form the antigen-binding point of the antibody. The amino acids that constitute the FR or CDR regions can be determined by comparing the amino acid sequence of antibodies of the same type.

[0074] The present invention comprises not only complete antibodies, but also immunologically active antibody fragments or fusion proteins formed by antibodies with other sequences. Consequently, the present invention also comprises fragments, derivatives and analogues of said antibodies.

[0075] Within the scope of the present invention, antibodies comprise murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric monoclonal antibodies Petition 870250087810, dated 09 / 29 / 2025, page 35 / 101 21 / 71 and humanized antibodies, which comprise both human and non-human components, can be obtained through standard DNA recombination techniques and are all useful antibodies. Chimeric antibodies are molecules in which different segments originate from distinct animal species, such as chimeric antibodies possessing variable regions derived from murine monoclonal antibodies and constant regions from human immunoglobulins (see, for example, U.S. Patent 4,816,567 and U.S. Patent 4,816,397, which are incorporated herein by reference in full). Humanized antibodies are antibody molecules derived from non-human species, containing one or more complementarity-determining regions (CDRs) originating from non-human species, in addition to structural frames derived from human immunoglobulin molecules (see U.S. Patent 5,585,089, incorporated herein by reference in full).These chimeric and humanized monoclonal antibodies can be prepared using DNA recombination techniques known in the art.

[0076] In the present invention, the antibody can be monospecific, bispecific, trispecific or more multispecific.

[0077] In the present invention, the antibody of the present invention also comprises its conserved variants, that is, peptides formed by replacing up to 10, preferably up to 8, more preferably up to 5, ideally up to 3 amino acids with amino acids of similar or analogous properties, with respect to the amino acid sequence of the antibody of the present invention. These conserved variant peptides are preferably Petition 870250087810, dated 09 / 29 / 2025, page 36 / 101 22 / 71 generated by amino acid substitutions according to Table A. Petition 870250087810, dated 09 / 29 / 2025, page 37 / 101 23 / 71 Table A Initial residue Representative substitution Substituiç ão preferenci al Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Lys; Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; But; No; Phe Leu Leu (L) Ile; Val; But; No; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Leu; Val; Ile; No; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; But; Phe; Alas Leu Anti-BDCA2 Antibody

[0078] In the present invention, said antibody is an anti-BDCA2 antibody. The present invention provides a highly specific and high-affinity antibody against BDCA2, comprising a heavy chain and a light chain, wherein said heavy chain contains an amino acid sequence from said variable region of the heavy chain (VH) and the light chain contains an amino acid sequence from said variable region of the light chain (VL).

[0079] Wherein, the said variable region of the heavy chain comprises the complementarity-determining regions of the heavy chain H-CDR1, H-CDR2 and H-CDR3, the amino acid sequence of H-CDR1 is shown in SYX1IH (No. Petition 870250087810, dated 09 / 29 / 2025, p. 38 / 101 24 / 71 ID SEQ: 30), the amino acid sequence of H-CDR2 is shown in TIYPGX2GDTSYNQKFKG (N° ID SEQ: 31), the amino acid sequence of H-CDR3 is shown in MGDNEYFDY (N° ID SEQ: 8), and the aforementioned variable region of the light chain comprises complementarity-determining regions of the L-CDR1, L-CDR2, L-CDR3 light chain; wherein, the amino acid sequence of L-CDR1 is shown in RASGNIHNYLA (N° ID SEQ: 9), the amino acid sequence of L-CDR2 is shown in DAETLAX3 (N° ID SEQ: 32), the amino acid sequence of L-CDR3 is shown in QHFWSTPYT (N° ID SEQ: 11); where X1 is I or L; X2 is selected from the group consisting of the amino acids: N, G, A, S, R; and / or X3 is selected from the group consisting of the amino acids: D, I or E.

[0080] Preferably, the amino acid sequence of H-CDR1 is shown in SYIIH (Seq ID No: 6), the amino acid sequence of H-CDR2 is shown in TIYPGXTIYPGNGDTSYNQKFKG (Seq ID No: 7), the amino acid sequence of H-CDR3 is shown in MGDNEYFDY (Seq ID No: 8), and said variable region of the light chain comprises complementarity-determining regions of the L-CDR1, L-CDR2, L-CDR3 light chains; wherein, the amino acid sequence of L-CDR1 is shown in RASGNIHNYLA (Seq ID No: 9), the amino acid sequence of L-CDR2 is shown in DAETLAD Petition 870250087810, dated 09 / 29 / 2025, p. 39 / 101 25 / 71 (Seq ID No.: 10), the amino acid sequence of L-CDR3 is shown in QHFWSTPYT (Seq ID No.: 11); wherein, in the above amino acid sequences, any of the amino acid sequences may further comprise a derived sequence that optionally contains the addition, omission, modification and / or substitution of at least one amino acid, while maintaining the binding affinity to BDCA2.

[0081] In another example of preferential selection, the sequence formed by the addition, omission, modification and / or substitution of at least one amino acid is preferably an amino acid sequence that exhibits homology or sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90% and ideally at least 95%.

[0082] Methods for determining sequence homology or identity that are well known to those with common skill in the technique include, but are not limited to: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputation: Computational and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computational Sequence Data Analysis, Part I, Griffin, AM and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). The preferred methods for determining homology should achieve the maximum Petition 870250087810, dated 09 / 29 / 2025, p. 40 / 101 26 / 71 correspondence between the tested sequences. The methods for determining homology are compiled into publicly available computer programs. Preferred computer program methods for determining homology between two sequences include, but are not limited to: the GCG software package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, SF et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith-Waterman algorithm can also be used to determine similarity.

[0083] Preferably, said antibodies comprise one or more of the following: full-length antibodies, antigen-binding domain protein fragments, bispecific antibodies, multispecific antibodies, monochain antibody fragments (scFv), single-domain antibodies (sdAb), and single-domain antibodies. They may also comprise monoclonal or polyclonal antibodies derived from the aforementioned antibodies. Said monoclonal antibody may be developed through various methods and technologies, including hybridoma technology, phage display technology, and single lymphocyte gene cloning technology. The primary approach involves the preparation of monoclonal antibodies from wild-type or transgenic mice through hybridoma technology.

[0084] The aforementioned full-length antibody protein is a full-length antibody protein. Petition 870250087810, dated 09 / 29 / 2025, page 41 / 101 27 / 71 conventional within the field, comprising variable regions of the heavy chain, variable regions of the light chain, constant region of the heavy chain and constant region of the light chain. Said variable region of the heavy chain and the variable region of the light chain of the protein, together with the constant region of the human heavy chain and the constant region of the human light chain, constitute a fully human full-length antibody protein. Preferably, said full-length antibody protein is IgG1, IgG2, IgG3 or IgG4.

[0085] The antibody (BDCA2-binding antibody) of the present invention may be a full-length protein (e.g., IgG1, IgG2a, IgG2b, or IgG2c) or a protein fragment containing an antigen-binding domain (e.g., Fab, F(ab'), sdAb, ScFv fragment).

[0086] The antibody (anti-BDCA2 antibody) of the present invention can be a wild-type protein or a mutant protein engineered with specific mutations to achieve particular effects, such as eliminating the effector function of the antibody through mutation.

[0087] The antibody of the present invention may be a double-chain or single-chain antibody and may be selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies and, most preferably, fully human antibodies.

[0088] The aforementioned antibody derivatives of the present invention may be monochain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2, or others. Petition 870250087810, dated 09 / 29 / 2025, p. 42 / 101 28 / 71 derivatives of antibodies known in the field, as well as any one or more of the IgA, IgD, IgE, IgG, IgM antibodies, or antibodies of other subclasses.

[0089] The aforementioned single-chain antibody is a conventional single-chain antibody within the art, comprising a variable heavy chain region, a variable light chain region and a short peptide of 15 to 20 amino acids.

[0090] Wherein the animal in question is preferably a mammal, such as a mouse.

[0091] As used in this document, “Fc variant” or “Fc variant” denotes a protein containing a modified Fc domain. The Fc variants of the present invention are defined by the amino acid modifications that constitute them. Thus, for example, N434S or 434S denotes an Fc variant having a serine substitution at position 434 relative to the parental Fc peptide, wherein the numbering follows the EU index. Similarly, M428L / N434S defines Fc variants with substitutions at positions M428L and N434S relative to the parental Fc polypeptide. The identity of the wild-type amino acids may be nonspecific, in which case the variant is designated 428L / 434S. Note that the order of the substitutions is arbitrarily provided; that is, for example, 428L / 434S denotes the same Fc variant as M428L / N434S, and so on. For all positions discussed here, the numbering follows the EU index. The EU index or EU numbering scheme, as in Kabat et al., refers to the EU antibody numbering system (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, incorporated herein by reference in its entirety). Modifications may include additions, omissions, modifications, and / or substitutions. Substitutions may include... Petition 870250087810, dated 09 / 29 / 2025, p. 43 / 101 29 / 71 include both naturally occurring and non-naturally occurring amino acids. Variants may contain non-natural amino acids. Examples include US6,586,207; WO98 / 48032; WO03 / 073238; US2004-0214988A1; WO05 / 35727A2; WO05 / 74524A2; JWChin et al., (2002), Journal of the American Chemical Society 124:9026-9027; JWChin and PGSchultz, (2002), ChemBioChem 11: 1135-1137; JWChin, et al., (2002), PICAS United States of America 99: 11020-11024; and L.Wang and PGSchultz, (2002), Chem. 1-10, all of which are incorporated by reference in their entirety.

[0092] The antibody of the present invention may be a chimeric antibody, a humanized antibody, or an antibody with grafting and / or CDR modification targeting BDCA2 (e.g., human BDCA2).

[0093] Additionally, the CDR regions of the mouse antibody are transplanted into a selected humanized model, replacing the CDR regions of the humanized model. The variable region of the heavy chain is then recombined with the human IgG1 constant region, and the variable region of the light chain with the human kappa constant region, producing a chimeric antibody. Based on the three-dimensional structure of this antibody, reverse mutations were introduced in buried residues, residues that interact directly with the CDR regions, and residues that significantly influence the conformation of the VL and VH chains of the antibody, resulting in multiple humanized antibodies.

[0094] In the preceding aspects of the present invention, the number of amino acids of said addition, omission, modification and / or substitution is preferably not greater than 40% of the total number of amino acids in the sequence. Petition 870250087810, dated 09 / 29 / 2025, page 44 / 101 30 / 71 of amino acids, most preferably not exceeding 35%, most preferably 1-33%, most preferably 5-30%, most preferably 10-25%, and most preferably 15-20%.

[0095] In the foregoing aspects of the present invention, more preferably, the number of amino acids of said addition, omission, modification and / or substitution may be 1-7, more preferably 1-5, more preferably 1-3 and more preferably 1-2.

[0096] Preferably, said humanized antibody comprises a variable region of the heavy chain and a variable region of the light chain, wherein, in relation to a murine antibody, said variable region of the heavy chain comprises a region determining heavy chain complementarity with the following amino acid sequence: the amino acid sequence of H-CDR1 is shown in SYX1IH, wherein X1 is I or L; and / or the amino acid sequence of H-CDR2 is shown in TIYPGX2GDTSYNQKFKG, wherein X2 is selected from the group consisting of the amino acids: N, G, A, S, R (preferably G); and / or said variable region of the light chain comprises regions determining light chain complementarity with the following amino acid sequence: The amino acid sequence of L-CDR2 is shown in DAETLAX3, where X3 is selected from the group consisting of the amino acids: D, I, or E; where X1, X2, and X3 are each independently parallel to I, N, and D.

[0097] In another example of preferential selection, the Petition 870250087810, dated 09 / 29 / 2025, page 45 / 101 31 / 71 The variable region of the heavy chain and the variable region of the light chain of the humanized antibody select heavy chain complementarity-determining regions and light chain complementarity-determining regions of the group: H-CDR1 is selected from the group: SYIIH (Seq ID No.: 6) SYLIH (N° ID SEQ:14) (2) H-CDR2 is selected from group: TIYPGNGDTSYNQKFKG (SEQ ID NO:7) TIYPGGGDTSYNQKFKG (SEQ ID NO:15) TIYPGAGDTSYNQKFKG (SEQ ID NO:16) TIYPGSGDTSYNQKFKG (SEQ ID NO:17) TIYPGRGDTSYNQKFKG (Seq ID No.: 18) (3) H-CDR3 is MGDNEYFDY (Seq ID No.: 8) (4) L-CDR1 is RASGNIHNYLA (Seq ID No.: 9) (5) L-CDR2 is selected from the group: DAETLAD (Seq ID No.: 10) DAETLAI (Seq ID No.: 19) DAETLAE (N° ID SEQ:20); (6) L-CDR3 is QHFWSTPYT (SEQ ID NO: 11).

[0098] In another example of preferential selection, the amino acid sequence of said variable region of the heavy chain comprises the amino acid sequences shown in SEQ ID No. 3, 12, 21, 22, 23, 24 or 25, or exhibits 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with it.

[0099] In another example of preferential selection, the amino acid sequence of the aforementioned variable region of Petition 870250087810, dated 09 / 29 / 2025, p. 46 / 101 32 / 71 light chain comprises the amino acid sequence shown in SEQ ID No. 5, 13, 26 or 27, or exhibits at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with it.

[00100] In another example of preferential selection, the amino acid sequence of the variable region of the heavy chain (VH) and / or the amino acid sequence of the variable region of the light chain (VL) of the antibody targeting BDCA2 are presented in Table B below: Table B Antibody Name Sequence Identification VH Sequence Identification VL 23C10A6B5 3 5 Humanized Antibody 230 12 13 230IL-NG-DE 22 27 230NG-DE 24 27 230IL-DE 21 27 230IL-DI 21 26 230IL-NG-DI 22 26 230IL-NR-DI 23 26 230IL-NR-DE 23 27 230NG-DI 24 26 230NR-DI 25 26 230NR-DE 25 27 Recombinant Protein

[00101] The present invention also provides a recombinant protein comprising the CDR1 heavy chain (H-CDR1), the CDR2 heavy chain (H-CDR2) and the CDR3 heavy chain (H-CDR3) of the BDCA2 antibody of the first aspect of the present invention. Petition 870250087810, dated 09 / 29 / 2025, p. 47 / 101 33 / 71 and / or the CDR1 light chain (L-CDR1), CDR2 light chain (L-CDR2) and CDR3 light chain (L-CDR3).

[00102] The said recombinant protein further comprises a constant region of the heavy chain of said antibody and / or a constant region of the light chain of said antibody, wherein the constant region of the heavy chain of said antibody is conventional in the art, preferably a constant region of the heavy chain of said antibody derived from a mouse or a constant region of the heavy chain of said antibody derived from a human, and more preferably a constant region of the heavy chain of said antibody derived from a human. The said constant region of said light chain of said antibody is conventional in the art, preferably being a constant region of the light chain of said antibody derived from a mouse or a constant region of the light chain of said antibody derived from a human, more preferably a constant region of the light chain of said antibody derived from a human.

[00103] The recombinant protein in question is a conventional protein in the art, preferably a full-length antibody protein, antigen-binding domain protein fragments, bispecific antibodies, multispecific antibodies, monochain antibody fragments (scFv), single-domain antibodies (sdAb), and single-domain antibodies. They may also comprise monoclonal or polyclonal antibodies derived from the aforementioned antibodies. The monoclonal antibody in question may be developed through various methods and technologies, including hybridoma technology, phage display technology, and cloning technology. Petition 870250087810, dated 09 / 29 / 2025, page 48 / 101 34 / 71 unique lymphocyte genes. The main approach involves the preparation of monoclonal antibodies from wild-type or transgenic mice using hybridoma technology.

[00104] The aforementioned full-length antibody protein is a conventional full-length antibody protein within the field, comprising variable heavy chain regions, variable light chain regions, a constant heavy chain region, and a constant light chain region. The aforementioned variable heavy chain region and variable light chain region of the protein, together with the constant human heavy chain region and the constant human light chain region, constitute a fully human full-length antibody protein. Preferably, the aforementioned full-length antibody protein is IgG1, IgG2, IgG3, or IgG4.

[00105] The aforementioned monochain antibody is a conventional monochain antibody within the art, comprising a variable region of the heavy chain, a variable region of the light chain, and a short peptide of 15 to 20 amino acids.

[00106] The aforementioned antigen-binding fragment is a conventional antigen-binding fragment in the art, comprising an Fd fragment of a variable region of the light chain, a constant region of the light chain, and a constant region of the heavy chain. Preferably, the aforementioned antigen-antibody binding domain protein fragment comprises Fab and F(ab').

[00107] The aforementioned single-domain antibody is a conventional single-domain antibody within the art, comprising a variable region of the heavy chain and a Petition 870250087810, dated 09 / 29 / 2025, page 49 / 101 35 / 71 constant region of the heavy chain.

[00108] The aforementioned single-domain antibody is a conventional single-domain antibody in the art, comprising only the variable region of the heavy chain.

[00109] The expression vector, referred to as the method for preparing the recombinant protein, is a conventional method in the art. The said preparation method is preferably: isolation from a recombinant expression construct expressing the protein, or obtaining the protein sequence by artificial synthesis. The method of isolating and obtaining from a recombinant expression transformant expressing the said protein is preferably as follows: a nucleic acid molecule encoding the said protein and exhibiting a point mutation is cloned into a recombinant vector; the resulting recombinant vector is transformed into a transformant to obtain a recombinant expression transformant; and the said recombinant protein is isolated and purified by culturing the recombinant expression transformant. Nucleic Acid

[00110] The present invention also provides a nucleic acid that encodes the above antibody (e.g., anti-BDCA2 antibody) or recombinant protein, or the variable region of the heavy chain or the variable region of the light chain of the anti-BDCA2 antibody.

[00111] The aforementioned nucleic acid preparation method comprises conventional methods within the technique, preferably including the following steps: obtaining the nucleic acid molecule that encodes the aforementioned Petition 870250087810, dated 09 / 29 / 2025, p. 50 / 101 36 / 71 protein through gene cloning techniques, or obtaining the nucleic acid molecule that encodes said protein through artificial synthesis of the complete sequence.

[00112] Skilled individuals will recognize that the nucleotide sequence encoding the amino acid sequence of the above protein can be suitably modified by introducing substitutions, deletions, alterations, insertions, or additions to provide a polynucleotide homolog. The polynucleotide homologs of the present invention can be produced by substituting, deleting, or adding one or more nucleotides within the gene encoding the protein sequence, provided that antibody activity is maintained. Vector

[00113] The present invention also provides a recombinant expression vector comprising said nucleic acid.

[00114] Wherein, the said recombinant expression vector can be obtained by conventional methods within the art, namely by linking the nucleic acid molecule of the present invention to various expression vectors. The said expression vector can be any conventional vector within the art, provided it is capable of accommodating the aforementioned nucleic acid molecule. The said vector may advantageously comprise: various plasmids, miniplasmids, phages or viral vectors, etc.

[00115] The present invention further provides a recombinant expression transformant comprising the Petition 870250087810, dated 09 / 29 / 2025, p. 51 / 101 37 / 71 referred to as recombinant expression vector.

[00116] The method for preparing said recombinant expression transformant is conventional in the art, advantageously comprising: transforming said recombinant expression vector into host cells. Said host cell may be any conventional host cell within the art, provided that it satisfies the requirements for stable self-replication of said recombinant expression vector and permits said effective expression of the nucleic acid it carries. Preferably, said host cell is E. coli TG1 or E. coli BL21 (for monochain antibody or Fab antibody expression), or HEK293 or CHO cells (for full-length IgG antibody expression). Transformation of said recombinant expression plasmid into host cells yields the preferred recombinant expression transformant of the present invention.whereby the transformation method employed is a conventional method within the art, preferably chemical transformation, thermal shock transformation or electroporation. Antibody Preparation

[00117] The sequence of the antibody DNA molecules or fragment thereof of the present invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences for the light and heavy chains can be fused to form a single-chain antibody.

[00118] Once the relevant sequence is obtained, it can be produced in large quantities using methods Petition 870250087810, dated 09 / 29 / 2025, page 52 / 101 38 / 71 recombinants. This typically involves cloning it into a vector, transferring it to cells, and then isolating the sequence from the proliferated host cell using conventional methods.

[00119] Furthermore, the relevant sequence, particularly when the fragment length is short, can be synthesized using artificial synthesis methods. Typically, longer sequences are obtained by first synthesizing multiple small fragments and subsequently linking them together.

[00120] Currently, it is entirely feasible to obtain, solely through chemical synthesis, the aforementioned DNA sequences that encode the antibodies (or their fragments or derivatives) of the present invention. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[00121] The present invention also relates to vectors comprising the appropriate DNA sequences mentioned above and suitable promoters or control sequences. These vectors can be used to transform appropriate host cells to enable protein expression.

[00122] Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Preferred animal cells include (but are not limited to): CHO-S, HEK-293 cells. Petition 870250087810, dated 09 / 29 / 2025, page 53 / 101 39 / 71

[00123] Typically, the transformed host cells are cultured under suitable conditions to express the antibody of the present invention. The antibody of the present invention is then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion-exchange chromatography, hydrophobic chromatography, size exclusion chromatography, or affinity chromatography, which are conventional separation and purification methods known to those skilled in the field.

[00124] The resulting monoclonal antibody can be characterized by conventional means. For example, the binding specificity of the monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). The binding affinity of the monoclonal antibody can, for example, be determined by Scatchard analysis, as described by Munson et al., Anal. Biochem., 107: 220 (1980).

[00125] The antibody of the present invention can be expressed within cells, on cell membranes, or secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various methods that utilize its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional repurification procedures, treatment with protein precipitation agents (salting methods), centrifugation, permeation lysis, Petition 870250087810, dated 09 / 29 / 2025, page 54 / 101 40 / 71 Ultrasonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations thereof. Drug-antibody conjugate (ADC)

[00126] The present invention also provides antibody-drug conjugates (ADCs) based on the antibodies of the present invention.

[00127] Typically, said antibody-drug conjugate comprises said antibody and an effector molecule, wherein said antibody is conjugated to said effector molecule, preferably by chemical conjugation. Wherein said effector molecule is preferably a drug with therapeutic activity. Furthermore, said effector molecule may be one or more toxins, a chemotherapeutic agent, a small molecule drug, or a radionuclide.

[00128] The antibody of the present invention can be coupled to said effector molecule by means of a coupling agent. Examples of said coupling agent may be any one or a plurality of non-selective coupling agents, ligands using carboxyl groups, peptide chains, or ligands using disulfide bonds. Said non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde. Said coupling agents using carboxylic groups may comprise any one or a combination of cis-aconitane anhydride type coupling agents. Petition 870250087810, dated 09 / 29 / 2025, p. 55 / 101 41 / 71 (as cis-aconitane anhydride) and acylhydrazone-type coupling agents (where the coupling site is an acylhydrazone).

[00129] Certain residues in the antibody (such as Cys or Lys) are used for binding to various functional groups, including imaging reagents (e.g., chromophores and fluorophores), diagnostic reagents (e.g., contrast agents for magnetic resonance imaging and radioisotopes), stabilizers (e.g., polyethylene glycol polymers), and therapeutics. The antibody can be conjugated to functional agents to form antibody-functional agent conjugates. The functional agents (e.g., drugs, detection reagents, stabilizers) are covalently linked to the antibody. The functional agents can be linked directly to the antibody or indirectly through ligands.

[00130] Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, an ADC comprises a ligand positioned between the drug and the antibody. The ligand may be degradable or non-degradable. Degradable ligands typically degrade readily in intracellular environments, such as at the target site, thereby releasing the drug from the antibody. Suitable degradable ligands include, for example, enzymatically degradable ligands, such as ligands containing peptides degradable by intracellular proteases (e.g., lysosomal or endosomal proteases), or ligands containing glycans, such as ligands containing glucuronides degradable by glucuronidases. Peptide-based ligands may 'comprises', for example, Petition 870250087810, dated 09 / 29 / 2025, page 56 / 101 42 / 71 dipeptides, such as valine-cucurbituril, phenylalanine-lysine, or valine-alanine. Other suitable degradable ligands include, for example, pH-sensitive ligands (e.g., ligands hydrolyzed at pH < 5.5, such as hydrazone ligands) and ligands degraded under reducing conditions (e.g., disulfide ligands). Non-degradable ligands typically release the drug under conditions where the antibody is proteolytically cleaved.

[00131] Prior to binding to the antibody, the ligand possesses reactive functional groups capable of reacting with specific amino acid residues, with coupling being carried out via these reactive functional groups.Specific reactive functional groups for thiol are preferred and include: for example, maleimide derivatives; halogenated amides (e.g., iodinated, brominated or chlorinated); halogenated esters (e.g., iodinated, brominated or chlorinated); halogenated methyl ketones (e.g., iodinated, brominated or chlorinated); benzyl halides (e.g., iodinated, brominated or chlorinated); vinyl sulfoxides, pyridine disulfides; mercury derivatives, such as 3,6-bis(methylmercury)dioxane, wherein the counter-ion is acetate, chloride or nitrate; and polymethylenedimethylsulfide thiosulfate. The ligand may comprise, for example, a maleimide linked to the antibody via a thiobutyridine.

[00132] The drug can be any cytotoxic agent, cell growth inhibitor, or immunosuppressant. In some forms of implementation, the ligand connects the antibody and the drug, where the drug has a functional group capable of forming a bond with the ligand. By Petition 870250087810, dated 09 / 29 / 2025, page 57 / 101 43 / 71 For example, the drug may possess an amino group, carboxyl group, thiol group, hydroxyl group, or ketone group capable of binding to the ligand. When the drug is directly bound to the ligand, the drug possesses a reactive active group prior to its binding to the antibody.

[00133] Useful drug classes include, for example, antimicrotulin drugs, DNA groove-binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemotherapeutic sensitizing agents, topoisomerase inhibitors, and vinca alkaloids. Particularly useful examples of classes of cytotoxic drugs comprising, for example, DNA groove-binding agents, DNA alkylating agents and microtubule inhibitors, typical cytotoxic drugs comprising, for example, auristatins, camptothecins, doxorubicin / duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or drugs containing benzodiazepines (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines) and vinca alkaloids.

[00134] In the present invention, the drug ligand can be used to form an ADC in a single step. In other embodiments, bifunctional ligand compounds can be used to form ADCs through a two- or multi-step method. For example, the cysteine ​​residue reacts with the reactive part of the ligand in the first step and, in a subsequent step, the functional group in the ligand reacts with the drug part to form the ADC. Petition 870250087810, dated 09 / 29 / 2025, page 58 / 101 44 / 71

[00135] Typically, the functional group on the ligand is selected to facilitate the specific reaction with a suitable reactive moiety in the drug moiety. As a non-limiting example, an azide-based moiety can be employed to react specifically with a reactive alkyne group in the drug moiety. The drug is covalently linked to the ligand via a 1,3-dipolar cycloaddition between the azide and alkyne groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reacting with hydrazides and alkoxyamines); phosphines (suitable for reacting with azides); isocyanates and isothiocyanates (suitable for reacting with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reactions with amines and alcohols). These and other binding strategies, such as those described in Bioconjugation Technology, Second Edition (Elsevier), are well known to experts in the field.Experts in the technical field will understand that, for selective reactions between the drug moiety and the ligand, when a complementary pair of reactive functional groups is selected, each member of that complementary pair can be employed for either the ligand or the drug.

[00136] The present invention also provides methods for preparing ADCs, which may further comprise: coupling the antibody to the drug-binding compound under conditions sufficient to form an antibody-drug conjugate (ADC).

[00137] In certain embodiments, the method of the present invention comprises: conjugating the antibody to a bifunctional linker compound under conditions sufficient to Petition 870250087810, dated 09 / 29 / 2025, page 59 / 101 45 / 71 to form an antibody-ligand conjugate. In these embodiments, the method of the present invention further comprises: conjugating the antibody-ligand conjugate to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody through the ligand.

[00138] In some embodiments, the antibody-drug conjugate (ADC) molecule is shown by the following molecular formula: in which: Ab is an antibody, LU is a ligand; D is a drug; and the subscript p is a value selected between 1 and 8. Pharmaceutical Composition

[00139] The present invention further provides a composition. In another example of preferred selection, said composition is a pharmaceutical composition containing the aforementioned antibody or its active fragment or its fusion protein or its ADC or corresponding immune cells, together with a pharmacologically acceptable vector. Typically, these substances can be formulated in a non-toxic, inert, and pharmacologically acceptable aqueous vector, wherein the pH is generally about 5-8, more preferably about 6-8, although the pH may vary depending on the nature of the substances being formulated and the condition being treated.

[00140] The prepared pharmaceutical composition can be administered by conventional routes, it includes, but is not Petition 870250087810, dated 09 / 29 / 2025, pp. 60 / 101 46 / 71 limiting to: intratumoral, intraperitoneal, intravenous or topical administration. Typically, the preferred routes of administration for the pharmaceutical compositions in the present invention are injection or oral administration. The injection administration preferably comprises intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous routes. The pharmaceutical composition may be in various conventional pharmaceutical formulations, preferably in solid, semi-solid or liquid form, and may be aqueous solutions, non-aqueous solutions or suspensions. More preferably, it may be in the form of tablets, capsules, granules, injections or infusions.

[00141] The antibody described in the invention herein can also be used in cell therapy where the nucleotide sequence is expressed intracellularly, such as in the aforementioned antibody immunotherapy with chimeric antigen receptor T cells (CAR-T).

[00142] The pharmaceutical composition of this invention is a pharmaceutical composition for preventing and / or treating diseases associated with abnormal expression or function of BDCA2.

[00143] The pharmaceutical composition of the present invention can be used directly to bind to BDCA2 protein molecules, thus being applicable for preventing and treating diseases such as tumors.

[00144] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99% by weight, preferably 0.01-90% by weight, more preferably 0.1-80% by weight) of the monoclonal antibody. Petition 870250087810, dated 09 / 29 / 2025, pp. 61 / 101 The pharmaceutical composition of the present invention consists of the aforementioned 47 / 71 (or its conjugate) and a pharmacologically acceptable carrier or excipient. Such carriers comprise (but are not limited to): saline solution, buffers, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation must be adapted to the method of administration. The pharmaceutical composition of the present invention can be prepared in injectable form, for example, by conventional methods using physiological saline or aqueous solutions containing glucose and other excipients. Pharmaceutical compositions, such as injections or solutions, must be manufactured under aseptic conditions. The dosage of the active ingredient is therapeutically effective, for example, approximately 1 microgram / kilogram of body weight to approximately 5 milligrams / kilogram of body weight per day. Furthermore, the peptides of the present invention can be used in combination with other therapeutic agents.

[00145] In the present invention, preferably, the pharmaceutical composition of the present invention further comprises one or more pharmaceutical vectors. The pharmaceutical vectors are conventional vectors within the art, and can be any suitable physiologically or pharmacologically acceptable excipient. The pharmaceutical excipients are conventional pharmaceutical excipients in the field, preferably including pharmacologically acceptable diluents, fillers or diluents, etc. More preferably, the pharmaceutical composition comprises 0.01 to 99.99% of the aforementioned protein and 0.01 to 99.99% of the pharmaceutical vector, wherein the aforementioned Petition 870250087810, dated 09 / 29 / 2025, page 62 / 101 48 / 71 percents represent mass percentages relative to the aforementioned pharmaceutical composition.

[00146] In the present invention, the said dosage of the pharmaceutical composition is preferably said effective dosage, which is an amount capable of alleviating or delaying the progression of disease, degenerative or traumatic conditions. Said effective dosage can be determined individually and will be based, in part, on considerations of the symptoms to be treated and the desired outcome. Experts in the field can determine the effective dosage employing the aforementioned factors, such as on an individual basis, and conducting experiments that do not exceed conventional practice.

[00147] When administering the pharmaceutical composition, a safe and effective dose of the conjugate is given to a mammal, wherein said safe and effective dose is typically at least approximately 10 micrograms per kilogram of body weight and, in most cases, does not exceed approximately 50 milligrams per kilogram of body weight. Preferably, the dose is approximately 10 micrograms per kilogram of body weight to approximately 20 milligrams per kilogram of body weight. Naturally, specific dosages should also take into account factors such as the route of administration and the patient's health status, which fall within the scope of the expertise of a qualified professional.

[00148] The present invention provides the application of the above-mentioned pharmaceutical composition in the preparation of a drug to prevent and / or treat diseases associated with the abnormal expression or function of BDCA2. Preferably, the Petition 870250087810, dated 09 / 29 / 2025, pp. 63 / 101 49 / 71 diseases associated with the aforementioned abnormal expression or function of BDCA2 are inflammatory diseases or autoimmune diseases. Applications

[00149] The present invention also provides uses for the antibodies, antibody-drug conjugates (ADCs), recombinant proteins and / or immune cells of the present invention, for example, in the preparation of diagnostic preparations or drugs.

[00150] Preferably, the aforementioned drug is intended for the prevention and / or treatment of diseases associated with the abnormal expression or function of BDCA2.

[00151] In the present invention, the diseases associated with abnormal BDCA2 expression or function are those conventionally recognized in the field as being related to the aforementioned abnormalities. Preferably, the diseases associated with abnormal BDCA2 expression or function are inflammatory or autoimmune diseases, including, but not limited to, systemic lupus erythematosus, cutaneous lupus erythematosus, discoid lupus, lupus nephritis, cutaneous lupus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type 1 diabetes, dermatomyositis and polymyositis, or combinations thereof. The main advantages of the present invention comprise (1) The mouse-derived anti-BDCA2 antibody of the present invention exhibits superior affinity compared with the positive control antibody Litifilimab; the humanized and modified anti-BDCA2 antibody also has enhanced affinity. (2) The BDCA2 antibody of the present invention inhibits Petition 870250087810, dated 09 / 29 / 2025, pp. 64 / 101 50 / 71 effectively stimulates the secretion of IFN-α and IgM by CpG-A stimulated PBMCs, demonstrating potential as a therapeutic agent for autoimmune diseases.

[00152] The present invention is further described in the context of specific implementation examples. It should be understood that these implementation examples are provided to illustrate the present invention and are not intended to limit the scope of the present invention. Experimental methods not specified in detail in the following implementation examples were generally performed under routine conditions, as referred to in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or in accordance with the manufacturer's recommendations. Unless otherwise indicated, percentages and quantities are calculated by weight. Implementation Example 1: Preparation and screening of animals immunized with antigen and hybridomas (i) Preparation of the antigen protein and positive control antibody

[00153] The human BDCA2 extracellular domain sequence (BDCA2-ECD) used as an antigen was obtained from the UniProt database (Entry: Q8WTT0), with the amino acid sequence shown in SEQ ID No.: 1. A 6*His tag was added to its C-terminus and constructed in the pcDNA 3.4 expression vector. HEK-293F cells were transfected and, after 5 days of expression, the cell culture supernatant was collected and purified to obtain the BDCA2-His protein. Similarly, after replacing the aforementioned 6*His tag with the human IgG1 Fc sequence and transfecting HEK-293F cells, the BDCA2-Fc protein was obtained after expression and purification. Petition 870250087810, dated 09 / 29 / 2025, pages 65 / 101 51 / 71

[00154] The positive control antibody Litifilimab (BIIB059,24F4A) was obtained from patent CN111961134A. After synthesis of the genes for the variable region of the heavy chain and the variable region of the light chain, these were recombined with the constant regions of the human IgG1 heavy chain and the kappa light chain, respectively. Each was constructed in the pcDNA 3.4 expression vector, expressed using the HEK-293F system and purified by protein A affinity chromatography. (i) Immunization of mice

[00155] Balb / c mice were routinely immunized with human BDCA2-His protein (produced internally, purity >95%) expressed in the 293F mammalian cell line. On day 1, soluble human BDCA2-His protein was emulsified with Freund's complete adjuvant and administered by multipoint subcutaneous injection to Balb / c mice (100 μg of human BDCA2-His per mouse in 0.5 mL). On day 14, soluble human BDCA2-His protein was emulsified with Freund's incomplete adjuvant and administered by subcutaneous injection to Balb / c mice (50 μg of human BDCA2-His / mouse / 0.5 mL). On day 28, soluble human BDCA2-His protein was emulsified with Freund's incomplete adjuvant and administered by subcutaneous injection to Balb / c mice (50 μg of human BDCA2-His / mouse / 0.5 mL). Three weeks later, soluble human BDCA2-His protein (50 μg / mouse / 0.2 mL) was administered intraperitoneally as a challenge dose.Three to four days after the challenge, the spleens of the mice were harvested for fusion experiments. (i) Preparation and selection of hybridoma cells

[00156] 4 days after final immunization, splenocytes Petition 870250087810, dated 09 / 29 / 2025, pp. 66 / 101 52 / 71 mouse cells were fused with SP2 / 0 mouse myeloma cells via PEG fusion using standard hybridoma protocols. The fused cells were uniformly suspended in complete medium, defined as RPMI 1640-GLUMAX supplemented with 1% penicillin-streptomycin, 20% FBS (fetal bovine serum), and 1x HAT. The fused cells were seeded at 3 χ¹⁰⁴ cells / 200 μL per well in 60 χ¹⁶ well plates. After 712 days, the supernatants were collected, and hybridoma wells exhibiting positive binding activity to human BDCA2 were selected by ELISA.

[00157] The ELISA method for screening hybridoma wells positive for human BDCA2 binding activity is as follows: Dilute BDCA2-Fc in PBS buffer to 1 μg / mL. 100 μL per well are added to the plate and incubated overnight at 4 °C. The next day, the supernatant is discarded. 5% skimmed milk powder is added and the plate is blocked at 37 °C for 1 hour. The plate is washed 3 times with PBST and stored. The hybridoma supernatant collected sequentially from the blocked plate is added at 100 μL per well and incubated at 37 °C for 1 hour. The plate is washed 3 times with PBST, HRP-labeled goat mouse IgG secondary antibody is added, and it is incubated at 37 °C for 30 minutes. After 3 washes with PBST, the residual liquid is dried on absorbent paper. 100 μL of TMB are added to each well and incubated at room temperature (20±5 °C) in the dark for 5 minutes. The substrate reaction is stopped by adding 50 μL of 2M H2SO4 stopping solution to each well.The OD value at 450 nm is read using a microplate reader to analyze the binding capacity of the antibody that binds to BDCA2. Petition 870250087810, dated 09 / 29 / 2025, pp. 67 / 101 53 / 71 Hybridoma cell lines obtained from whole medium containing serum are expanded and selected. The cells are centrifuged and transferred to serum-free Hybridoma-SFM medium at a density of 1-2 χ¹⁰⁶ cells / mL. They are cultured for one week at 37 °C with 8% CO₂. The culture supernatant is centrifuged to collect it. It is purified by protein G affinity chromatography to obtain the anti-human BDCA2 monoclonal antibody protein. Through screening, 30 hybridoma cell lines were obtained. 1.4 Binding capacity of the antibody that binds to human BDCA2-Fc

[00158] The binding capacity of the antibody that binds to human BDCA2-Fc was determined by enzyme-linked immunosorbent assay (ELISA). The specific method is as follows:

[00159] The BDCA2-Fc protein was diluted to 1 μg / mL in PBS buffer, with 100 μL per well added to the plate and incubated overnight at 4 °C. The plate was then blocked with 5% skimmed milk powder and incubated at 37 °C for 1 hour; after washing the plate 3 times with PBST, an 11-fold serial dilution of laboratory-prepared mouse anti-BDCA2 antibody in 1% BSA-PBS buffer was added, starting with 10 μg / mL. 1% BSA-PBS was used as a blank control. 100 μL per well was added to the pre-coated BDCA2-Fc plate and incubated at 37 °C for one hour; the plate was washed 3 times with PBST, the HRP-labeled secondary antibody (goat IgG anti-mouse) was added and incubated at 37 °C for 3 minutes.After washing the plate 3 times with PBST, the residual liquid was dried as much as possible with absorbent paper. 100 μL of TMB was added to each well. Store at room temperature (20±5 °C) and protected from light during storage. Petition 870250087810, dated 09 / 29 / 2025, pp. 68 / 101 After 54 / 71 minutes, the substrate reaction was stopped by adding 50 μL of 2 M H2SO4 stopping solution to each well. The optical density (OD) value at 450 nm was read using a microplate reader to analyze the binding capacity of the antibody that binds to human BDCA2. The data obtained were analyzed using GraphPad Prism 9 software for adjustment. The results are shown in Figures 1A-1B.

[00160] Figures 1A-1B demonstrate that most murine antibodies exhibit strong binding activity to the target antigen BDCA2-Fc, with EC50 values ​​as shown in Tables 1-2. Table 1: EC50 Antibody (ng / mL) EC50 Antibody (ng / mL) 2B7E5 14.69 17G7D3 107.2 2E3F6 18.85 20E10F5 32.65 2D12H3 21.86 21B10A3 22.51 11G11C12 16.86 21E1A3 9.28 27G9G9 24.19 21E5C8 25.41 39E3B2 23.59 26C3F1 28.01 44H3A7 16.44 35E12F1 31.31 52G1A3 28.15 36F2A1 63.8 52H10D11 28.2 39F4D1 15.79 106G8F8 25.34 55G10A9 26.19 24F4A 34.61 Petition 870250087810, dated 09 / 29 / 2025, pp. 69 / 101 55 / 71 Table 2: Antibody EC50 (ng / mL) 16G1H3 21.34 23C10A6B5 20.14 36A6C5E2 138.3 39F6C9 24.55 40H1F3G8 49.29 48A5H1F10 19.54 52E5D3 26.54 136B4H2E3 26.51 144A4G1A1 83.4 146D6A9E5 25.33 24F4A 46.27 1.5 Determination of the binding affinity of murine antibodies that bind to 293FT-BDCA2

[00161] The present implementation example used fluorescence-activated cell screening (FACS) to determine the binding affinity of murine antibodies that bind to 293FT-BDCA2.

[00162] This experiment used 293FT-BDCA2 cells (a genetically modified cell line constructed in the laboratory using lentivirus to express high levels of human BDCA2) as target cells. After counting, the cells were washed once with 1% BSA-PBS. A density of 2 χ¹⁰⁵ cells per well was seeded at 100 μg per well; centrifugation was performed to remove the supernatant. Mouse anti-human BDCA2 antibody was diluted in 1% BSA-PBS from 10 μg / mL in a series of 3-fold serial dilutions to produce 11 gradients. 1% BSA-PBS was used as a blank control. 100 μg were taken from each dilution to resuspend the 293FT-BDCA2 cells in the wells. It was incubated at 4°C for 1 hour. The cells should be washed twice with PBS to remove Petition 870250087810, dated 09 / 29 / 2025, pp. 70 / 101 56 / 71 unbound mouse antibody and then resuspended in 100 μL per well. PE-labeled anti-mouse secondary antibody (purchased from Jackson, catalog number 715116150) should be diluted 1:500 and incubated at 4 °C for 1 hour. Cells were washed twice with PBS to remove unbound secondary antibodies and then resuspended in 200 μL of PBS. The binding affinity of the murine antibody to these cells was determined by flow cytometry. The data obtained were analyzed and fitted using GraphPad Prism 9 software, with the results shown in Figures 2A-2B.

[00163] The results indicate that most murine antibodies bind specifically to BDCA2 on the antibody in 293FT cells, with EC50 values ​​as shown in Tables 3-4. Table 3: Antibody EC50 (ng / mL) Antibody C50(ng / mL) 2B7E5 72.08 17G7D3 5251 2E3F6 306.4 20E10F5 257.3 2D12H3 139.5 21B10A3 124.7 11G11C12 87.09 21E1A3 85.94 27G9G9 198.5 21E5C8 108.2 39E3B2 78.75 26C3F1 78.28 44H3A7 103.1 35E12F1 44.23 52G1A3 114.8 36F2A1 349.7 52H10D11 248.8 39F4D1 135 . 6 106G8F8 1348 55G10A9 92.82 24F4A 103.4 Petition 870250087810, dated 09 / 29 / 2025, pp. 71 / 101 57 / 71 Table 4: Antibody EC50 (ng / mL) 16G1H3 72.7 23C10A6B5 83.53 36A6C5E2 4787 39F6C9 81.91 40H1F3G8 410.1 48A5H1F10 172 . 6 52E5D3 272.3 136B4H2E3 94.27 144A4G1A1 31337 146D6A9E5 17724 24F4A 124.2 Example of implementation 2: Humanization of a murine anti-BDCA2 monoclonal antibody (i) Determination of the sequence of the variable region of the murine anti-BDCA2 monoclonal antibody

[00164] The 23C10A6B5 hybridoma clone was selected as the candidate antibody. Total RNA was extracted from the corresponding hybridoma monoclonal cell line using Trizol (acquired from Life Technologies). The mRNA was reverse transcribed into cDNA using a reverse transcription kit (acquired from Takara). PCR was performed using combined primers reported in the literature (Antibody Engineering, Volume 1, edited by Roland Kontermann and Stefan Dübel, with primer sequences obtained on page 323). The resulting PCR products were sequenced and analyzed using the Kabat database, confirming the obtained sequences as the sequences of the variable region of the murine antibody. The sequence information is as follows:

[00165] The complete gene sequence for the variable region of the heavy chain is 354 bp long, encoding 118 amino acid residues. The nucleotide sequence is shown in No. Petition 870250087810, dated 09 / 29 / 2025, pp. 72 / 101 58 / 71 ID SEQ: 2, and the amino acid sequence is shown in N° ID SEQ: 3;

[00166] The complete gene sequence for the variable region of the light chain is 321 bp long, encoding 107 amino acid residues. The nucleotide sequence is shown in SEQ ID No.: 4, and the amino acid sequence is shown in SEQ ID No.: 5. (i) Construction of the human-murine chimeric antibody and validation of its activity

[00167] The sequences of the variable region of the murine heavy chain and the variable region of the light chain (SEQ ID No.: 3 and SEQ ID No.: 5) obtained in implementation example 2.1 were fused, respectively, to the constant region of the human IgG1 heavy chain and the constant region of the kappa light chain to construct the human-mouse chimeric antibody C-23C10. Its binding activity to the BDCA2 antigen and its inhibitory effect on CpG-A-induced IFN-α secretion by PBMCs were then validated.

[00168] The binding activity assay method was that described in implementation example 1.4, with the following modifications: the coated antigen BDCA2-Fc was replaced by BDCA2-his at a coating concentration of 1 μg / mL, and the secondary antibody was changed to anti-human Fc HRP (SinoBiological, Catalog No. SSA001) at a working dilution of 1:3000.

[00169] The experimental results are shown in Figure 3, demonstrating that C-23C10 exhibits superior binding activity to the BDCA2 antigen compared to the positive control 24F4A.

[00170] CpG-A (CpG ODNs Class A) refers to Petition 870250087810, dated 09 / 29 / 2025, pp. 73 / 101 59 / 71 oligodeoxyribonucleotides containing CpG palindromic sequences, capable of stimulating substantial IFN-α secretion from pDCs. This implementation example used PBMCs to evaluate the inhibitory effect of the candidate antibody on CpG-A-induced IFN-α secretion in PBMCs.

[00171] CpG-A was diluted to 8 μg / mL (final concentration of 2 μg / mL) in RPMI 1640 + 3% FBS medium and added to a 96-well flat-bottom plate at 50 μL per well. The antibody was diluted to 16 nM in RPMI 1640 + 3% FBS, followed by serial 5-fold dilutions (final concentration of 4 nM, 5-fold dilution) and added to the CpG-A wells at 50 μL per well. CpG-A and antibody were co-incubated for 1 hour at 37 °C in a 5% CO2 incubator. PBMC cells were centrifuged and counted, then adjusted to a density of 500,000 cells per well in the aforementioned 96-well plate (100 μL per well). The plates were incubated overnight at 37 °C in an incubator with 5% CO2. The cells were centrifuged and the supernatant was collected for measurement of H-IFNa content.

[00172] The experimental results in Figure 4 demonstrate that C-23C10 effectively inhibits IFNα secretion by CpG-A stimulated PBMCs, exhibiting superior activity to the positive control 24F4. (i) Humanization of the murine anti-BDCA2 monoclonal antibody

[00173] Analysis of amino acid sequences in the variable regions of the heavy and light chains of 23C10A6B5 identified three antigenic complementarity-determining regions (ADRs) and four frames (FRs) according to the Petition 870250087810, dated 09 / 29 / 2025, pp. 74 / 101 60 / 71 Kabat rules. The amino acid sequences of the complementarity-determining regions of the heavy chain are as follows: HCDR1: SYIIH (Seq ID No.: 6) HCDR2: TIYPGNGDTSYNQKFKG (SEQ ID NO: 7) and HCDR3: MGDNEYFDY (Seq ID No.: 8). The amino acid sequences of the complementarity-determining region of the light chain were LCDR1: RASGNIHNYLA (N° ID SEQ: 9), LCDR2: DAETLAD (N° ID SEQ: 10) e LCDR3: QHFWSTPYT (N° ID SEQ: 11).

[00174] By performing homology comparisons in NCBI IgBlast with human IgG germline sequences, IGHV1-46*01 was selected as a heavy chain CDR graft template, and IGKV1-NL1*01 as a light chain CDR graft template. The CDR regions of the 23C10A6B5 antibody were grafted onto the selected humanized templates, replacing the CDR regions of the human templates. The variable region of the heavy chain was then recombined with the human IgG1 constant region, while the variable region of the light chain was recombined with the human kappa constant region. In parallel, based on the three-dimensional structure of this antibody, reverse mutations were introduced in residues that directly interact with the CDR regions and in residues that significantly influence the conformation of the antibody's VL and VH chains. Thus, multiple humanized antibodies were generated.Through affinity screening, multiple humanized antibodies were identified, residues that interact directly with the CDR regions, and those that significantly influence the conformation of the VL and VH of the antibody. Petition 870250087810, dated 09 / 29 / 2025, pp. 75 / 101 61 / 71 antibody. Multiple humanized antibodies were obtained and, by affinity screening, the sequence of the variable region of the heavy chain (SEQ ID No.: 12) and the sequence of the variable region of the light chain (SEQ ID No.: 13) of humanized antibody 230 were determined. Example of implementation 3: Maturation of the affinity of the humanized antibody 230

[00175] Using AlphaFold2 software, the complex structure of antibody 230 and antigen BDCA2-his was predicted, and the interaction interface was analyzed. A mutant library was constructed by introducing single-point saturation mutations at key interaction sites. Through ELISA affinity screening, mutation sites capable of increasing the affinity of antibody 230 were identified. The experimental methods followed the implementation example, with the following modifications: the antigen coating plate used BDCA2-his instead of BDCA2-Fc at a concentration of 0.1 μg / mL (equivalent to 10 ng per well). The secondary antibody used was human anti-Fc HRP (SinoBiological, Catalog No. SSA001) at a working dilution of 1:3000.

[00176] The results are shown in Figure 5. Experimental results indicate that the mutation from I to L in the CDR1 heavy chain: SYIIH (Seq ID No.: 6) produces SYLIH (Seq ID No.: 14). The mutation from N in the CDR2 heavy chain: TIYPGNGDTSYNQKFKG (Seq ID No.: 7) to G, A, S, and R, respectively, produces TIYPGGGDTSYNQKFKG (Seq ID No.: 15). TIYPGAGDTSYNQKFKG (SEQ ID NO: 16) TIYPGSGDTSYNQKFKG (SEQ ID NO: 17) Petition 870250087810, dated 09 / 29 / 2025, pp. 76 / 101 62 / 71 TIYPGRGDTSYNQKFKG (SEQ ID No.: 18) (antibodies 230-I33L, 230-N55G, 230-N55A, 230-N55S, and 230-N55R), respectively, all effectively increasing the affinity of antibody 230.

[00177] Furthermore, the substitution of residue D in the CDR2 light chain: DAETLAD (SEQ ID No.: 10) for I or E resulted in DAETLAI (SEQ ID No.: 19) and DAETLAE (SEQ ID No.: 20). These mutations maintained comparable affinity to 230, showing an average expression increase greater than 30%. The remaining saturation mutant strains exhibited affinity and expression levels comparable to those of the 230 progenitor.

[00178] Selective combinations of the aforementioned CDR mutations were integrated with the IgG1 constant region to construct the antibodies 230IL-DI, 230IL-DE, 230IL-NG-DI, 230IL-NG-DE, 230IL-NR-DI, 230IL-NR-DE, 230NG-DI, 230NG-DE, 230NR-DI and 230NR-DE.Wherein, the heavy chains of antibodies 230IL-DI and 230IL-DE are identical, with the sequence of the variable region of the heavy chain as shown in No. ID SEQ:21; the heavy chains of antibodies 230IL-NG-DI and 230IL-NG-DE are identical, with the sequence of the variable region of the heavy chain as shown in No. ID SEQ:22; the heavy chains of antibodies 230IL-NR-DI and 230IL-NR-DE are identical, with the sequence of the variable region of the heavy chain as shown in No. ID SEQ:23; the heavy chains of antibodies 230NG-DI and 230NG-DE are identical, with the sequence of the variable region of the heavy chain as shown in No. ID SEQ:24; the heavy chains of antibodies 230NR-DI and 230NR-DE are identical; the variable region of the heavy chain is shown in No. ID SEQ:25.

[00179] The light chains of the antibodies 230IL-DI, 230IL-NG-DI, 230IL-NR-DI, 230NG-DI and 230NR-DI are identical, Petition 870250087810, dated 09 / 29 / 2025, pp. 77 / 101 63 / 71 with the variable region sequence of the light chain as shown in SEQ ID No.: 26; the light chains of antibodies 230IL-DE, 230IL-NG-DE, 230IL—NR—DE, 230NG-DE and 230NR-DE are identical, with the variable region sequence of the light chain shown as SEQ ID No.: 27.

[00180] Using the ELISA assay method described above, the affinity of antibodies 230IL-DI, 230IL-DE, 230IL-NG-DI, 230IL-NG-DE, 230IL-NR-DI, 230IL-NR-DE, 230NG-DI, 230NG-DE, 230NR-DI and 230NR-DE for the BDCA2-his antigen protein.

[00181] The results are shown in Figure 6. The experimental results indicate that: the affinity of the 10 mutant strains for the antigen protein is higher than that of the parental monoclonal antibody 230. Example of implementation 4: Determination of the kinetic dissociation constant (KD)

[00182] The FC region of the heavy chain of antibodies 230IL-NG-DE and 230-NG-DE (also referred to as 230NG-DE) of implementation example 3 was designed in accordance with patents CN200880123009.4 and CN200480018985.5, in parallel, introducing the M428L / N434S and G236A / S239D / I332E mutations to construct antibody 237011 (heavy chain sequence as SEQ ID No.: 28, variable region sequence of the light chain as SEQ ID No.: 27) and antibody 2391 (heavy chain sequence as SEQ ID No.: 29, variable region sequence of the light chain as SEQ ID No.: 27).

[00183] Using the Biacore 8K molecular interaction analyzer, the dissociation kinetics of the antibody binding to BDCA2-his was determined by the capture method. Antibodies were diluted to 1 μg / mL in 1x HBS-N buffer (pH 7.0) containing 5 mM CaCl2 and 0.02% Tween 20, and Petition 870250087810, dated 09 / 29 / 2025, pp. 78 / 101 64 / 71 captured using a Protein A chip. The antigen was diluted in 1x HBS-N buffer (pH 7.0) containing 5 mM CaCl2 and 0.02% Tween 20. Six concentration gradients, with a maximum concentration of 12.5 nM, were defined for antibody binding. Dissociation was measured in 1x HBS-N buffer (pH 7.0) containing 5 mM CaCl2 and 0.02% Tween 20.

[00184] The experimental results are shown in Table 5, demonstrating that candidate antibodies 237011 and 2391 exhibit higher affinity for the BDCA2-his antigen than the positive control antibody 24F4A. Table 5: Affinity dissociation constants Mobile phase Stationary phase KD (M) ka (1 / Ms) Kd (1 / s) BDCA2-his 237011 2.59E-11 2.52E+07 6.52E-04 BDCA2-his 2391 3.57E-11 2.44E+07 8.74E-04 BDCA2-his 230 7 . 68E-11 1.70E+07 1.31E-03 BDCA2-his 24F4A 8.70E-11 1.39E+06 1.21E-04 Note: KD denotes affinity constant; ka denotes binding rate constant; kd denotes dissociation rate constant. Implementation Example 5: Inhibition of CpG-A-induced IFNα secretion in PBMCs

[00185] The inhibitory effect of the humanized candidate antibody on CpG-A-induced IFNa secretion in PBMCs was verified using the experimental method referred to in implementation example 2.2.

[00186] The experimental results in Figure 7 demonstrate that the antibodies 237011, 2391, 230-IL-NG—DE and 230-NG—DE effectively inhibited CpG-A stimulated IFN-α secretion in PBMCs, exhibiting significantly superior activity compared to the positive control antibody 24F4A. Petition 870250087810, dated 09 / 29 / 2025, pp. 79 / 101 65 / 71 Implementation Example 6: Inhibition of IgM secretion by PBMCs induced by CpG-A

[00187] To validate the inhibitory effect of humanized candidate antibodies on CpG-A-induced IgM secretion by PBMCs, the experimental method was as follows: PBMC cells were resuspended in RPMI 1640 + 10% FBS medium, adjusted to a cell density of 5 χ 106 cells / mL, and 100 μΣ per well were added to a 3599 plate. 50 μΣ of CpG-A (CpG2216) was added to each well, reaching a final concentration of 2 μg / mL. The antibody was diluted in medium and 50 μΣ was added to each well, reaching a final concentration of 3000 nM. First, 100 μΣ of cells are incubated with 50 μΣ of antibody at 37 °C, 5% CO2 for 1 hour, then 50 μΣ of CPG-A is added and placed in an incubator at 37 °C, 5% CO2. After 7 days of incubation, centrigation is performed to collect the cell supernatant and measure the IgM content in the supernatant.

[00188] The experimental results in Figure 8 demonstrate that the antibodies 237011, 2391, 230-IΣ-NG-DE, and 230-NG-DE effectively inhibited CpG-A-stimulated IgM secretion in PBMCs, exhibiting significantly superior activity compared to the positive control antibody 24F4A. Implementation Example 7: IFNα Inhibition in Mice

[00189] To validate the inhibitory effect of the humanized antibody on IFN-α secretion in mice, and given that the antibody does not cross-react with the homologous BDCA2 protein in mice, in vivo experiments were performed using mice with a reconstituted humanized immune system, established through the injection of human huCD34+ embryonic stem cells (mice Petition 870250087810, dated 09 / 29 / 2025, pages 80 / 101 66 / 71 with HuHSC-NCG humanized immune system, acquired from GemPharmatech Co., Ltd.

[00190] The experimental protocol was as follows: On day 0, test mice were weighed and randomly assigned to 3 groups based on body weight: the model group (PBS-treated group), the 24F4A-treated group, and the 237011-treated group. That afternoon, each group received intraperitoneal injections according to the protocol described in Table 6. 18 hours after administration (Day 1), all 3 groups received an intravenous injection into the caudal vein of CpG-A at a dose of 500 μg / 250 μL per mouse. 24 hours after CpG-A administration, blood was collected from the retro-orbital venous plexus. After coagulation and centrifugation, serum was collected for ELISA detection of human IFNa concentration. Table 6: Specific administration protocol for each group Group | Dose | Route of administration | Frequency / times of administration | Number of animals | Group 1 | CpG-A + PBS | N / A | Intraperitoneal injection | Single dose | 6 | Group 2 | CpG-A + 24F4A 10 mg / kg | Intraperitoneal injection | Single dose | 7 | Group 3 | CpG-A + 237011 10 mg / kg | Intraperitoneal injection | Single dose | 7

[00191] The experimental results shown in Figure 9 indicate that the 237011 administration group significantly suppressed IFNα activity in mice more effectively than the 24F4A administration group. Petition 870250087810, dated 09 / 29 / 2025, pp. 81 / 101 67 / 71

[00192] The sequences of the present invention are as follows: Human BDCA2 extracellular domain (BDCA2-ECD) SEQ ID No.: 1 NFMYSKTVKRLSKLREYQQYHPSLTCVMEGKDIEDWSCCPTPWTSFQSSCYFISTG MQSWTKSQKNCSVMGADLVVINTREEQDFIIQNLKRNSSYFLGLSDPGGRRHWQWV DQTPYNENVTFWHSGEPNNLDERCAIINFRSSEEWGWNDIHCHVPQKSICKMKKIYI

[00193] Nucleotide sequence of the variable region of the heavy chain 23C10A6B5 SEQ ID No.: 2 CAGGTGCAACTACAGCAGCCTGGGGCTGACCTGGTGAAGCCCTGGGGCCTCAGTGAT GATGTCCTGCAAGGCTTCTGGATACACATTTACCAGTTACATTATTCACTGGGTAA AACAGACACCTGGACAGGGCCTGGAATGGATTGGAACTATTTATCCAGGAAATGGTGAT GATACTTCCTACAATCAGAAATTCAAAGGCAAGGCCACATTGACTGCAGACAAATC CTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCT ATTACTGTGCAAGAATGGGGGATAACGAATACTTTGACTACTGGGGCCAAGGAACC ACTCTCCATCTCCATCATTC Heavy chain variable region amino acid sequence 23C10A6B5 SEQ ID No: 3 QVQLQQPGADLVKPGASVMMSCKASGYTFTSYIIHWVKQTPGQGLEWIGTIYPGNG DTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARMGDNEYFDYWGQGT TLTVSS

[00194] Light Chain Variable Region Nucleotide Sequence 23C10A6B5 SEQ ID No.: 4 GACATCCAGATGACTCAGTCTCCAGCCCTCCCTATCTGCATCTGTGGGAGAAACTGT CACCATCACATGTCGAGCAAGTGGAAATATTCAATTAGCATGGTATCAGC AGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATGCAGAAACCTTAGCAGAT GGTGTGCCATCAAGGTCCAGTGGCAGTGGATCCGGAACACATATTCTCTCAATAT CAACAGCCTGCAGCCTGAAGATTTTGGGAGTTTTTACTGTCAACATTTTTGGAGTA CTCCGTACACGTTCGGAGGGGGACCAAGCTGGAAATAAA

[00195] Light Chain Variable Region Amino Acid Sequence 23C10A6B5 SEQ ID No.: 5 DIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYDAETLAD Petition 870250087810, of 29 / 09 / 2025, p. 82 / 101 68 / 71 GVPSRFSGSGSGTQYSLNINSLQPEDFGSFYCQHFWSTPYTFGGGTKLEIK HCDR1 of 23C10A6B5: SYIIH (SEQ ID No: 6) HCDR2 of 23C10A6B5: TIYPGNGDTSYNQKFCG (SEQ ID NO: 7) HCDR3 of 23C10A6B5: MGDNEYFDY (SEQ ID No: 8) LCDR1 of 23C10A6B5: RASGNIHNYLA (No. ID SEQ: 9) LCDR2 of 23C10A6B5: DAETLAD (SEQ ID No: 10) LCDR3 of 23C10A6B5: QHFWSTPYT (SEQ ID No: 11) Variable region of the humanized antibody heavy chain 230 Sequence ID No.: 12 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYIIHWVRQAPGQGLEWMGTIYPGNG DTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARMGDNEYFDYWGQGT LVTVSS

[00196] Variable region of the humanized antibody light chain 230 SEQ ID No.: 13 DIQMTQSPSSLSASVGDRVTITCRAGNIHNYLAWYQQKPGKAPKLLLYDAETLAD GVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWSTPYTFGGGTKVEIK 230 Antibody-IL Heavy chain CDR1 No. ID SEQ: 14 SYLIH 230 Antibody-NG Heavy chain CDR2 No. ID SEQ: 15 TIYPGGGDTSYNQKFKG 230 antibody-NA Heavy chain CDR2 No. ID SEQ: 16 TIYPGAGDTSYNQKFKG 230 antibody-NS Heavy chain CDR2 No. ID SEQ: 17 TIYPGSGDTSYNQKFKG 230 Antibody-NR Heavy chain CDR2 ID SEQ: 18 TIYPGRGDTSYNQKFKG 230 Antibody-DI Light chain CDR2 N° ID SEQ: 19 DAETLAI 230 Antibody-DE Light chain CDR2 N° ID SEQ: 20 DAETLAE

[00197] Variable region of the heavy chain 230IL-DI No. ID SEQ: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYLIHWVRQAPGQGLEWMGTIYPGNG Petition 870250087810, dated 09 / 29 / 2025, pp. 83 / 101 69 / 71 DTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARMGDNEYFDYWGQGT LVTVSS

[00198] Variable region of the heavy chain 230IL-NG-DI SEQ ID No.: 22 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYLIHWVRQAPGQGLEWMGTIYPGGG DTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARMGDNEYFDYWGQGT LVTVSS

[00199] Variable region of the heavy chain 230IL-NR-DI No. ID SEQ: 23 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYLIHWVRQAPGQGLEWMGTIYPGRG DTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARMGDNEYFDYWGQGT LVTVSS

[00200] Variable region of the heavy chain 230NG-DI No. ID SEQ: 24 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYIIHWVRQAPGQGLEWMGTIYPGGG DTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARMGDNEYFDYWGQGT LVTVSS

[00201] Variable region of the heavy chain 230NR-DI No. ID SEQ: 25 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYIIHWVRQAPGQGLEWMGTIYPGRG DTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARMGDNEYFDYWGQGT LVTVSS

[00202] Variable region of the 230IL-DI light chain No. ID SEQ: 26 DIQMTQSPSSLSASVGDRVTITCRASGNIHNYLAWYQQKPGKAPKLLLYDAETLAI GVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWSTPYTFGGGTKVEIK Variable region of the light chain 230IL-DE No. ID SEQ: 27 DIQMTQSPSSLSASVGDRVTITCRAGNIHNYLAWYQQKPGKAPKLLLYDAETLAE GVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWSTPYTFGGGTKVEIK

[00203] Antibody heavy chain 237011 SEQ ID No: 28 Petition 870250087810, dated 09 / 29 / 2025, pp. 84 / 101 70 / 71 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYLIHWVRQAPGQGLEWMGTIYPGGG DTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARMGDNEYFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK

[00204] Heavy chain of antibody 2391 SEQ ID NO: 29 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYIIHWVRQAPGQGLEWMGTIYPGGG DTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYCARMGDNEYFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLAGPDVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLPGK HCDR1 antibody against BDCA2 SYX1IH, where X1 is I or L (Seq ID No.: 30) HCDR2 of the anti-BDCA2 antibody TIYPGX2GDTSYNQKFKG, where X2 is N or G or A or S or R (Seq ID No.: 31) HCDR2 of the anti-BDCA2 antibody DAETLAX3, where X3 is I or E (Seq ID No.: 32)

[00205] All literature cited in this invention is incorporated by reference into the present application, as if each were cited individually as a reference. Furthermore, it should be understood that, after reading the preceding description of this application, those skilled in the art may make various modifications or alterations to the present invention, and such equivalent forms are equally encompassed in this application. Petition 870250087810, dated 09 / 29 / 2025, pp. 85 / 101 71 / 71 scope defined by the attached claims. Petition 870250087810, dated 09 / 29 / 2025, pp. 86 / 101

Claims

1 / 6 CLAIMS 1. Antibody or antigen-binding fragment thereof for binding to BDCA2, characterized in that it comprises a variable heavy chain region and a variable light chain region, wherein said variable heavy chain region comprises H-CDR1, H-CDR2 and H-CDR3 heavy chain complementarity-determining regions, wherein the H-CDR1 amino acid sequence is shown in SYX1IH (SEQ ID No: 30), the H-CDR2 amino acid sequence is shown in TIYPGX2GDTSYNQKFKG (SEQ ID No: 31), the H-CDR3 amino acid sequence is shown in MGDNEYFDY (SEQ ID No: 8), and said variable light chain region comprises L-CDR1, L-CDR2, L-CDR3 light chain complementarity-determining regions; wherein, the amino acid sequence of L-CDR1 is shown in RASGNIHNYLA (Seq ID No: 9), the amino acid sequence of L-CDR2 is shown in DAETLAX3 (Seq ID No: 32), the amino acid sequence of L-CDR3 is shown in QHFWSTPYT (Seq ID No: 11);where X1 is I or L; X2 is selected from the group consisting of the amino acids: N, G, A, S, R; and / or X3 is selected from the group consisting of the amino acids: D, I, or E.

2. Antibody or antigen-binding fragment thereof, according to claim 1, characterized in that X1, X2, X3 are selected from the group: X1 is I, X2 is N, X3 is D; Petition 870250087810, dated 09 / 29 / 2025, page 87 / 101 2 / 6 X1 is L, X2 is N, X3 is I; X1 is L, X2 is N, X3 is E; X1 is L, X2 is G, X3 is I; X1 is L, X2 is G, X3 is E; X1 is L, X2 is R, X3 is I; X1 is L, X2 is R, X3 is E; X1 is I, X2 is G, X3 is I; X1 is I, X2 is G, X3 is E; X1 is I, X2 is R, X3 is I; or X1 is I, X2 is R, X3 is E.

3. Antibody or antigen-binding fragment thereof, according to claim 1, characterized in that said variable region of the heavy chain comprises the heavy chain complementarity-determining regions H-CDR1, H-CDR2, H-CDR3, wherein the amino acid sequence of H-CDR1 is shown in SYIIH (Seq ID No: 6), the amino acid sequence of H-CDR2 is shown in TIYPGXTIYPGNGDTSYNQKFKG (Seq ID No: 7), the amino acid sequence of H-CDR3 is shown in MGDNEYFDY (Seq ID No: 8), and said variable region of the light chain comprises light chain complementarity-determining regions L-CDR1, L-CDR2, L-CDR3; wherein, the amino acid sequence of L-CDR1 is shown in RASGNIHNYLA (Seq ID No.: 9), the amino acid sequence of L-CDR2 is shown in DAETLAD (Seq ID No.: 10), the amino acid sequence of L-CDR3 is shown in QHFWSTPYT (Seq ID No.: 11); Petition 870250087810, dated 09 / 29 / 2025, p.88 / 101 3 / 6 where, in the amino acid sequences above, any of the amino acid sequences may also comprise a derived sequence that optionally contains the addition, omission, modification and / or substitution of at least one amino acid, while maintaining the binding affinity to BDCA2.

4. Antibody that binds to BDCA2 or its antigen-binding fragment, according to claim 1, characterized in that the amino acid sequence of the variable region of the heavy chain of said antibody that binds to BDCA2 or its antigen-binding fragment is shown in SEQ ID No. 3, while the amino acid sequence of the variable region of the light chain is shown in SEQ ID No. 5; or the amino acid sequence of the variable region of the heavy chain of said antibody that binds to BDCA2 or its antigen-binding fragment is shown as SEQ ID No. 12, while the amino acid sequence of the variable region of the light chain is shown in SEQ ID No. 13; or the amino acid sequence of the variable region of the heavy chain of said antibody that binds to BDCA2 or the antigen-binding fragment thereof is selected as No. ID SEQ: 21, 22, 23, 24 or 25, while the amino acid sequence of the variable region of the light chain is selected from No. ID SEQ: 26 or 27; 5. An antibody or antigen-binding fragment thereof, according to claim 1, characterized in that the antibody is selected from the group of: antibodies of animal origin, chimeric antibodies, humanized antibodies, fully human antibodies, or combinations thereof.

6. A recombinant protein, characterized in that said recombinant protein comprises: Petition 870250087810, dated 29 / 09 / 2025, p. 89 / 101 4 / 6 (i) Said antibody that binds to BDCA2 or an antigen-binding fragment thereof according to any of claims 1 to 5; and (ii) optionally, a labeling sequence to facilitate expression and / or purification.

7. Nucleotide molecule, characterized in that said nucleotide molecule encodes said antibody that binds to BDCA2 or an antigen-binding fragment thereof according to any of claims 1 to 5.

8. Expression vector, characterized in that said expression vector contains said nucleotide molecules as per claim 7.

9. Host cell, characterized in that said host cell contains said expression vector as per claim 8.

10. Method for preparing said antibody that binds to BDCA2 or its antigen-binding fragment, according to any one of claims 1 to 5, characterized in that the method comprises the following steps: a) culturing said host cell as per claim 9, under expression conditions to express said antibody that binds to BDCA2 or its antigen-binding fragment; b) separating and purifying said antibody that binds to BDCA2 or its antigen-binding fragment.

11. Composition, characterized in that the composition contains said antibody that binds to BDCA2 or an antigen-binding fragment thereof according to any of claims 1 to 5, and a pharmacologically acceptable vector. Petition 870250087810, dated 29 / 09 / 2025, pp. 90 / 101 5 / 6 12. Application of said antibody that binds to BDCA2 or antigen-binding fragment thereof, according to any one of claims 1 to 5, or of said composition, according to claim 11, in the preparation of a medicament for the treatment of inflammatory or autoimmune diseases.

13. Application of said antibody, according to claim 12, characterized in that the inflammatory or autoimmune diseases are selected from the following group: systemic lupus erythematosus, cutaneous lupus erythematosus, discoid lupus, lupus nephritis, cutaneous lupus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type 1 diabetes, dermatomyositis and polymyositis.

14. Antibody-drug conjugate, characterized in that the antibody-drug conjugate contains: (a) an antibody moiety, said antibody moiety comprising an antibody or the antigen-binding fragment thereof according to any of claims 1 to 5; and (b) a coupling moiety conjugated to said antibody moiety, said coupling moiety selected from the group: a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme or a combination thereof.

15. CAR construct, characterized in that the scFv fragment of the antigen-binding region of the monoclonal antibody of said CAR construct is a binding domain that specifically binds to BDCA2, and in which the variable region of the heavy chain of said scFv comprises: heavy chain complementarity-determining regions. Petition 870250087810, dated 09 / 29 / 2025, p.91 / 101 6 / 6 H-CDR1, H-CDR2 and H-CDR3, wherein the amino acid sequence of H-CDR1 is shown in SYIIH (Seq ID No.: 6), the amino acid sequence of H-CDR2 is shown in TIYPGNGDTSYNQKFKG (Seq ID No.: 7), the amino acid sequence of H-CDR3 is shown in MGDNEYFDY (Seq ID No.: 8), and the variable region of the light chain of said scFv comprises: complementarity-determining regions of the light chain L-CDR1, L-CDR2 and L-CDR3, wherein the said amino acid sequence of L-CDR1 is shown in RASGNIHNYLA (Seq ID No.: 9), the amino acid sequence of L-CDR2 is shown in DAETLAD (Seq ID No.: 10), and the amino acid sequence of L-CDR3 is shown in QHFWSTPYT (Seq ID No.: 11). ID SEQ: 11); wherein, in the amino acid sequences above, any of the amino acid sequences may also comprise a derived sequence that optionally contains the addition, omission, modification and / or substitution of at least one amino acid, while maintaining the binding affinity to BDCA2.Petition 870250087810, dated 09 / 29 / 2025, pp. 92-101.

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