Anti-BDCA2 antibody

Abstract

We provide an anti-BDCA2 antibody. This anti-BDCA2 antibody can bind to the BDCA2 antigen with high specificity and affinity, and can effectively inhibit CpG-A from stimulating PBMCs to secrete IFNα and IgM, and is expected to be used as a therapeutic agent for autoimmune diseases.

Description

Technical Field

[0001] The present invention relates to the field of antibodies. Specifically, it relates to anti-BDCA2 antibodies.

Background Art

[0002] Type I interferon (IFN-I) is an inflammatory mediator mainly produced by plasmacytoid dendritic cells (pDC) and is a component of the innate immune system. The type I IFN gene is strictly regulated. Usually, in healthy individuals, the production of IFN-α is hardly detected. However, in patients suffering from many systemic autoimmune diseases, persistent type I IFN production and increased expression of IFN-α regulatory genes are observed. IFN-α activates both the innate and adaptive immune systems. When an individual has a genetic predisposition to enhanced IFN-α production and prominent IFN-α response, tolerance is disrupted and antibodies against self-antigens are produced. These antibodies interact with self-antigens to form ICs, stimulating the synthesis of IFN-α by pDC. Then, they stimulate B cells, promote production, and cause a vicious cycle of persistent IFN-α production and autoimmune reactions.

[0003] Blood dendritic cell antigen 2 (BDCA2), also called CLEC4C or CD303, is specifically expressed only in pDC. When BDCA2 is activated, it activates the SYK protein, leading to the activation of a complex composed of BLNK, BTK, and PLCγ2, mobilizes intracellular Ca 2+ and inhibits the release of inflammatory cytokines such as IFN-I and IL-6 mediated by the TLR7 and TLR9 signaling pathways.

[0004] BDCA2-targeted drugs begin by intervening in the function of pDC cells, rapidly internalizing BDCA2 from the pDC surface and indirectly inhibiting the release of IFN-I, inflammatory cytokines, and other upstream stimuli. Furthermore, since BDCA2 signaling is not affected by IFN-I or other inducing stimuli, BDCA2-targeted therapies offer a new treatment option for autoimmune diseases. However, conventional BDCA2-targeted drugs still have many shortcomings, and the development of new anti-BDCA2 antibodies is needed in this field. [Overview of the Initiative]

[0005] The objective of this invention is to provide an anti-BDCA2 antibody. In a first aspect of the present invention, an antibody or antigen-binding fragment that binds to BDCA2 is provided, characterized by comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain complementarity determining regions H-CDR1, H-CDR2, and H-CDR3, where, The amino acid sequence of H-CDR1 is shown in SYX1IH (SEQ ID NO:30). The amino acid sequence of H-CDR2 is shown as TIYPGX2GDTSYNQKFKG (SEQ ID NO:31). The amino acid sequence of H-CDR3 is shown in MGDNEYFDY (SEQ ID NO: 8), and The aforementioned light chain variable region includes light chain complementarity determination regions L-CDR1, L-CDR2, and L-CDR3, where, 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). Here, X1 is I or L, X2 is selected from the group of amino acids consisting of N, G, A, S, and R, and / or X3 is selected from the group of amino acids consisting of D, I, or E.

[0006] In another preferred embodiment, X1, X2, X3 are X1 is I, X2 is N, and X3 is D. X1 is L, X2 is N, and X3 is I. X1 is L, X2 is N, and X3 is E. X1 is L, X2 is G, and X3 is I. X1 is L, X2 is G, and X3 is E. X1 is L, X2 is R, and X3 is I. X1 is L, X2 is R, and X3 is E. X1 is I, X2 is G, X3 is I. X1 is I, X2 is G, and X3 is E. X1 is I, X2 is R, X3 is I, or X1 is I, X2 is R, and X3 is E. To be selected from a group.

[0007] In another preferred embodiment, the heavy chain variable region includes heavy chain complementarity determination regions H-CDR1, H-CDR2, and H-CDR3, where, 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 aforementioned light chain variable region includes light chain complementarity determination regions L-CDR1, L-CDR2, and L-CDR3, where, 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). Here, any of the amino acid sequences in the above amino acid sequence includes a derivative sequence in which at least one arbitrarily selected amino acid is added, deleted, modified, and / or substituted, thereby maintaining the binding affinity of BDCA2.

[0008] In another preferred embodiment, the ratio (F1 / F0) of the affinity F1 of the derivative antibody to BDCA2 to the affinity F0 of the corresponding non-derivative antibody to BDCA2 is 0.5 to 4, preferably 0.7 to 1.5, and more preferably 0.8 to 1.2.

[0009] In another preferred embodiment, the number of amino acids added, deleted, modified and / or substituted is 1 to 5 (for example, 1 to 3, preferably 1 to 2, more preferably 1).

[0010] In another preferred embodiment, the derivative sequence comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain complementarity determining region having the following amino acid sequence: The amino acid sequence is shown as SYX1IH for H-CDR1, where X1 is I or L and / or H-CDR2 whose amino acid sequence is shown as TIYPGX2GDTSYNQKFKG, where X2 is selected from the group of amino acids consisting of N, G, A, S, R (preferably G), and / or The light chain variable region includes a light chain complementarity determination region having the following amino acid sequence: The amino acid sequence of DAETLAX3 is L-CDR2, where X3 is selected from the group of amino acids consisting of D, I, or E. Here, X1, X2, and X3 are independent of each other and are not simultaneously I, N, or D.

[0011] In another preferred embodiment, in the derivative sequence, X1, X2, and X3 are X1 is L, X2 is N, and X3 is I. X1 is L, X2 is N, and 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, is selected from the group.

[0012] In another preferred embodiment, in the derivative sequence, the heavy chain variable region is the H-CDR1 with the amino acid sequence shown as SYLIH (SEQ ID NO:14), the H-CDR2 with the amino acid sequence shown as TIYPGGGDTSYNQKFKG (SEQ ID NO:15), the H-CDR3 with the amino acid sequence shown as MGDNEYFDY (SEQ ID NO:8), and the light chain variable region is the L-CDR1 with the amino acid sequence shown as RASGNIHNYLA (SEQ ID NO:9), the L-CDR2 with the amino acid sequence shown as DAETLAE (SEQ ID NO:20), the L-CDR3 with the amino acid sequence shown as QHFWSTPYT (SEQ ID NO:11).

[0013] In another preferred embodiment, in the derivative sequence, the heavy chain variable region is the H-CDR1 with the amino acid sequence shown as SYIIH (SEQ ID NO:6), the H-CDR2 with the amino acid sequence shown as TIYPGGGDTSYNQKFKG (SEQ ID NO:15), The amino acid sequence includes H-CDR3 as shown in MGDNEYFDY (SEQ ID NO: 8), and The aforementioned light chain variable region is L-CDR1, whose amino acid sequence is RASGNIHNYLA (SEQ ID NO:9), The amino acid sequence of L-CDR2 shown in DAETLAE (SEQ ID NO:20) and It contains L-CDR3, whose amino acid sequence is QHFWSTPYT (SEQ ID NO: 11).

[0014] In another preferred embodiment, in the derivative sequence, the heavy chain variable region is The amino acid sequence of H-CDR1 shown in SYLIH (SEQ ID NO:14) and H-CDR2, whose amino acid sequence is TIYPGNGDTSYNQKFKG (SEQ ID NO:7), The amino acid sequence includes H-CDR3 as shown in MGDNEYFDY (SEQ ID NO: 8), and The aforementioned light chain variable region is L-CDR1, whose amino acid sequence is RASGNIHNYLA (SEQ ID NO:9), The amino acid sequence of L-CDR2 shown in DAETLAE (SEQ ID NO:20) and It contains L-CDR3, whose amino acid sequence is QHFWSTPYT (SEQ ID NO: 11).

[0015] In another preferred embodiment, the antibody is a mouse antibody, a chimeric antibody, or a humanized antibody.

[0016] In another preferred embodiment, the heavy chain variable region of the antibody further comprises a human-derived framework region, and / or the light chain variable region of the antibody further comprises a human-derived framework region.

[0017] In another preferred embodiment, the heavy chain variable region of the antibody further comprises a mouse-derived framework region, and / or the light chain variable region of the antibody further comprises a mouse-derived framework region.

[0018] In another preferred embodiment, the heavy chain steady region is of human origin, and / or the light chain steady region is of human origin.

[0019] In another preferred embodiment, the heavy chain constant region is the human antibody heavy chain IgG1 constant region, and the light chain constant region is the human antibody light chain kappa constant region.

[0020] In another preferred embodiment, the heavy chain steady region includes an Fc segment.

[0021] In another preferred embodiment, the Fc segment is an Fc variant, and the modified Fc variants include the following mutations: M428L / N434S, G236A / S239D / I332E, S239D / I332E / A330L, S239D / I332E / G236A, S298A, A330L, I332E, E333A, and / or K334A. Here, the residue numbers in the Fc mutant correspond to the EU index numbers in Kabat.

[0022] In another preferred embodiment, the antibody is selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, or combinations thereof.

[0023] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0024] In another preferred embodiment, the antibody is either a full-length antibody protein or an antigen-binding fragment.

[0025] In another preferred embodiment, the antibody is a bispecific antibody or a multispecific antibody.

[0026] In another preferred embodiment, the antibody is in the form of a drug conjugate.

[0027] In another preferred embodiment, the affinity between the antibody and the antigen BDCA2 is 1E-9M or less, preferably 1E-10M or less, and more preferably 1E-11M or less.

[0028] In another preferred embodiment, the BDCA2 is human BDCA2, preferably the extracellular domain of human BDCA2.

[0029] In another preferred embodiment, the sequence of the human BDCA2 extracellular domain is shown as SEQ ID NO:1.

[0030] In another preferred embodiment, the antigen-binding fragment comprises a Fab fragment, an F(ab')2 fragment, an Fv fragment, and an scFv.

[0031] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment that binds to BDCA2 is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:5.

[0032] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment that binds to BDCA2 is shown in SEQ ID NO:12, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:13.

[0033] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment that binds to BDCA2 is selected from SEQ ID NO: 21, 22, 23, 24, or 25, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 26, or 27.

[0034] In another preferred embodiment, the amino acid sequence of the heavy chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with the amino acid sequence shown in SEQ ID NO. 3, 12, 21, 22, 23, 24, or 25.

[0035] In another preferred embodiment, the amino acid sequence of the light chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with the amino acid sequence shown in SEQ ID NO. 5, 13, 26, or 27.

[0036] In a second aspect of the present invention, (i) an antibody or antigen-binding fragment that binds to BDCA2 according to the first aspect of the present invention, (ii) A recombinant protein comprising an optionally selected tag sequence that supports expression and / or purification.

[0037] In another preferred embodiment, the tag array includes 6His tags.

[0038] In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.

[0039] In another preferred embodiment, the recombinant protein is a monomer, dimer, or polymer.

[0040] A third aspect of the present invention provides a nucleotide molecule characterized by encoding an antibody that binds to BDCA2 as described in the first aspect of the present invention or an antigen-binding fragment thereof.

[0041] In another preferred embodiment, the nucleotide sequence encoding the heavy chain variable region of the nucleotide molecule is shown in SEQ ID NO:2, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO:4.

[0042] A fourth aspect of the present invention provides an expression vector characterized by containing the nucleotide molecule described in the third aspect of the present invention.

[0043] A fifth aspect of the present invention provides a host cell characterized by containing the expression vector described in the fourth aspect of the present invention.

[0044] In a sixth aspect of the present invention, a) A step of culturing host cells according to the fifth aspect of the present invention under expression conditions to express an antibody that binds to BDCA2 or an antigen-binding fragment thereof, The present invention provides a method for preparing an antibody or antigen-binding fragment that binds to BDCA2 according to a first aspect of the present invention, comprising the steps of: b) isolating and purifying the antibody or antigen-binding fragment that binds to BDCA2 according to a);

[0045] A seventh aspect of the present invention provides a composition comprising an antibody or antigen-binding fragment thereof that binds to BDCA2 as described in the first aspect of the present invention, and a pharmaceutically acceptable vector.

[0046] An eighth aspect of the present invention provides the use of an antibody or antigen-binding fragment that binds to BDCA2 as described in the first aspect of the present invention, or a composition as described in the seventh aspect of the present invention, in the preparation of a drug for treating an inflammatory disease or an autoimmune disease. In another preferred embodiment, the inflammatory or autoimmune disease is selected from systemic lupus erythematosus, cutaneous lupus erythematosus, discoid lupus erythematosus, lupus nephritis, cutaneous lupus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type 1 diabetes mellitus, dermatomyositis, and polymyositis.

[0047] In the ninth aspect of the present invention, the scFv segment of the monoclonal antibody antigen-binding region is a binding region that specifically binds to BDCA2, and the heavy chain variable region of the scFv is 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), and the amino acid sequence of H-CDR3 is shown in MGDNEYFDY (SEQ ID NO: 8), and it contains the heavy chain complementarity determining regions H-CDR1, H-CDR2, and H-CDR3, and The light chain variable region of the scFv is, The present invention provides a CAR construct comprising light chain complementarity determining regions L-CDR1, L-CDR2, and L-CDR3, wherein any of the amino acid sequences in the above amino acid sequences are shown in RASGNIHNYLA (SEQ ID NO: 9), L-CDR2 is shown in DAETLAD (SEQ ID NO: 10), and L-CDR3 is shown in QHFWSTPYT (SEQ ID NO: 11), and wherein any of the above amino acid sequences include a derivative sequence that can maintain the binding affinity of BDCA2 by adding, deleting, modifying and / or substituting at least one arbitrarily selected amino acid.

[0048] In another preferred embodiment, the derivative sequence comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain complementarity determining region having the following amino acid sequence: The amino acid sequence is H-CDR1 shown in SYX1IH, where X1 is I or L, and / or H-CDR2 whose amino acid sequence is shown as TIYPGX2GDTSYNQKFKG, where X2 is selected from the group of amino acids consisting of N, G, A, S, R (preferably G), and / or The light chain variable region includes a light chain complementarity determination region having the following amino acid sequence: The amino acid sequence of DAETLAX3 is L-CDR2, where X3 is selected from the group of amino acids consisting of D, I, or E. Here, X1, X2, and X3 are independent of each other and are not simultaneously I, N, or D.

[0049] A tenth aspect of the present invention provides recombinant immune cells characterized by expressing the exogenous CAR construct described in the ninth aspect of the present invention.

[0050] In the 11th aspect of the present invention, (a) an antibody moiety comprising an antibody that binds to BDCA2 according to the first aspect of the present invention or an antigen-binding fragment thereof, (b) The present invention provides an antibody-drug conjugate comprising a binding site selected from the group consisting of a measurable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0051] In a twelfth aspect of the present invention, (1) The step of contacting the sample in vitro with the antibody or antigen-binding fragment described in the first aspect of the present invention, or the antibody-drug conjugate described in the eleventh aspect of the present invention, (2) A method for in vitro testing of BDCA2 protein in a sample is provided, characterized by comprising the step of measuring whether an antigen-antibody complex is formed, and the formation of the complex indicates the presence of BDCA2 protein in the sample.

[0052] A thirteenth aspect of the present invention provides a method for preventing and / or treating BDCA2-related diseases, comprising administering to a subject requiring assistance an antibody conjugated to BDCA2 as described in the first aspect of the present invention or its antigen-binding fragment, a composition as described in the seventh aspect of the present invention, recombinant immune cells as described in the tenth aspect of the present invention, or an antibody-drug conjugate as described in the eleventh aspect of the present invention, or a combination thereof.

[0053] In another preferred embodiment, the BDCA2-related disease is selected from inflammatory or autoimmune diseases, including but not limited to systemic lupus erythematosus, cutaneous lupus erythematosus, discoid lupus erythematosus, lupus nephritis, cutaneous lupus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type 1 diabetes, dermatomyositis, and polymyositis or a combination thereof.

[0054] It should be understood that each of the above technical features of the present invention and each of the technical features specifically described below (for example, in the examples) can be combined with each other within the scope of the present invention to constitute novel or preferred technical solutions. Due to space limitations, each will not be explained in detail here. [Brief explanation of the drawing]

[0055] [Figure 1A] Figure 1A shows the binding ability of mouse antibodies to the human BDCA2-Fc protein. [Figure 1B] Figure 1B shows the binding ability of mouse antibodies to the human BDCA2-Fc protein. [Figure 2A] Figure 2A shows the measurement of the binding affinity of the mouse antibody to the stable expression cell line 293FT-BDCA2. [Figure 2B] Figure 2B shows the measurement of the binding affinity of the mouse antibody to the stable expression cell line 293FT-BDCA2. [Figure 3] Figure 3 shows the binding ability of human-mouse chimeric antibodies to the human BDCA2-his protein. [Figure 4] Figure 4 shows the inhibitory effect of human-mouse chimeric antibodies on IFNa secretion by CpG-A-induced PBMCs. [Figure 5] Figure 5 shows the results of ELISA screening to determine the effect of different mutation sites on the affinity of 230 antibodies. [Figure 6] Figure 6 shows the results of ELISA analysis of the affinity of anti-BDCA2 antibody mutants for the BDCA2-his antigen protein. [Figure 7] Figure 7 shows the results of analyzing the inhibitory effect of candidate antibodies on IFNa secretion in CpG-A-induced PBMCs using PBMCs. [Figure 8] Figure 8 shows the inhibitory effect of candidate antibodies on IFNa secretion by CpG-A-induced PBMCs. [Figure 9] Figure 9 shows the inhibitory effect of candidate antibodies on IFNa secretion in mice. [Modes for carrying out the invention]

[0056] As a result of extensive and thorough research and screening, the inventors unexpectedly discovered a series of mouse antibodies that exhibit excellent binding activity to the target antigen BDCA2-Fc. Further selection of candidate antibodies, humanization, and CDR modification of the antibodies resulted in humanized antibodies that showed affinity and expression levels equivalent to or higher than the parent antibodies. The antibodies of the present invention effectively inhibit IFNa and IgM secretion from PBMCs induced by CpG-A stimulation, and their activity is significantly superior to that of the positive control antibody litifirimab. Based on this, the present invention was completed.

[0057] term Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this invention pertains.

[0058] The term "approximately" refers to a value or composition within a tolerance range for a particular value or composition as determined by those skilled in the art, which may depend on the measurement method, measurement value, or composition.

[0059] In this specification, the terms “contain” or “include” may be open, semi-closed, or closed. In other words, the terms also include the meaning of “essentially consisting of” or “consisting of.”

[0060] antibody In this specification, the terms “antibody” or “immunoglobulin” refer to a heterotetrameric glycoprotein of approximately 150,000 daltons having identical structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is attached to a heavy chain by one covalent disulfide bond, with different disulfide numbers between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has intrachain disulfide bonds at regular intervals. Each heavy chain has a variable region (VH) at one end, followed by several constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other, with the constant region of the light chain corresponding to the first constant region of the heavy chain, and the variable region of the light chain corresponding to the variable region of the heavy chain. Certain amino acid residues form an interface between the light chain variable region and the heavy chain variable region.

[0061] In this specification, the term "variable" refers to a difference in the sequence of a portion of the variable region of an antibody, which in turn determines the binding affinity and specificity of different antibodies to specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three fragments of the light and heavy chain variable regions, called complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portion of the variable region is called the framework region (FR). The four FR regions in the native heavy and light chain variable regions basically exhibit a β-sheet structure connected by three CDRs that form a connecting loop, and can sometimes form a partial β-sheet structure. The CDRs of each chain are held in close proximity to each other via the FR region and, together with the CDR 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 region does not directly participate in the binding of the antibody to the antigen, but exhibits various effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0062] The "light chains" of vertebrate antibodies (immunoglobulins) can be classified into two distinct classes (called κ and λ) based on the amino acid sequence of their constant region. Immunoglobulins are further classified into different classes based on the amino acid sequence of their heavy chain constant region. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which are further classified into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional arrangements of the different classes of immunoglobulins are well known to those skilled in the art.

[0063] Generally, the antigen-binding properties of an antibody are described by three specific regions located within the variable regions of the heavy and light chains, namely the variable regions (CDRs), which are further divided into four framework regions (FRs). The amino acid sequences of these four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a loop structure and are spatially aligned by β-sheets formed between the FRs, with the heavy chain CDR and the corresponding light chain CDR constituting the antigen-binding site of the antibody. The amino acids constituting the FR or CDR regions can be identified by comparing the amino acid sequences of similar antibodies.

[0064] The present invention includes not only complete antibodies, but also antibody fragments with immunoactivity or fusion proteins formed with antibodies and other sequences. Therefore, the present invention also includes the aforementioned antibody fragments, derivatives, and analogs.

[0065] In this invention, antibodies include mouse, chimeric, humanized, or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies containing human and non-human portions, can be obtained by standard DNA recombination techniques and are all useful antibodies. A chimeric antibody is a molecule in which different parts originate from different animal species, for example, a chimeric antibody having a variable region of a mouse-derived monoclonal antibody and a constant region of human immunoglobulin (see, for example, U.S. Patents 4,816,567 and 4,816,397, the entirety of which is incorporated herein by reference). A humanized antibody is a non-human antibody having one or more complementarity-determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule (see U.S. Patent 5,585,089, the content of which is incorporated herein by reference). These chimeric and humanized monoclonal antibodies can be prepared by DNA recombination techniques well known to those skilled in the art.

[0066] In this invention, the antibody may have single specificity, bispecificity, triplicate specificity, or more specificity.

[0067] In the present invention, the antibody of the present invention also includes its conserved variants, in which up to 10 amino acids, preferably up to 8, more preferably up to 5, and most preferably up to 3, are replaced with amino acids having similar or analogous properties compared to the amino acid sequence of the antibody of the present invention, thereby forming a polypeptide. These conserved variant polypeptides are preferably produced by amino acid substitution according to Table A. [Table A]

[0068] Anti-BDCA2 antibody In the present invention, the antibody is an anti-BDCA2 antibody. The present invention provides an antibody that exhibits high specificity and high affinity for BDCA2, which includes a heavy chain and a light chain, wherein the heavy chain includes the amino acid sequence of the heavy chain variable region (VH), and the light chain includes the amino acid sequence of the light chain variable region (VL).

[0069] Here, the heavy chain variable region includes the heavy chain complementarity determination regions H-CDR1, H-CDR2, and H-CDR3. The amino acid sequence of H-CDR1 is shown in SYX1IH (SEQ ID NO:30). The amino acid sequence of H-CDR2 is shown as TIYPGX2GDTSYNQKFKG (SEQ ID NO:31). The amino acid sequence of H-CDR3 is shown in MGDNEYFDY (SEQ ID NO: 8), and The aforementioned light chain variable region includes light chain complementarity determination regions L-CDR1, L-CDR2, and L-CDR3, where, 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). Here, X1 is I or L, X2 is selected from the group of amino acids consisting of N, G, A, S, and R, and / or X3 is selected from the group of amino acids consisting of D, I, or E.

[0070] 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 TIYPGNGDTSYNQKFKG (SEQ ID NO: 7). The amino acid sequence of H-CDR3 is shown in MGDNEYFDY (SEQ ID NO: 8), and The aforementioned light chain variable region includes light chain complementarity determination regions L-CDR1, L-CDR2, and L-CDR3, where, 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). Here, any of the amino acid sequences in the above amino acid sequence includes a derivative sequence in which at least one arbitrarily selected amino acid is added, deleted, modified, and / or substituted, thereby maintaining the binding affinity of BDCA2.

[0071] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or substituting at least one amino acid is preferably an amino acid sequence having at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably 95% homology or sequence identity.

[0072] Methods for determining sequence homology or identity that are well known to those skilled in the art include: Computational Molecular Biology, edited by Lesk, AM, Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, edited by Smith, DW, Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, edited by Griffin, AM and Griffin, HG, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, edited by Gribskov, M. and Develeux, J., M Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied This includes, but is not limited to, Math., 48:1073 (1988). The preferred method for measuring homology is to obtain the greatest match between the sequences being measured. Methods for measuring homology are implemented in publicly available computer programs. Preferred computer program methods for measuring homology between two sequences include, but are not limited to, the GCG program package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., 1990). The public can obtain the BLASTX program (BLAST manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990) from NCBI and other sources. The known Smith-Waterman method is also applicable to homology measurement.

[0073] Preferably, the antibodies described herein include one or more full-length antibody proteins, antigen-antibody binding domain protein fragments, bispecific antibodies, multispecific antibodies, single-chain antibody fragments (scFv), single-domain antibodies (sdAb), and single-domain antibodies (Signle-domain antibodies), as well as monoclonal or polyclonal antibodies produced from such antibodies. The monoclonal antibodies can be developed using a variety of methods and techniques, including hybridoma technology, phage display technology, and single-lymphocyte gene cloning technology. The mainstream approach is to produce monoclonal antibodies from wild-type or transgenic mice using hybridoma technology.

[0074] The full-length antibody protein described above is a conventional full-length antibody protein in the art and includes a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region. The heavy chain variable region and light chain variable region of the protein, together with the human heavy chain constant region and the human light chain constant region, constitute a complete human full-length antibody protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3, or IgG4.

[0075] The antibody (anti-BDCA2 antibody) in the present invention may be a full-length protein (such as IgG1, IgG2a, IgG2b, or IgG2c), or a protein fragment containing an antigen-antibody binding domain (such as Fab,F(ab'), sdAb, or ScFv fragment).

[0076] The antibody (anti-BDCA2 antibody) in this invention may be a wild-type protein, or it may be a mutant that has undergone specific mutations to achieve a particular effect, such as a mutation that removes the effector function of the antibody.

[0077] 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, and humanized antibodies, preferably humanized antibodies and human-animal chimeric antibodies, and more preferably fully humanized antibodies.

[0078] The antibody derivatives described in the present invention may include single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2, or other antibody derivatives known in the art, as well as any one or more antibodies of IgA, IgD, IgE, IgG, and IgM antibodies or other subtypes.

[0079] The aforementioned single-chain antibody is a conventional single-chain antibody in this field, comprising a heavy chain variable region, a light chain variable region, and a short peptide consisting of 15 to 20 amino acids.

[0080] Here, the animal is preferably a mammal such as a mouse.

[0081] In this specification, the terms “Fc variant” or “mutant Fc” refer to proteins that contain modifications to the Fc domain. The Fc variants of the present invention are defined based on the amino acid modifications that constitute them. For example, N434S or 434S is an Fc variant having a serine substitution at position 434 relative to the parent Fc polypeptide, and its number follows the EU index. Similarly, M428L / N434S defines an Fc variant having the M428L and N434S substitutions. The wild-type amino acid may be nonspecific relative to the parent Fc polypeptide, in which case the variant is called 428L / 434S. The order of substitutions is arbitrary, i.e., 428L / 434S is the same Fc variant as M428L / N434S. All position numbers discussed herein follow the EU index. This refers to the EU Index or the EU Index number in Kabat or the EU antibody number in the EU numbering system (Edelman et al., 1969, ProcNatlAcadSciUSA63:78-85, the entire text is incorporated herein by reference). Modifications may be additions, deletions, or substitutions. Substitutions may be natural and non-natural amino acids. They may contain amino acids. Mutants 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, which are incorporated herein by reference in their entirety.

[0082] The antibodies of the present invention may be chimeric antibodies, humanized antibodies, CDR-implanted antibodies, and / or modified antibodies targeting BDCA2 (e.g., human BDCA2).

[0083] Furthermore, the CDR region of the mouse antibody is transplanted into a selected humanized template, replacing the CDR region of the humanized template. The heavy chain variable region is further recombined with the human IgG1 constant region, and the light chain variable region is recombined with the human kappa chain constant region to obtain a chimeric antibody. Based on the three-dimensional structure of the antibody, reverse mutations are performed on embedding residues, residues that directly interact with the CDR region, and residues that significantly affect the conformation of the antibody's VL and VH regions to obtain multiple humanized antibodies.

[0084] In the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably not more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably not more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, and more preferably 15-20%.

[0085] In the present invention, more preferably, the number of amino acids added, deleted, modified and / or substituted may be 1 to 7, more preferably 1 to 5, more preferably 1 to 3, and more preferably 1 to 2.

[0086] Preferably, the humanized antibody includes a heavy chain variable region and a light chain variable region, and compared to a mouse antibody, the heavy chain variable region includes a heavy chain complementarity determining region having the following amino acid sequence: The amino acid sequence is H-CDR1 shown in SYX1IH, where X1 is I or L, and / or H-CDR2 whose amino acid sequence is shown as TIYPGX2GDTSYNQKFKG, where X2 is selected from the group of amino acids consisting of N, G, A, S, R (preferably G), and / or The light chain variable region includes a light chain complementarity determination region having the following amino acid sequence: The amino acid sequence of DAETLAX3 is L-CDR2, where X3 is selected from the group of amino acids consisting of D, I, or E. Here, X1, X2, and X3 are independent of each other and are not simultaneously I, N, or D.

[0087] In another preferred embodiment, the heavy chain variable region and light chain variable region of the humanized antibody include a heavy chain complementarity determining region and a light chain complementarity determining region selected from the following group: (1) H-CDR1 is selected from the following group: SYIIH (SEQ ID NO:6) SYLIH (SEQ ID NO:14) (2) H-CDR2 is selected from the following 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) Selected from the group below L-CDR2: DAETLAD(SEQ ID NO:10) DAETLAI (SEQ ID NO:19) DAETLAE(SEQ ID NO:20); (6) L-CDR3 is QHFWSTPYT (SEQ ID NO: 11).

[0088] In another preferred embodiment, the amino acid sequence of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO. 3, 12, 21, 22, 23, 24, or 25, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity thereto.

[0089] In another preferred embodiment, the amino acid sequence of the light chain variable region includes the amino acid sequence shown in SEQ ID NO. 5, 13, 26, or 27, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity thereto.

[0090] In another preferred embodiment, the heavy chain variable region (VH) amino acid sequence and / or light chain variable region (VL) amino acid sequence of the antibody targeting BDCA2 are shown in Table B below: [Table B]

[0091] Recombinant protein The present invention also provides recombinant proteins comprising the heavy chain CDR1 (H-CDR1), heavy chain CDR2 (H-CDR2), and heavy chain CDR3 (H-CDR3) of the BDCA2 antibody described in a first aspect of the present invention, and / or the light chain CDR1 (L-CDR1), light chain CDR2 (L-CDR2), and light chain CDR3 (L-CDR3) of the BDCA2 antibody.

[0092] The recombinant protein further comprises an antibody heavy chain constant region and / or an antibody light chain constant region, wherein the antibody heavy chain constant region is a conventional, preferably rat antibody heavy chain constant region or human antibody heavy chain constant region, more preferably human antibody heavy chain constant region. The antibody light chain constant region is a conventional, preferably rat light chain antibody constant region or human antibody light chain constant region, more preferably human antibody light chain constant region.

[0093] The recombinant protein is a conventional protein in the field, preferably one or more of a full-length antibody protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single-chain antibody fragment (scFv), a single-domain antibody (sdAb), and a single-domain antibody (Signle-domain antibody), as well as a monoclonal or polyclonal antibody produced from such antibody. The monoclonal antibody can be developed using a variety of methods and techniques, including hybridoma technology, phage display technology, and single lymphocyte gene cloning technology. The mainstream method is to produce monoclonal antibodies from wild-type mice or transgenic mice using hybridoma technology.

[0094] The full-length antibody protein described above is a conventional full-length antibody protein in the art and includes a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region. The heavy chain variable region and light chain variable region of the protein, together with the human heavy chain constant region and the human light chain constant region, constitute a complete human full-length antibody protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3, or IgG4.

[0095] The aforementioned single-chain antibody is a conventional single-chain antibody in this field, comprising a heavy chain variable region, a light chain variable region, and a short peptide consisting of 15 to 20 amino acids.

[0096] The aforementioned antigen-antibody binding protein fragment is a conventional antigen-antibody binding protein fragment in the art, comprising a light chain variable region, a light chain constant region, and an Fd segment of the heavy chain constant region. Preferably, the antigen-antibody binding domain protein fragment is Fab and F(ab').

[0097] The aforementioned single-domain antibody is a conventional single-domain antibody in the field, comprising a heavy chain variable region and a heavy chain constant region.

[0098] The aforementioned single-domain antibody is a conventional single-domain antibody in this field, containing only the heavy chain variable region.

[0099] Here, the method for preparing the recombinant protein is a conventional preparation method in the field. Preferably, the preparation method includes isolating the protein from a transformant that recombinantly expresses the protein, or obtaining the protein by artificially synthesizing the protein sequence. Preferably, isolation from a transformant that recombinantly expresses the protein is performed by cloning a nucleic acid molecule encoding the protein and having point mutations into a recombinant vector, transforming the transformant using the obtained recombinant vector to obtain a recombinant-expressing transformant, and then culturing the obtained recombinant-expressing transformant to isolate and purify the recombinant protein.

[0100] nucleic acid The present invention also provides nucleic acids encoding the heavy chain variable region or light chain variable region of the antibody (e.g., an anti-BDCA2 antibody), recombinant protein, or anti-BDCA2 antibody.

[0101] The method for preparing nucleic acids is a conventional method in the field and preferably includes the step of obtaining a nucleic acid molecule encoding a protein by gene cloning technology, or the step of obtaining a nucleic acid molecule encoding a protein by artificial whole sequence synthesis.

[0102] Those skilled in the art will understand that polynucleotide homologs can be obtained by appropriately substituting, deleting, modifying, inserting, or adding to the base sequence encoding the amino acid sequence of a protein. The polynucleotide homolog of the present invention can be prepared by substituting, deleting, or adding one or more bases in the gene encoding the protein sequence while maintaining antibody activity.

[0103] vector The present invention also provides a recombinant expression vector comprising the nucleic acid.

[0104] Here, the recombinant expression vector can be obtained by conventional methods in the art, i.e., by linking the nucleic acid molecule described in the present invention to various expression vectors. The expression vector may be any conventional vector in the art, as long as it can support the nucleic acid molecule. Preferably, the vector includes various plasmids, cosmids, phages, or viral vectors.

[0105] The present invention also provides a recombinant expression transformant comprising the recombinant expression vector.

[0106] Here, the method for preparing the recombinant expression transformant is a conventional preparation method in the art, and preferably, it can be produced by transforming a host cell with the recombinant expression vector. The host cell may be any conventional host cell in the art, as long as it can stably replicate the recombinant expression vector and efficiently express the nucleic acid it carries. Preferably, the host cell is E. coli TG1 or E. coli BL21 cell (expressing a single-chain antibody or Fab antibody), or HEK293 or CHO cell (expressing a full-length IgG antibody). The preferred recombinant expression transformant of the present invention is obtained by transforming a host cell with the recombinant expression plasmid. Here, the transformation method is a conventional transformation method in the art, preferably a chemical transformation method, a heat shock method, or an electroporation method.

[0107] Antibody preparation The DNA molecule sequences of the antibody or fragment of the present invention can be obtained by conventional techniques such as amplification using PCR or genome library screening. Furthermore, single-chain antibodies can be formed by fusing the coding sequences of the light chain and heavy chain.

[0108] Once the desired sequence is obtained, it can be acquired in large quantities by recombination. This is usually done by cloning it into a vector, transplanting it into cells, and then isolating the desired sequence from the proliferated host cells using conventional methods.

[0109] Furthermore, especially when the fragment length is short, it is possible to synthesize the sequence through artificial synthesis. In many cases, very long fragments can be obtained by synthesizing several smaller fragments and ligating them.

[0110] Currently, DNA sequences encoding the protein (or its fragment or derivative thereof) of the present invention are readily available through chemical synthesis. These DNA sequences can be introduced into various existing DNA molecules (or vectors, etc.) or cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0111] The present invention also relates to vectors comprising a suitable DNA sequence and a suitable promoter or regulatory sequence as described above. These vectors can be used to transform suitable host cells so that they can express proteins.

[0112] The host cell may be a prokaryotic cell such as a bacterial cell; or a lower eukaryotic cell such as a yeast cell; or a higher eukaryotic cell such as a mammalian cell. Preferred animal cells include, but are not limited to, CHO-S and HEK-293 cells.

[0113] Typically, transformed host cells are cultured under conditions suitable for the expression of the antibody of the present invention. The antibody of the present invention can then be purified using conventional immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, and conventional separation and purification methods known to those skilled in the art.

[0114] The obtained monoclonal antibodies can be identified by conventional methods. For example, the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). The binding affinity of monoclonal antibodies can be measured, for example, by Scatchard analysis as described by Munson et al., Anal. Biochem., 107:220 (1980).

[0115] The antibodies of the present invention may be expressed intracellularly or at the cell membrane, or secreted extracellularly. If necessary, recombinant proteins can be isolated and purified by various isolation methods utilizing their physical, chemical, and other properties. These methods are known to those skilled in the art. Examples of these methods include, but are not limited to, conventional regeneration processes, treatment with protein precipitants (salting-out), centrifugation, osmotic bacterial disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), various other liquid chromatography techniques, and combinations thereof.

[0116] Antibody-drug conjugate (ADC) The present invention also provides antibody-drug conjugates (ADCs) based on the antibodies of the present invention.

[0117] Typically, the antibody-drug conjugate comprises an antibody and an effector molecule, the antibody being bound to the effector molecule, preferably chemically. Here, preferably, the effector molecule is a drug having therapeutic activity. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic agent, a small molecule drug, or a radionuclide.

[0118] The antibody of the present invention and the effector molecule may be coupled via a coupling agent. The coupling agent may be one or more of the following: a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, or a coupling agent using a disulfide bond. The non-selective coupling agent refers to a compound that covalently bonds the effector molecule and the antibody, such as glutaraldehyde. The carboxyl group coupling agent may be one or more of the following: a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) or an acylhydrazone coupling agent (where the coupling site is an acylhydrazone).

[0119] Specific residues on antibodies (such as Cys and Lys) are used for binding to functional groups, including imaging reagents (such as chromophores and fluorescent groups), diagnostic reagents (such as MRI contrast agents and radioisotopes), stabilizers (such as glycol polymers), and therapeutic agents. Antibodies can form antibody-functional agent complexes by binding to functional agents. Functional agents (drugs, measurement reagents, stabilizers, etc.) are bound to antibodies (covalently). Functional agents can be bound to antibodies directly or indirectly via linkers.

[0120] Antibodies can form antibody-drug conjugates (ADCs) by binding to drugs. Typically, ADCs contain a linker between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers are typically readily degraded in the intracellular environment, for example, by degradation at the desired site, thereby releasing the drug from the antibody. Suitable degradable linkers include enzymatically degradable linkers, such as linkers containing peptidyl groups that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or carbohydrate linkers containing glucuronides that can be degraded by glucuronidases. Peptidyl group linkers may include dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze when the pH is below a certain level, such as hydrazone) and linkers that degrade under reducing conditions (e.g., disulfide linkage linkers). Typically, non-degradable linkers release drugs under conditions where the antibody is hydrolyzed by a protease.

[0121] Before binding to the antibody, the linker has an activating group that can react with a specific amino acid residue, and binding is achieved via this activating group. Sulfhydryl-specific activating groups are preferred and include, for example, maleimide compounds, halogenated amides (e.g., iodized, brominated, or chlorinated), halogenated esters (e.g., iodized, brominated, or chlorinated), halogenated methyl ketones (e.g., iodized, brominated, or chlorinated), benzyl halides (e.g., iodized, brominated, or chlorinated), vinyl sulfones, pyridyl disulfide, mercury derivatives such as 3,6-di-(mercurymethyl)dioxane whose counterion is an acetate, chloride, or nitrate, and polymethylenedimethyl sulfide thiosulfonates. The linker may include, for example, a maleimide that binds to the antibody via thiosuccinimide.

[0122] The drug may be any cytotoxic, cell proliferation inhibitory, or immunosuppressive drug. In some embodiments, the linker binds the antibody to the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug may have an amino group, carboxyl group, sulfhydryl group, hydroxyl group, or carbonyl group capable of forming a bond with the linker. In the case of a drug directly bound to the linker, the drug has a reactive group before binding to the antibody.

[0123] Useful drug types include, for example, antitubulins, DNA minor groove binders, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, and vinca alkaloids. Particularly useful examples of cytotoxic drugs include DNA supraclude binders, DNA alkylating agents, and tubulin inhibitors. Typical examples of cytotoxic drugs include auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, oxazolidinobenzodiazepines), and vinca alkaloids.

[0124] In this invention, a drug-linker is used to form an ADC in a simple one-step process. In other embodiments, a bifunctional linker compound is used to form an ADC in a two-step or multi-step process. For example, in step 1, a cysteine ​​residue reacts with the reactive portion of the linker, and in a subsequent step, the functional group of the linker reacts with the drug to form an ADC.

[0125] Typically, functional groups on the linker are selected to facilitate specific reactions with appropriate reactive groups on the drug moiety. As a non-limiting example, an azide-based moiety is used for specific reactions with reactive alkynyl groups on the drug moiety. The drug is covalently bonded to the linker via a 1,3-dipolar cycloaddition reaction between the azide and the alkynyl group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reactions with hydrazides and alkoxyamines), phosphines (suitable for reactions with azides), isocyanates and isothiocyanates (suitable for reactions with amines and alcohols), and activated esters such as N-succinimidyl esters (suitable for reactions with amines and alcohols). These and other bonding strategies are well known to those skilled in the art, as described in *Studies in Biomaterials*, 2nd edition (Elsevier). Those skilled in the art will understand that when complementary reactive functional groups are selected for selective reactions between the drug moiety and the linker, each member of the complementary pair can be used in both the linker and the drug.

[0126] The present invention also provides a method for preparing an antibody-drug conjugate (ADC), which may further comprise conjugating an antibody with a drug-linker compound under conditions sufficient to form an ADC.

[0127] In some embodiments, the method of the present invention involves conjugating an antibody with a bifunctional linker compound under conditions sufficient to form an antibody-linker complex. In these embodiments, the method of the present invention further involves conjugating the antibody-linker complex to a drug moiety under conditions sufficient to covalently bond the drug moiety to the antibody via the linker.

[0128] In these embodiments, the antibody-drug conjugate (ADC) is represented by the following molecular formula. [ka] During the ceremony, Ab stands for antibody, LU stands for linker, D is a drug The subscript 'p' is a value chosen from 1 to 8.

[0129] Pharmaceutical composition The present invention also provides compositions. In preferred embodiments, the composition is a drug-pharmaceutical composition comprising the antibody or its activated fragment or fusion protein, or its ADC or corresponding immune cell, and a pharmaceutically acceptable vector. Typically, these substances may be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous vector medium, in which case the pH may typically be about 5 to 8, preferably about 6 to 8, and the pH may vary depending on the properties of the substances being formulated and the condition to be treated.

[0130] The formulated pharmaceutical composition can be administered by conventional routes, including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration. Typically, preferred routes of administration for the pharmaceutical composition described in the present invention are by injection or oral administration. Preferably, the injection route includes intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection. The pharmaceutical composition may be in various conventional dosage forms in the art, preferably in solid, semi-solid, or liquid form, and may be an aqueous solution, non-aqueous solution, or suspension, more preferably a tablet, capsule, granule, injection, or infusion.

[0131] Furthermore, the antibody described in the present invention may also be a cell therapy drug expressed in cells by a nucleotide sequence, such as the antibody for chimeric antigen receptor T cell immunotherapy (CAR-T).

[0132] The pharmaceutical composition described in the present invention is a pharmaceutical composition for preventing and / or treating diseases associated with BDCA2 expression or dysfunction.

[0133] Since the pharmaceutical composition of the present invention can directly bind to the BDCA2 protein molecule, it can be used for the prevention and treatment of diseases such as tumors.

[0134] The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, particularly preferably 0.1-80 wt%) of the monoclonal antibody (or its complex) described in the present invention and a pharmaceutically acceptable vector or excipient. Such vectors include, but are not limited to, sodium chloride, buffer, glucose, water, glycerin, ethanol, and combinations thereof. The formulation needs to be adapted to the dosage form. The pharmaceutical composition of the present invention can be prepared, for example, by conventional methods as an aqueous solution containing physiological saline or glucose and other excipients, and can be made into an injectable preparation. Pharmaceutical compositions such as injectable preparations and solutions should preferably be prepared under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example, about 1 μg / kg body weight to about 5 mg / kg body weight per day. Furthermore, the polypeptide of the present invention can also be used in combination with other therapeutic agents.

[0135] Preferably, in the present invention, the pharmaceutical composition described in the present invention further comprises one or more pharmaceutically usable vectors. The pharmaceutically usable vectors are conventional pharmaceutically usable vectors in the art and may be any suitable pharmaceutically acceptable pharmaceutical additives. The pharmaceutically acceptable additives are conventional pharmaceutically acceptable pharmaceutical additives in the art and preferably include pharmaceutically acceptable excipients, fillers or diluents. More preferably, the pharmaceutical composition comprises 0.01 to 99.99% of the protein and 0.01 to 99.99% of the pharmaceutically usable vector, wherein the percentages are by mass percentages of the pharmaceutical composition.

[0136] Preferably, in the present invention, the dosage of the pharmaceutical composition is an effective amount that can alleviate or delay the progression of a disease, degenerative disease, or debilitating disease. The effective amount can be determined on an individual basis, taking into consideration the symptoms to be treated and the desired outcome. Those skilled in the art can determine the effective amount by using the above-mentioned factors, such as individual-based testing, and conducting experiments that do not exceed the usual range.

[0137] When using a drug composition, a safe and effective amount of immune complexes should be administered to the mammal, where a safe and effective amount is typically at least about 10 μg / kg body weight, and in most cases about 50 mg / kg body weight or less, preferably about 10 μg / kg body weight to about 20 mg / kg body weight. Of course, the specific dosage should take into account further factors such as the mode of administration and the patient's health condition, all of which are within the scope of a skilled physician's expertise.

[0138] The present invention provides the use of pharmaceutical compositions in the preparation of agents for preventing and / or treating diseases associated with BDCA2 expression or dysfunction. Preferably, the diseases associated with BDCA2 expression or dysfunction are inflammatory diseases or autoimmune diseases.

[0139] application The present invention also provides the use of the antibodies, antibody-drug conjugates (ADCs), recombinant proteins, and / or immune cells of the present invention. For example, they can be used in the preparation of diagnostic formulations or pharmaceutical preparations.

[0140] Preferably, the agent is an agent for preventing and / or treating diseases associated with BDCA2 expression or dysfunction.

[0141] In the present invention, the diseases associated with BDCA2 expression or dysfunction are conventional diseases in the art associated with BDCA2 expression or dysfunction. Preferably, the diseases associated with BDCA2 expression or dysfunction include, but are not limited to, systemic lupus erythematosus, cutaneous lupus erythematosus, discoid lupus erythematosus, lupus nephritis, cutaneous lupus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type 1 diabetes mellitus, dermatomyositis, and polymyositis or combinations thereof, inflammatory diseases or autoimmune diseases.

[0142] The main advantages of this invention are as follows: (1) The mouse anti-BDCA2 antibody of the present invention has excellent affinity, which is superior to the positive control antibody litifirimab, and the anti-BDCA2 antibody modified after humanization also has higher affinity. (2) The BDCA2 antibody of the present invention can effectively inhibit IFNa and IgM secretion by PBMCs induced by CpG-A stimulation and is expected to be a therapeutic agent for autoimmune diseases.

[0143] The present invention will be described in more detail below, in accordance with specific embodiments. It should be understood that this is used solely to illustrate the present invention and not to limit its scope. In the following embodiments, experimental methods not specifically indicated follow conventional conditions, such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or manufacturer-recommended conditions. Unless otherwise specified, percentages and quantities are on a weight basis.

[0144] Example 1: Preparation and screening of antigen-immunized animals and hybridomas 1.1 Preparation of antigen protein and positive control antibody The human BDCA2 extracellular domain (BDCA2-ECD) sequence used as an antigen was obtained from the UniProt database (Entry: Q8WTT0), and its amino acid sequence is shown in SEQ ID NO: 1. A 6×His tag was added to its N-terminus, and it was incorporated into a pcDNA 3.4 expression vector. HEK-293F cells were transfected with this vector and expressed for 5 days. After expression, the cell culture supernatant was collected and purified to obtain the BDCA2-his protein. Similarly, the 6×His tag was replaced with the Fc sequence of human IgG1, and HEK-293F cells were transfected with this protein. Expression and purification were then performed to obtain the TL1A-Fc protein.

[0145] The heavy and light chain sequences of the positive control antibody litifirimab (BIIB059,24F4A) were obtained from patent CN111961134A. The heavy chain variable region and light chain variable region genes of this antibody were synthesized, then recombined with human heavy chain IgG1 and light chain Kappa constant region, respectively. These were then incorporated into pcDNA 3.4 expression vectors, expressed using the HEK-293F system, and purified using Protein A affinity chromatography.

[0146] 1.2 Mouse Immunity Balb / c mice were immunized using human BDCA2-His protein (in-house produced, purity >95%) expressed in mammalian cell 293F as usual. On day 1, soluble human BDCA2-His protein was emulsified with complete Freund's adjuvant and subcutaneously injected into multiple sites in Balb / c mice (human BDCA2-His 100 μg / mouse / 0.5 mL). On day 14, soluble human BDCA2-His protein was emulsified with complete Freund's adjuvant and subcutaneously injected into Balb / c mice (human BDCA2-His 50 μg / mouse / 0.5 mL). On day 28, soluble human BDCA2-His protein was emulsified with complete Freund's adjuvant and subcutaneously injected into Balb / c mice (human BDCA2-His 50 μg / mouse / 0.5 mL). Three weeks later, the mice were stimulated by intraperitoneal injection of 50 μg / 0.2 mL of soluble human BDCA2-His protein. Three to four days later, the mouse spleens were harvested and fusion experiments were performed.

[0147] 1.3 Preparation and screening of hybridoma cells Three to four days after final immunization of mice, PEG fusion of mouse spleen cells and mouse myeloma cells SP2 / 0 was performed using standard hybridoma technology. The fused cells were homogeneously suspended in complete medium consisting of 1% penicillin-streptomycin, 20% fetal bovine serum, and 1*HAT supplemented with RPMI1640-GLUMAX. The fused cells were then divided into 3*10 4 Individual cells were seeded at 200 μL / well into 60 96-well plates. The supernatant was collected after 7–12 days, and hybridoma wells positive for human TL1A binding activity were screened using ELISA.

[0148] The screening method for human-to-human BDCA2 binding activity-positive hybridoma wells using ELISA is as follows: BDCA2-Fc was diluted to 1 μg / mL using PBS buffer and added to a 100 μL / well plate, where it was incubated overnight at 4°C. The supernatant was discarded the next day, 5% skim milk powder was added, the plate was blocked at 37°C for 1 hour, and the plate was washed three times with PBST. The hybridoma supernatant collected in 100 μL / wells was sequentially added to the blocked plate and left at 37°C for 1 hour. The plate was washed three times with PBST, HRP-labeled goat anti-mouse IgG secondary antibody was added, and the plate was left at 37°C for 30 minutes. After washing the plate three times with PBST, any remaining droplets were wiped off as much as possible on absorbent paper, 100 μL / well of TMB was added, and the plate was left at room temperature (20±5°C) in the dark for 5 minutes. The substrate reaction was stopped by adding 2M H2SO4 stop solution at 50 μL / well, and the OD value at 450 nm was read using a microplate reader to analyze the binding ability of the test antibody to the target antigen BDCA2. Hybridoma cell lines obtained by screening were grown in serum-containing complete medium, centrifuged, and then replaced with serum-free culture medium, Hybridoma-SFM medium, and the cell density was increased to 1-2 × 10⁶. 6The cells were cultured at 8% CO2 and 37°C for 1 week. The culture supernatant was collected by centrifugation and purified by Protein G affinity chromatography to obtain anti-human BDCA2 monoclonal antibody protein. A total of 30 hybridoma cell lines were obtained by screening.

[0149] 1.4 Binding ability of mouse antibodies to human BDCA2-Fc protein The binding ability of mouse antibodies to human BDCA2-Fc protein was analyzed using enzyme-linked immunosorbent assay (ELISA). The specific method is as follows: BDCA2-Fc protein was diluted to 1 μg / mL using PBS buffer and added to a 100 μL / well plate, where it was incubated overnight at 4°C. The plate was then blocked with 5% skim milk powder and incubated at 37°C for 1 hour. After washing the plate three times with PBST, laboratory-prepared anti-human BDCA2 mouse antibody was serially diluted 3-fold from 10 μg / mL to an 11-fold gradient using 1% BSA-PBS buffer. 1% BSA-PBS was used as a blank control and added to a BDCA2-Fc plate pre-coated with 100 μL / well, where it was incubated at 37°C for 1 hour. The plate was washed three times with PBST, HRP-labeled goat anti-mouse IgG secondary antibody was added, and the mixture was left at 37°C for 30 minutes. After washing the plate three more times with PBST, any remaining droplets were wiped off as much as possible on absorbent paper, 100 μL / well of TMB was added, and the mixture was left at room temperature (20±5°C) in the dark for 5 minutes. The substrate reaction was stopped by adding 2M H2SO4 stop solution at 50 μL / well, and the OD value at 450 nm was read using a microplate reader to analyze the binding ability of the test antibody to the target antigen, human BDCA2-Fc. The obtained data were fitted and analyzed using GraphPad Prism 9 software. The results are shown in Figures 1A-1B.

[0150] As can be seen from Figures 1A and 1B, most mouse antibodies showed good binding activity to the target antigen BDCA2-Fc, and the EC50 values ​​are shown in Tables 1 and 2. [Table 1] [Table 2]

[0151] 1.5 Measurement of binding affinity of mouse antibody to the stable expression cell line 293FT-BDCA2 In this example, the binding affinity of a mouse antibody to target cells 293FT-BDCA2 was measured using the fluorescence-activated cell sorting (FACS) method.

[0152] In this experiment, 293FT-BDCA2 (a modified cell line highly expressing human BDCA2, constructed in the laboratory using a lentiviral vector) was used as the target cell, counted, and washed once with 1% BSA-PBS. 2 × 10 5 Cells were plated in 100 μL / well and the supernatant was removed by centrifugation. Anti-human BDCA2 mouse antibody was serially diluted 3-fold in 11 gradients from 10 μg / mL using 1% BSA-PBS. 100 μL of each was resuspended in 293FT-BDCA2 cell wells using 1% BSA-PBS as a blank control and incubated at 4°C for 1 hour. To remove unbound mouse antibody, cells were washed twice with PBS, then resuspended in 100 μL / well, and PE-labeled anti-mouse secondary antibody (purchased from Jackson, catalog number 715116150) was diluted 1:500 and incubated at 4°C for 1 hour. To remove unbound mouse antibody, cells were washed twice with PBS, then resuspended in 200 μL of PBS, and the binding affinity of the mouse antibody to the cells was measured using a flow cytometer. The obtained data were fitted and analyzed using GraphPad Prism 9 software. The results are shown in Figures 2A and 2B.

[0153] As a result, most mouse antibodies were able to specifically bind to BDCA2 on the surface of 293FT cells, and the EC50 values ​​are shown in Tables 3 and 4. [Table 3] [Table 4]

[0154] Example 2 Humanization of mouse anti-human BDCA2 monoclonal antibody 2.1 Identification of Variable Region Sequences of Mouse Anti-Human BDCA2 Monoclonal Antibodies Hybridoma clone 23C10A6B5 was selected as a candidate antibody, and total RNA was extracted from the corresponding hybridoma monoclonal cell line using Trizol (purchased from Life Technologies). The mRNA was reverse transcribed into cDNA using a reverse transcription kit (purchased from Takara), and PCR was performed using a combination primer reported in the literature (《Antibody Engineering》Volume 1, Edited by Roland Kontermann and Stefan Dubel, primer sequences are on page 323). The resulting PCR products were sequenced and analyzed in the Kabat database, confirming that the obtained sequences were the variable region sequences of the mouse antibody. The sequence information is as follows: The heavy chain variable region gene sequence is 354 bp in length, encoding 118 amino acid residues. The nucleotide sequence is shown in SEQ ID NO:2, and the amino acid sequence is shown in SEQ ID NO:3. The light chain variable region gene sequence is 321 bp in length and codes for 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.

[0155] 2.2 Construction and validation of human-mouse chimeric antibodies The mouse heavy chain variable region and light chain variable region sequences (SEQ ID NO:3 and SEQ ID NO:5) obtained in Example 2.1 were ligated to the human IgG1 heavy chain constant region and the kappa-type light chain constant region, respectively, to construct the human-mouse chimeric antibody C-23C10. The binding activity to the antigen BDCA2 and the inhibitory effect on IFNa secretion by CpG-A-induced PBMCs were then verified.

[0156] Binding activity was measured in the same manner as in Example 1.4, but the coating antigen BDCA2-Fc was replaced with BDCA2-his, the coating concentration was set to 1 μg / mL, and the secondary antibody used was anti-human Fc HRP (SinoBiological, catalog number SSA001) at a working concentration of 1:3000.

[0157] The experimental results are shown in Figure 3, where the binding activity of C-23C10 to the BDCA2 antigen is superior to that of the positive control 24F4A.

[0158] CpG-A (CpG ODNs Class A) is an oligodeoxynucleotide with a CpG palindrome sequence that stimulates massive secretion of IFNa from pDC cells. In this example, the inhibitory effect of a candidate antibody on IFNa secretion from CpG-A-induced PBMCs was measured using PBMCs.

[0159] CpG-A was diluted to 8 μg / mL (final concentration 2 μg / mL) using RPMI 1640 + 3% FBS medium and added to a flat-bottomed 96-well plate at 50 μL / well. Antibody was diluted to 16 nM (final concentration 4 nM, 5-fold dilution) using RPMI 1640 + 3% FBS medium and added to the CpG-A well at 50 μL / well. CpG-A and antibody were incubated in a 37°C, 5% CO2 incubator for 1 hour. PBMC cells were centrifuged and counted to adjust cell density, and 500,000 cells / well were inoculated into the 96-well plate, making a total of 100 μL / well, and incubated overnight in a 37°C, 5% CO2 incubator. The cell supernatant was collected by centrifugation and the H-IFNa content in the supernatant was measured.

[0160] The experimental results are shown in Figure 4, where C-23C10 effectively inhibits IFNa secretion from PBMCs induced by CpG-A stimulation, and its activity is superior to that of the positive control 24F4.

[0161] 2.3 Humanization of mouse anti-human BDCA2 monoclonal antibodies The amino acid sequence of the 23C10A6B5 heavy / light chain variable region was analyzed, and three antigen complementarity-determining regions (CDRs) and four framework regions (FRs) were determined according to Kabat rules. Here, the amino acid sequence of the heavy chain complementarity-determining region is: HCDR1:SYIIH (SEQ ID NO:6) HCDR2:TIYPGNGDTSYNQKFKG(SEQ ID NO:7) and The sequence is HCDR3:MGDNEYFDY (SEQ ID NO: 8), and the amino acid sequence of the light chain complementarity determining region is: LCDR1:RASGNIHNYLA(SEQ ID NO:9), LCDR2:DAETLAD(SEQ ID NO:10) and LCDR3:QHFWSTPYT (SEQ ID NO:11).

[0162] By comparing homology with human IgG germline sequences (Germline) using NCBI IgBlast, IGHV1-46*01 was selected as the heavy chain CDR transplantation template and IGKV1-NL1*01 as the light chain CDR transplantation template. The CDR region of the 23C10A6B5 antibody was transplanted into the selected humanized template, replacing the CDR region of the humanized template. The heavy chain variable region was recombined with the human IgG1 constant region, and the light chain variable region was recombined with the human kappa chain constant region. Furthermore, based on the three-dimensional structure of the antibody, reverse mutations were performed on embedding residues, residues that directly interact with the CDR region, and residues that significantly affect the conformation of the antibody's VL and VH regions, yielding multiple humanized antibodies. Affinity screening determined the heavy chain variable region sequence (SEQ ID NO: 12) and light chain variable region sequence (SEQ ID NO: 13) of humanized antibody 230.

[0163] Example 3: Affinity maturation of humanized antibody 230 The complex structure of the 230 antibody and the BDCA2-his antigen was predicted using AlphaFold2 software, and the interaction interface was analyzed. A mutant library was constructed by introducing single-point saturation mutations at the major interaction sites. Mutation sites that improve the affinity of the 230 antibody were identified by ELISA affinity screening. The experimental method was the same as in Example 1.4, but the coating antigen BDCA2-Fc was replaced with BDCA2-his, the coating concentration was set to 0.1 μg / mL, i.e., 10 ng / well, and the secondary antibody used was anti-human Fc HRP (SinoBiological, catalog number SSA001) at a working concentration of 0.1:3000.

[0164] The results are shown in Figure 5. According to the experimental results, heavy chain CDR1: [ka] It mutates to obtain the SYLIH (SEQ ID NO:14) mutation, and heavy chain CDR2: [ka] It mutated into, TIYPGGGDTSYNQKFKG(SEQ ID NO:15), TIYPGAGDTSYNQKFKG(SEQ ID NO:16), TIYPGSGDTSYNQKFKG(SEQ ID NO:17), The TIYPGRGDTSYNQKFKG (SEQ ID NO:18) mutation was obtained (corresponding antibodies were 230-I33L, 230-N55G, 230-N55A, 230-N55S, and 230-N55R, respectively). All of these effectively improve the affinity of the 230 antibody.

[0165] Furthermore, light chain CDR2: [ka] Alternatively, mutations to E were obtained, yielding DAETLAI (SEQ ID NO: 19) and DAETLAE (SEQ ID NO: 20). The mutants maintained similar affinity to 230, but their average expression levels increased by more than 30%, and the remaining saturated mutants showed similar affinity and expression levels to the parent antibody 230.

[0166] Selected mutations in the aforementioned CDR region were combined and ligated to the constant region of the IgG1 subtype to construct 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. Here, the heavy chains of the 230IL-DI and 230IL-DE antibodies are the same, and the heavy chain variable region sequence is shown in SEQ ID NO:21. The heavy chains of the 230IL-NG-DI and 230IL-NG-DE antibodies are the same, and the heavy chain variable region sequence is shown in SEQ ID NO:22. The heavy chains of the 230IL-NR-DI and 230IL-NR-DE antibodies are the same, and the heavy chain variable region sequence is shown in SEQ ID NO:23. The heavy chains of the 230NG-DI and 230NG-DE antibodies are the same, and the heavy chain variable region sequence is shown in SEQ ID NO:24. The heavy chains of the 230NR-DI and 230NR-DE antibodies are the same, and the heavy chain variable region sequence is shown in SEQ ID NO:25.

[0167] The light chains of the 230IL-DI, 230IL-NG-DI, 230IL-NR-DI, 230NG-DI, and 230NR-DI antibodies are the same, and the light chain variable region sequence is shown in SEQ ID NO:26. The light chains of the 230IL-DE, 230IL-NG-DE, 230IL-NR-DE, 230NG-DE, and 230NR-DE antibodies are the same, and the light chain variable region sequence is shown in SEQ ID NO:27.

[0168] Based on the aforementioned ELISA test method, the affinity of 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 antibodies to the BDCA2-his antigen protein was investigated.

[0169] The results are shown in Figure 6. The experimental results indicated that the affinity of the 10 mutant strains for the antigen protein was higher than that of the parent monoclonal antibody 230.

[0170] Example 4: Measurement of affinity dissociation constant KD value Based on patents CN200880123009.4 and CN200480018985.5, the M428L / N434S and G236A / S239D / I332E mutations were introduced into the heavy chain FC region of the 230IL-NG-DE and 230-NG-DE (also known as 230NG-DE) antibodies of Example 3 to construct the 237011 antibody (heavy chain sequence shown in SEQ ID NO:28, light chain variable region sequence shown in SEQ ID NO:27) and the 2391 antibody (heavy chain sequence shown in SEQ ID NO:29, light chain variable region sequence shown in SEQ ID NO:27).

[0171] Kinetic parameters for the binding and dissociation of candidate antibody proteins and the antigen BDCA2-his were measured using a capture method with a Biacore 8K intermolecular interaction analyzer. The antibody was diluted to 1 μg / mL using 1*HBS-N, pH 7.0 buffer containing 5 mM CaCl2, 0.02% Tween 20, and the core antibody was captured with a Protein A tip. The antigen was diluted with 1*HBS-N, pH 7.0 buffer containing 5 mM CaCl2, 0.02% Tween 20, bound to the antibody in a 6-step concentration gradient with a maximum concentration of 12.5 nM, and dissociated in 1*HBS-N, pH 7.0 buffer containing 5 mM CaCl2, 0.02% Tween 20.

[0172] The experimental results are shown in Table 5. The affinity of candidate antibodies 237011 and 2391 for the antigen BDCA2-his is superior to that of the positive control antibody 24F4A. [Table 5] Note: KD represents the affinity constant, ka represents the association rate constant, and kd represents the dissociation rate constant.

[0173] Example 5: Inhibitory effect of CpG-A-induced PBMC on IFNa secretion The inhibitory effect of a humanized candidate antibody on IFNa secretion from CpG-A-induced PBMCs was investigated. See Example 2.2 for experimental procedures.

[0174] The experimental results are shown in Figure 7. Candidate antibodies 237011, 2391, 230IL-NG-DE, and 230-NG-DE were all able to inhibit IFNa secretion from PBMCs stimulated by CpG-A, and their activity was significantly superior to that of the positive control antibody 24F4A.

[0175] Example 6: Inhibitory effect of CpG-A-induced PBMC on IFNa secretion The inhibitory effect of a humanized candidate antibody on IFNa secretion by CpG-A-induced PBMCs was investigated. The experimental method was as follows: PBMC cells were resuspended in RPMI 1640 + 10% FBS medium, and the cell density was set to 5 × 10⁶. 6 The solution was adjusted to 1 / ml and added to a 3599 plate at 100 μL / well. 50 μL / well of CPG-A (CpG2216) was added to achieve a final concentration of 2 μg / mL. The antibody was diluted with culture medium to 50 μL / well and a final concentration of 3000 nM. 100 μL of cells and 50 μL of antibody were incubated at 37°C, 5% CO2 for 1 hour, then 50 μL of CPG-A was added and the mixture was placed in a 37°C, 5% CO2 incubator. After incubation for 7 days, the cell supernatant was collected by centrifugation, and the IgM content in the supernatant was measured.

[0176] The experimental results are shown in Figure 8. Candidate antibodies 237011, 2391, 230IL-NG-DE, and 230-NG-DE can all inhibit IgM secretion from PBMCs stimulated by CpG-A, and their activity is significantly superior to that of the positive control antibody 24F4A.

[0177] Example 7: Inhibitory effect on IFNa in mice The inhibitory effect of a candidate humanized antibody on IFNa secretion in mice was investigated. Since the candidate antibody did not cross-react with the BDCA2 homologous protein in mice, animal in vivo experiments were conducted using reconstituted humanized immune system mice (HuHSC-NCG immune system humanized mice, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) constructed by injection of human huCD34+ embryonic stem cells.

[0178] The experimental design was as follows: On day 0, the body weight of the target mice was measured, and they were randomly divided into three groups based on body weight: a model group (PBS administration group), a 24F4A administration group, and a 237011 administration group. On the afternoon of the same day, each group received intraperitoneal administration according to the administration plan in Table 6. 18 hours after administration (day 1), all three groups of mice were injected via tail vein at a dose of 500 μg / 250 μL / mouse. 24 hours after CpG-A injection, blood was collected from the posterior orbital venous plexus, and after coagulation and centrifugation, serum was collected and the human IFNa concentration was measured by ELISA. [Table 6] The experimental results are shown in Figure 9. The inhibitory effect on IFNa activity in mice administered 237011 was significantly better than that in the 24F4A-administered group. The arrangement of this invention is as follows: Human BDCA2 extracellular domain (BDCA2-ECD) SEQ ID NO:1 NFMYSKTVKRLSKLREYQQYHPSLTCVMEGKDIEDWSCCPTPWTSFQSSCYFISTGMQSWTKSQKNCSVMGADLVVINTREEQDFIIQNLKRNSSYFLGLSDPGGRRHWQWVDQTPYNENVTFWHSGEPNNLDERCAIINFRSSEEWGWNDIHCHVPQKSICKMKKIYI 23C10A6B5 Heavy Chain Variable Region Nucleotide Sequence SEQ ID NO:2 CAGGTGCAACTACAGCAGCCTGGGGCTGACCTGGTGAAGCCTGGGGCCTCAGTGATGATGTCCTGCAAGGCTTCTGGATACACATTTACCAGTTACATTATTCACTGGGTAAAACAGACACCTGGACAGGGCCTGGAATGGATTGGAACTATTTATCCAGGAAATGGTGATACTTCC TACAATCAGAAATTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAATGGGGGATAACGAATACTTTGACTACTGGGGCCAAGGAACCACTCTCACAGTCTCCTCA 23C10A6B5 Heavy Chain Variable Region Amino Acid Sequence SEQ ID NO:3 QVQLQQPGADLVKPGASVMMSCKASGYTFTSYIIHWVKQTPGQGLEWIGTIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARMGDNEYFDYWGQGTTLTVSS 23C10A6B5 Light Chain Variable Region Nucleotide Sequence SEQ ID NO:4 GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGGAAATATTCACAATTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATGATGCAGAAACCT TAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCCGGAACACAATATTCTCTCAATATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTTTTACTGTCAACATTTTTGGAGTACTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA 23C10A6B5 Light chain variable region amino acid sequence SEQ ID NO:5 DIQMTQSPASLSASVGETTVTITCRASGNIHNYLAWYQQKQGKSPQLLVYDAETLADGVPSRFSGSGSGTQYSLNINSLQPEDFGSFYCQHFWSTPYTFGGGTKLEIK HCDR1:SYIIH of 23C10A6B5 (SEQ ID NO:6) 23C10A6B5's HCDR2:TIYPGNGDTSYNQKFKG (SEQ ID NO:7) 23C10A6B5's HCDR3:MGDNEYFDY (SEQ ID NO:8) 23C10A6B5's LCDR1:RASGNIHNYLA (SEQ ID NO:9) LCDR2 of 23C10A6B5:DAETLAD (SEQ ID NO:10) 23C10A6B5's LCDR3:QHFWSTPYT (SEQ ID NO:11) SEQ ID NO:12 of the heavy chain variable region of humanized antibody 230 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYIIHWVRQAPGQGLEWMGTIYPGNGDTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARMGDNEYFDYWGQGTLVTVSS SEQ ID NO:13 of the light chain variable region of humanized antibody 230 DIQMTQSPSSLSASVGDRVTITCRASGNIHNYLAWYQQKPGKAPKLLLYDAETLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWSTPYTFGGGTKVEIK 230 antibody-IL heavy chain CDR1 SEQ ID NO:14 SYLIH 230 Antibody-NG Heavy Chain CDR2 SEQ ID NO:15 TIYPGGGDTSYNQKFKG 230 Antibody-NA Heavy Chain CDR2 SEQ ID NO:16 TIYPGAGDTSYNQKFKG 230 Antibody-NS Heavy Chain CDR2 SEQ ID NO:17 TIYPGSGDTSYNQKFKG 230 Antibody-NR Heavy Chain CDR2 SEQ ID NO:18 TIYPGRGDTSYNQKFKG 230 Antibody-DI Light Chain CDR2 SEQ ID NO:19 DAETLAI 230 Antibody-DE Light Chain CDR2 SEQ ID NO:20 DAETLAE 230IL-DI Heavy Chain Variable Region SEQ ID NO:21 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYLIHWVRQAPGQGLEWMGTIYPGNGDTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARMGDNEYFDYWGQGTLVTVSS 230IL-NG-DI Heavy Chain Variable Region SEQ ID NO:22 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYLIHWVRQAPGQGLEWMGTIYPGGGDTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARMGDNEYFDYWGQGTLVTVSS 230IL-NR-DI Heavy Chain Variable Region SEQ ID NO:23 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYLIHWVRQAPGQGLEWMGTIYPGRGDTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARMGDNEYFDYWGQGTLVTVSS 230NG-DI Heavy Chain Variable Region SEQ ID NO:24 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYIIHWVRQAPGQGLEWMGTIYPGGGDTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARMGDNEYFDYWGQGTLVTVSS 230NR-DI Heavy Chain Variable Region SEQ ID NO:25 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYIIHWVRQAPGQGLEWMGTIYPGRGDTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARMGDNEYFDYWGQGTLVTVSS Variable region of 230IL-DI light chain, SEQ ID NO:26 DIQMTQSPSSLSASVGDRVTITCRASGNIHNYLAWYQQKPGKAPKLLLYDAETLAIGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWSTPYTFGGGTKVEIK Variable region of 230IL-DE light chain, SEQ ID NO:27 DIQMTQSPSSLSASVGDRVTITCRASGNIHNYLAWYQQKPGKAPKLLLYDAETLAEGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWSTPYTFGGGTKVEIK Heavy chain of antibody 237011, SEQ ID NO:28 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYLIHWVRQAPGQGLEWMGTIYPGGGDTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARMGDNEYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK Heavy chain of antibody 2391, SEQ ID NO:29 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYIIHWVRQAPGQGLEWMGTIYPGGGDTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARMGDNEYFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK The anti-BDCA2 antibody HCDR1 SYX1IH, where X1 is either I or L (SEQ ID NO: 30) The anti-BDCA2 antibody HCDR2 TIYPGX2GDTSYNQKFKG,X2 is either N, G, A, S, or R (SEQ ID NO: 31). The anti-BDCA2 antibody LCDR2 DAETLAX3,X3 is either I or E (SEQ ID NO: 32).

[0179] All documents relating to the present invention are cited herein by reference, so that each document may be cited independently. Furthermore, after reading the foregoing, those skilled in the art may make various variations and modifications to the present invention, and it should be understood that equivalent forms of these variations are included within the scope of the claims of the present invention.

Claims

1. It includes a heavy chain variable region and a light chain variable region, the heavy chain variable region including heavy chain complementarity determination regions H-CDR1, H-CDR2, and H-CDR3, where, The amino acid sequence of H-CDR1 is SYX １ As shown in IH (SEQ ID NO: 30), The amino acid sequence of H-CDR2 is TIYPGX ２ As shown in GDTSYNQKFKG (SEQ ID NO: 31), The amino acid sequence of H-CDR3 is shown in MGDNEYFDY (SEQ ID NO: 8), and The light chain variable region includes light chain complementarity determination regions L-CDR1, L-CDR2, and L-CDR3, where, The amino acid sequence of L-CDR1 is shown in RASGNIHNYLA (SEQ ID NO: 9), The amino acid sequence of L-CDR2 is DAETLAX ３ (SEQ ID NO: 32) The amino acid sequence of L-CDR3 is shown in QHFWSTPYT (SEQ ID NO: 11), Here, X １ is I or L, X ２ is selected from amino acids in the group consisting of N, G, A, S, and R, and / or X ３ It is selected from amino acids in the group consisting of D, I, or E. An antibody or antigen-binding fragment thereof that binds to BDCA2, characterized in that

2. X １ 、 X ２ 、 X ３ are X １ is I and X ２ N is X ３ It is D, X １ L is X ２ N is X ３ is I, X １ L is X ２ N is X ３ E is, X １ L is X ２ G is X ３ is I, X １ L is X ２ G is X ３ E is, X １ L is X ２ R is X ３ is I, X １ L is X ２ R is X ３ E is, X １ is I and X ２ G is X ３ is I, X １ is I and X ２ G is X ３ E is, X １ is I and X ２ R, X ３ is I, or X １ is I and X ２ R is X ３ E is The antibody or antigen-binding fragment according to claim 1, characterized by being selected from a group.

3. The heavy chain variable region includes heavy chain complementarity determination regions H-CDR1, H-CDR2, and H-CDR3, where, The amino acid sequence of H-CDR1 is shown in SYIIH (SEQ ID NO: 6), The amino acid sequence of H-CDR2 is shown as TIYPGNGDTSYNQKFKG (SEQ ID NO: 7), The amino acid sequence of H-CDR3 is shown in MGDNEYFDY (SEQ ID NO: 8), and The light chain variable region includes light chain complementarity determination regions L-CDR1, L-CDR2, and L-CDR3, where, 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), The antibody or antigen-binding fragment according to claim 1, characterized in that any amino acid sequence in the amino acid sequence includes a derivative sequence in which at least one arbitrarily selected amino acid is added, deleted, modified, and / or substituted, thereby maintaining the binding affinity of BDCA2.

4. The BDCA2-binding antibody or antigen-binding fragment according to claim 1, characterized in that the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment that binds to BDCA2 is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 5, or the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment that binds to BDCA2 is shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 13, or the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment that binds to BDCA2 is selected from SEQ ID NO: 21, 22, 23, 24 or 25, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 26 or 27.

5. The antibody or its antigen-binding fragment according to claim 1, characterized in that the antibody is selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, or combinations thereof.

6. (i) an antibody that binds to BDCA2 according to any one of claims 1 to 5 or an antigen-binding fragment thereof, (ii) Recombinant protein comprising an optionally selected tag sequence that supports expression and / or purification.

7. A nucleotide molecule characterized by encoding an antibody that binds to BDCA2 according to any one of claims 1 to 5 or an antigen-binding fragment thereof.

8. An expression vector characterized by comprising the nucleotide molecule described in claim 7.

9. A host cell characterized by containing the expression vector described in claim 8.

10. a) A step of culturing the host cells described in claim 9 under expression conditions to express an antibody that binds to BDCA2 or an antigen-binding fragment thereof, b) The step of isolating and purifying the antibody or antigen-binding fragment that binds to BDCA2 as described in a) A method for preparing an antibody or antigen-binding fragment that binds to BDCA2 according to any one of claims 1 to 5, characterized by including the following:

11. A composition comprising an antibody or antigen-binding fragment thereof that binds to BDCA2 according to any one of claims 1 to 5, and a pharmaceutically acceptable vector.

12. Use of an antibody or antigen-binding fragment that binds to BDCA2 according to any one of claims 1 to 5, or the composition according to claim 11, in the preparation of a drug for treating an inflammatory disease or an autoimmune disease.

13. The use according to claim 12, characterized in that the inflammatory disease or autoimmune disease is selected from systemic lupus erythematosus, cutaneous lupus erythematosus, discoid lupus erythematosus, lupus nephritis, cutaneous lupus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type 1 diabetes mellitus, dermatomyositis, and polymyositis.

14. (a) an antibody moiety comprising an antibody that binds to BDCA2 according to any one of claims 1 to 5 or an antigen-binding fragment thereof, (b) An antibody-drug conjugate comprising a binding site selected from the group consisting of a measurable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

15. The scFv segment of the monoclonal antibody antigen-binding region is a binding region that specifically binds to BDCA2, and the heavy chain variable region of the scFv is The amino acid sequence of H-CDR1 is shown as SYIIH (SEQ ID NO: 6), the amino acid sequence of H-CDR2 is shown as TIYPGNGDTSYNQKFKG (SEQ ID NO: 7), and the amino acid sequence of H-CDR3 is shown as MGDNEYFDY (SEQ ID NO: 8), and it includes the heavy chain complementarity determining regions H-CDR1, H-CDR2, and H-CDR3, and The light chain variable region of the scFv is, 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), and the amino acid sequence of L-CDR3 is shown in QHFWSTPYT (SEQ ID NO: 11), and it contains light chain complementarity determining regions L-CDR1, L-CDR2, and L-CDR3. Herein, the CAR construct is characterized in that any amino acid sequence in the above amino acid sequence includes a derivative sequence in which at least one arbitrarily selected amino acid is added, deleted, modified, and / or substituted, thereby maintaining the binding affinity of BDCA2.

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