Anti-BDCA2 antibody

Abstract

Provided is an anti-BDCA2 antibody. The anti-BDCA2 antibody can bind to a BDCA2 antigen with high specificity, has high affinity, and can effectively inhibit CpG-A from stimulating PBMCs to secrete IFNa and IgM, and thus is expected to serve as a drug for treating autoimmune diseases.

Description

Anti-BDCA2 antibody Technical Field

[0001] The present invention relates to the field of antibodies, and in particular, to an anti-BDCA2 antibody. Background Art

[0002] Type I interferon (IFN-I) is an inflammatory mediator produced primarily by plasmacytoid dendritic cells (pDCs) and is a component of the innate immune system. Type I IFN genes are tightly regulated. Normally, IFN-α production is barely detectable in healthy individuals. However, many patients with systemic autoimmune diseases show signs of persistent type I IFN production, and the expression of IFN-α regulatory genes is increased. IFN-α activates both the innate and adaptive immune systems. When individuals have a genetic predisposition that results in strong IFN-α production and / or a significant IFN-α response, tolerance is disrupted and antibodies against self-antigens are produced. These antibodies, together with the self-antigens, form interfering ICs, stimulating pDCs to synthesize IFN-α, and stimulating B cells to promote the production of autoantibodies, leading to a vicious cycle of persistent IFN-α production and autoimmune reactions.

[0003] Blood dendritic cell antigen 2 (BDCA2), also named CLEC4C or CD303, is specifically expressed only on pDCs. BDCA2 is triggered to activate SYK protein, leading to the activation of a complex composed of BLNK, BTK, and PLCγ2, which in turn mobilizes intracellular Ca 2+ , inhibiting the release of inflammatory factors mediated by TLR7 and TLR9 signaling pathways, such as IFN-I, IL-6, etc.

[0004] BDCA2-targeted drugs can intervene in pDC cell function, rapidly internalizing BDCA2 from the pDC surface and indirectly inhibiting the release of IFN-I and pro-inflammatory cytokines. Furthermore, BDCA2 signaling is unaffected by other IFN-I-inducing stimuli, making BDCA2 targeting a novel treatment option for autoimmune diseases. However, existing BDCA2-targeted drugs still have many shortcomings, and there is a need for new anti-BDCA2 antibodies in the field.

[0005] Summary of the Invention

[0006] The object of the present invention is to provide an anti-BDCA2 antibody.

[0007] In a first aspect of the present invention, an antibody or antigen-binding fragment thereof that binds to BDCA2 is provided, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain complementary determining regions H-CDR1, H-CDR2, and H-CDR3, wherein

[0008] The amino acid sequence of H-CDR1 is shown as SYX1IH (SEQ ID NO: 30).

[0009] The amino acid sequence of H-CDR2 is shown as TIYPGX2GDTSYNQKFKG (SEQ ID NO: 31).

[0010] The amino acid sequence of H-CDR3 is shown as MGDNEYFDY (SEQ ID NO: 8), and

[0011] The light chain variable region includes light chain complementary determining regions L-CDR1, L-CDR2, and L-CDR3; wherein,

[0012] The amino acid sequence of L-CDR1 is shown as RASGNIHNYLA (SEQ ID NO: 9),

[0013] The amino acid sequence of L-CDR2 is shown as DAETLAX3 (SEQ ID NO: 32).

[0014] The amino acid sequence of L-CDR3 is shown as QHFWSTPYT (SEQ ID NO: 11);

[0015] wherein X1 is I or L; X2 is selected from the group consisting of amino acids: N, G, A, S, R; and / or X3 is selected from the group consisting of amino acids: D, I or E.

[0016] In another preferred embodiment, X1, X2, and X3 are selected from the following groups:

[0017] X1 is I, X2 is N, and X3 is D;

[0018] X1 is L, X2 is N, and X3 is I;

[0019] X1 is L, X2 is N, and X3 is E;

[0020] X1 is L, X2 is G, and X3 is I;

[0021] X1 is L, X2 is G, and X3 is E;

[0022] X1 is L, X2 is R, and X3 is I;

[0023] X1 is L, X2 is R, and X3 is E;

[0024] X1 is I, X2 is G, and X3 is I;

[0025] X1 is I, X2 is G, and X3 is E;

[0026] X1 is I, X2 is R, and X3 is I; or

[0027] X1 is I, X2 is R, and X3 is E.

[0028] In another preferred embodiment, the heavy chain variable region includes heavy chain complementary determining regions H-CDR1, H-CDR2, and H-CDR3, wherein

[0029] The amino acid sequence of H-CDR1 is shown as SYIIH (SEQ ID NO: 6).

[0030] The amino acid sequence of H-CDR2 is shown as TIYPGNGDTSYNQKFKG (SEQ ID NO: 7).

[0031] The amino acid sequence of H-CDR3 is shown as MGDNEYFDY (SEQ ID NO: 8), and

[0032] The light chain variable region includes light chain complementary determining regions L-CDR1, L-CDR2, and L-CDR3; wherein,

[0033] The amino acid sequence of L-CDR1 is shown as RASGNIHNYLA (SEQ ID NO: 9),

[0034] The amino acid sequence of L-CDR2 is shown as DAETLAD (SEQ ID NO: 10).

[0035] The amino acid sequence of L-CDR3 is shown as QHFWSTPYT (SEQ ID NO: 11);

[0036] Any of the above amino acid sequences may also include derivative sequences that are optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain the BDCA2 binding affinity.

[0037] In another preferred embodiment, the ratio of the affinity F1 of the derivatized antibody for binding to BDCA2 to the affinity F0 of the corresponding non-derivatized antibody for binding to BDCA2 (F1 / F0) is 0.5-4, preferably 0.7-1.5, and more preferably 0.8-1.2.

[0038] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-5 (such as 1-3, preferably 1-2, more preferably 1).

[0039] In another preferred embodiment, the derivative sequence includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region includes a heavy chain complementarity determining region having the following amino acid sequence:

[0040] H-CDR1 with the amino acid sequence SYX1IH, wherein X1 is I or L; and / or

[0041] H-CDR2 with an amino acid sequence of TIYPGX2GDTSYNQKFKG, wherein X2 is selected from the group consisting of N, G, A, S, R (preferably G); and / or

[0042] The light chain variable region includes a light chain complementarity determining region having the following amino acid sequence:

[0043] L-CDR2 with an amino acid sequence as shown in DAETLAX3, wherein X3 is selected from the group consisting of amino acids D, I, or E;

[0044] Here, X1, X2, and X3 are each independently not I, N, or D at the same time.

[0045] In another preferred embodiment, in the derivative sequence, X1, X2, and X3 are selected from the following groups:

[0046] X1 is L, X2 is N, and X3 is I;

[0047] X1 is L, X2 is N, and X3 is E;

[0048] X1 is L, X2 is G, and X3 is I;

[0049] X1 is L, X2 is G, and X3 is E;

[0050] X1 is L, X2 is R, and X3 is I;

[0051] X1 is L, X2 is R, and X3 is E;

[0052] X1 is I, X2 is G, and X3 is I;

[0053] X1 is I, X2 is G, and X3 is E;

[0054] X1 is I, X2 is R, and X3 is I; or

[0055] X1 is I, X2 is R, and X3 is E.

[0056] In another preferred embodiment, in the derived sequence, the heavy chain variable region comprises

[0057] The amino acid sequence of H-CDR1 is shown in SYLIH (SEQ ID NO: 14),

[0058] H-CDR2 with the amino acid sequence TIYPGGGDTSYNQKFKG (SEQ ID NO: 15),

[0059] H-CDR3 with the amino acid sequence MGDNEYFDY (SEQ ID NO: 8), and

[0060] The light chain variable region comprises

[0061] The amino acid sequence of L-CDR1 is shown as RASGNIHNYLA (SEQ ID NO: 9),

[0062] The amino acid sequence of L-CDR2 is shown in DAETLAE (SEQ ID NO: 20),

[0063] The amino acid sequence of L-CDR3 is shown as QHFWSTPYT (SEQ ID NO: 11).

[0064] In another preferred embodiment, in the derived sequence, the heavy chain variable region comprises

[0065] The amino acid sequence of H-CDR1 is shown in SYIIH (SEQ ID NO: 6),

[0066] H-CDR2 with the amino acid sequence TIYPGGGDTSYNQKFKG (SEQ ID NO: 15),

[0067] H-CDR3 with the amino acid sequence MGDNEYFDY (SEQ ID NO: 8), and

[0068] The light chain variable region comprises

[0069] The amino acid sequence of L-CDR1 is shown as RASGNIHNYLA (SEQ ID NO: 9),

[0070] The amino acid sequence of L-CDR2 is shown in DAETLAE (SEQ ID NO: 20),

[0071] The amino acid sequence of L-CDR3 is shown as QHFWSTPYT (SEQ ID NO: 11).

[0072] In another preferred embodiment, in the derived sequence, the heavy chain variable region comprises

[0073] The amino acid sequence of H-CDR1 is shown in SYLIH (SEQ ID NO: 14),

[0074] The amino acid sequence of H-CDR2 is shown as TIYPGNGDTSYNQKFKG (SEQ ID NO: 7),

[0075] H-CDR3 with the amino acid sequence MGDNEYFDY (SEQ ID NO: 8), and

[0076] The light chain variable region comprises

[0077] The amino acid sequence of L-CDR1 is shown as RASGNIHNYLA (SEQ ID NO: 9),

[0078] The amino acid sequence of L-CDR2 is shown in DAETLAE (SEQ ID NO: 20),

[0079] The amino acid sequence of L-CDR3 is shown as QHFWSTPYT (SEQ ID NO: 11).

[0080] In another preferred embodiment, the antibody is a murine antibody, a chimeric antibody or a humanized antibody.

[0081] In another preferred example, the heavy chain variable region of the antibody further includes a human framework region, and / or the light chain variable region of the antibody further includes a human framework region.

[0082] In another preferred example, the heavy chain variable region of the antibody further includes a framework region of mouse origin, and / or the light chain variable region of the antibody further includes a framework region of mouse origin.

[0083] In another preferred embodiment, the heavy chain constant region is of human origin, and / or the light chain constant region is of human origin.

[0084] In another preferred embodiment, the heavy chain constant region is a human antibody heavy chain IgG1 constant region, and the light chain constant region is a human antibody light chain kappa constant region.

[0085] In another preferred embodiment, the heavy chain constant region comprises an Fc segment.

[0086] In another preferred embodiment, the Fc segment is an Fc variant, and the engineered Fc variant comprises the following mutations: M428L / N434S; G236A / S239D / I332E; S239D / I332E / A330L; S239D / I332E / G236A; S298A; A330L; I332E; E333A; and / or K334A;

[0087] The numbering of the residues in the Fc variant is that of the EU index as in Kabat.

[0088] In another preferred embodiment, the antibody is selected from the following group: animal-derived antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, or a combination thereof.

[0089] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0090] In another preferred embodiment, the antibody is a full-length antibody protein or an antigen-binding fragment.

[0091] In another preferred embodiment, the antibody is a bispecific antibody or a multispecific antibody.

[0092] In another preferred embodiment, the antibody is in the form of a drug conjugate.

[0093] In another preferred embodiment, the affinity of the antibody to the antigen BDCA2 is ≤1E-9M; preferably ≤1E-10M; more preferably ≤1E-11M.

[0094] In another preferred embodiment, the BDCA2 is human BDCA2; preferably, it is the extracellular domain of human BDCA2.

[0095] In another preferred embodiment, the sequence of the human BDCA2 extracellular domain is shown in SEQ ID NO: 1.

[0096] In another preferred embodiment, the antigen-binding fragment includes a Fab fragment, a F(ab')2 fragment, a Fv fragment, and a scFv.

[0097] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof 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.

[0098] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof 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.

[0099] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof 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.

[0100] 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.

[0101] 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.

[0102] In a second aspect of the present invention, a recombinant protein is provided, comprising:

[0103] (i) an antibody or antigen-binding fragment thereof that binds to BDCA2 according to the first aspect of the present invention; and

[0104] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0105] In another preferred embodiment, the tag sequence includes a 6His tag.

[0106] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0107] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

[0108] In a third aspect of the present invention, a nucleotide molecule is provided, which encodes the antibody or antigen-binding fragment thereof that binds to BDCA2 according to the first aspect of the present invention.

[0109] In another preferred embodiment, the nucleotide sequence encoding the heavy chain variable region of the nucleotide molecule is shown as SEQ ID NO: 2, and the nucleotide sequence encoding the light chain variable region is shown as SEQ ID NO: 4.

[0110] In the fourth aspect of the present invention, an expression vector is provided, wherein the expression vector contains the nucleotide molecule according to the third aspect of the present invention.

[0111] In the fifth aspect of the present invention, a host cell is provided, wherein the host cell contains the expression vector according to the fourth aspect of the present invention.

[0112] In a sixth aspect of the present invention, a method for preparing an antibody or antigen-binding fragment thereof that binds to BDCA2 according to the first aspect of the present invention is provided, the method comprising the following steps:

[0113] a) culturing the host cell according to the fifth aspect of the present invention under expression conditions to express the antibody or antigen-binding fragment thereof that binds to BDCA2;

[0114] b) isolating and purifying the antibody or antigen-binding fragment thereof that binds to BDCA2 described in a).

[0115] In a seventh aspect, the present invention provides a composition comprising the antibody or antigen-binding fragment thereof that binds to BDCA2 according to the first aspect of the present invention and a pharmaceutically acceptable carrier.

[0116] In an eighth aspect, the present invention provides use of the antibody or antigen-binding fragment thereof that binds to BDCA2 according to the first aspect of the present invention or the composition according to the seventh aspect of the present invention in the preparation of a medicament for treating inflammatory diseases or autoimmune diseases.

[0117] In another preferred embodiment, the inflammatory disease or autoimmune disease is selected from the group consisting of systemic lupus erythematosus, cutaneous lupus erythematosus, discoid lupus, lupus nephritis, cutaneous lupus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type I diabetes, dermatomyositis and polymyositis.

[0118] In a ninth aspect of the present invention, a CAR construct is provided, wherein the scFv segment of the monoclonal antibody antigen binding region of the CAR construct is a binding region that specifically binds to BDCA2, and the heavy chain variable region of the scFv comprises:

[0119] Heavy chain complementary determining regions H-CDR1, H-CDR2, and H-CDR3, wherein 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

[0120] The light chain variable region of the scFv comprises:

[0121] Light chain complementary determining regions L-CDR1, L-CDR2, and L-CDR3, wherein the amino acid sequence of L-CDR1 is shown as RASGNIHNYLA (SEQ ID NO: 9), the amino acid sequence of L-CDR2 is shown as DAETLAD (SEQ ID NO: 10), and the amino acid sequence of L-CDR3 is shown as QHFWSTPYT (SEQ ID NO: 11); wherein any of the above amino acid sequences further includes a derivative sequence that is optionally subjected to the addition, deletion, modification, and / or substitution of at least one amino acid and can retain BDCA2 binding affinity.

[0122] In another preferred embodiment, the derivative sequence includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region includes a heavy chain complementarity determining region having the following amino acid sequence:

[0123] H-CDR1 with the amino acid sequence SYX1IH, wherein X1 is I or L; and / or

[0124] H-CDR2 with an amino acid sequence of TIYPGX2GDTSYNQKFKG, wherein X2 is selected from the group consisting of N, G, A, S, R (preferably G); and / or

[0125] The light chain variable region includes a light chain complementarity determining region having the following amino acid sequence:

[0126] L-CDR2 with an amino acid sequence as shown in DAETLAX3, wherein X3 is selected from the group consisting of amino acids D, I, or E;

[0127] Here, X1, X2, and X3 are each independently not I, N, or D at the same time.

[0128] In the tenth aspect of the present invention, a recombinant immune cell is provided, wherein the immune cell expresses an exogenous CAR construct as described in the ninth aspect of the present invention.

[0129] In the eleventh aspect of the present invention, an antibody-drug conjugate is provided, wherein the antibody-drug conjugate comprises:

[0130] (a) an antibody portion comprising the antibody or antigen-binding fragment thereof according to the first aspect of the invention; and

[0131] (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0132] In a twelfth aspect of the present invention, a method for detecting BDCA2 protein in a sample in vitro is provided, the method comprising the steps of:

[0133] (1) contacting the sample in vitro with the antibody or antigen-binding fragment thereof according to the first aspect of the present invention or the antibody-drug conjugate according to the eleventh aspect of the present invention;

[0134] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of BDCA2 protein in the sample.

[0135] In a thirteenth aspect of the present invention, a method for preventing and / or treating a BDCA2-related disease is provided, comprising administering to a subject in need thereof the antibody or antigen-binding fragment thereof that binds to BDCA2 as described in the first aspect of the present invention, the composition as described in the seventh aspect of the present invention, the recombinant immune cell as described in the tenth aspect of the present invention, or the antibody-drug conjugate as described in the eleventh aspect of the present invention, or a combination thereof.

[0136] In another preferred embodiment, the BDCA2-related disease is selected from inflammatory diseases or autoimmune diseases, including but not limited to systemic lupus erythematosus, cutaneous lupus erythematosus, discoid lupus, lupus nephritis, cutaneous lupus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type I diabetes, dermatomyositis and polymyositis, or a combination thereof.

[0137] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0138] FIG1A-FIG1B show the binding ability of murine antibodies to human BDCA2-Fc protein.

[0139] FIG2A-FIG2B show the binding affinity determination of murine antibodies to the stably transfected cell line 293FT-BDCA2.

[0140] FIG3 shows the binding ability of human-mouse chimeric antibodies to human BDCA2-his protein.

[0141] FIG4 shows the inhibitory effect of human-mouse chimeric antibodies on the secretion of IFNa by PBMC induced by CpG-A.

[0142] FIG5 shows the ELISA screening of the effects of different mutation sites on the affinity of the 230 antibody.

[0143] FIG6 shows the affinity of anti-BDCA2 antibody mutants to BDCA2-his antigen protein detected by ELISA.

[0144] FIG7 shows the inhibitory effect of candidate antibodies on CpG-A-induced IFNa secretion by PBMCs using PBMCs.

[0145] FIG8 shows the inhibitory effect of candidate antibodies on CpG-A-induced IgM secretion by PBMC.

[0146] FIG9 shows the inhibitory effect of candidate antibodies on IFNα secretion in mice. DETAILED DESCRIPTION

[0147] After extensive and intensive research and extensive screening, the inventors unexpectedly discovered a series of murine antibodies with excellent binding activity to the target antigen BDCA2-Fc. Further candidate antibodies were selected and, through humanization and CDR modification, humanized antibodies were obtained with comparable or superior affinity and expression levels to the parental antibodies. The antibodies of the present invention effectively inhibit CpG-A-stimulated PBMC secretion of IFNα and IgM, with significantly superior activity compared to the positive control antibody litifilimab. This work was completed on this basis.

[0148] the term

[0149] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0150] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0151] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0152] Antibody

[0153] As used herein, the terms "antibody" or "immunoglobulin" are heterotetrameric glycoproteins of approximately 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0154] As used herein, the term "variable" refers to certain parts of the variable region in an antibody that are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-pleated configuration and are connected by three CDRs that form a connecting loop, and in some cases can form a partial β-pleated structure. The CDRs in each chain are closely together through the FR region and form the antigen-binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume 1, pages 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0155] The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two distinct classes, called kappa and lambda, based on the amino acid sequence of their constant regions. Immunoglobulins can be divided into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art.

[0156] Generally, an antibody's antigen-binding properties are described by three specific regions located in the variable regions of the heavy and light chains, known as the variable regions (CDRs). These regions are divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, spatially close to each other through the β-sheet formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antibody's antigen-binding site. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0157] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0158] In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human parts, can be obtained by standard DNA recombinant techniques, and they are all useful antibodies. A chimeric antibody is a molecule in which different parts are derived from different animal species, such as a chimeric antibody having a variable region from a mouse monoclonal antibody and a constant region from a human immunoglobulin (see, for example, U.S. Patent No. 4,816,567 and U.S. Patent No. 4,816,397, which are hereby incorporated by reference in their entirety). A humanized antibody refers to an antibody molecule derived from a non-human species, having one or more complementary determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Patent No. 5,585,089, which is hereby incorporated by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombinant techniques well known in the art.

[0159] In the present invention, the antibodies can be monospecific, bispecific, trispecific, or more multispecific.

[0160] In the present invention, the antibodies of the present invention also include conservative variants thereof, which refer to polypeptides in which no more than 10, preferably no more than 8, more preferably no more than 5, and most preferably no more than 3 amino acids are replaced with amino acids having similar or similar properties, compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by making amino acid substitutions according to Table A.

[0161] Table A

[0162] Anti-BDCA2 antibodies

[0163] In the present invention, the antibody is an anti-BDCA2 antibody. The present invention provides a highly specific and high-affinity antibody against BDCA2, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region (VH) amino acid sequence and the light chain comprises a light chain variable region (VL) amino acid sequence.

[0164] Wherein, the heavy chain variable region includes heavy chain complementary determining regions H-CDR1, H-CDR2, and H-CDR3,

[0165] The amino acid sequence of H-CDR1 is shown as SYX1IH (SEQ ID NO: 30).

[0166] The amino acid sequence of H-CDR2 is shown as TIYPGX2GDTSYNQKFKG (SEQ ID NO: 31).

[0167] The amino acid sequence of H-CDR3 is shown as MGDNEYFDY (SEQ ID NO: 8), and

[0168] The light chain variable region includes light chain complementary determining regions L-CDR1, L-CDR2, and L-CDR3; wherein,

[0169] The amino acid sequence of L-CDR1 is shown as RASGNIHNYLA (SEQ ID NO: 9),

[0170] The amino acid sequence of L-CDR2 is shown as DAETLAX3 (SEQ ID NO: 32).

[0171] The amino acid sequence of L-CDR3 is shown as QHFWSTPYT (SEQ ID NO: 11);

[0172] wherein X1 is I or L; X2 is selected from the group consisting of amino acids: N, G, A, S, R; and / or X3 is selected from the group consisting of amino acids: D, I or E.

[0173] Preferably,

[0174] The amino acid sequence of H-CDR1 is shown as SYIIH (SEQ ID NO: 6).

[0175] The amino acid sequence of H-CDR2 is shown as TIYPGNGDTSYNQKFKG (SEQ ID NO: 7).

[0176] The amino acid sequence of H-CDR3 is shown as MGDNEYFDY (SEQ ID NO: 8), and

[0177] The light chain variable region includes light chain complementary determining regions L-CDR1, L-CDR2, and L-CDR3; wherein,

[0178] The amino acid sequence of L-CDR1 is shown as RASGNIHNYLA (SEQ ID NO: 9),

[0179] The amino acid sequence of L-CDR2 is shown as DAETLAD (SEQ ID NO: 10).

[0180] The amino acid sequence of L-CDR3 is shown as QHFWSTPYT (SEQ ID NO: 11);

[0181] Any of the above amino acid sequences may also include derivative sequences that are optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain the BDCA2 binding affinity.

[0182] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or replacing at least one amino acid sequence is preferably an amino acid sequence with a homology or sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

[0183] Methods for determining sequence homology or identity that are well known to those of ordinary skill in the art include, but are not limited to, Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991, and Carillo, H. and Lipman, D., SIAM J. Applied Biology. Math., 48:1073 (1988). The preferred method for determining identity is to obtain the largest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining identity 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 BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith-Waterman algorithm can also be used to determine identity.

[0184] Preferably, the antibody described herein is 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 antibody or polyclonal antibody prepared from the above antibodies. The monoclonal antibody can be prepared by a variety of approaches and technologies, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. The mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice through hybridoma technology.

[0185] The full-length antibody protein is a conventional full-length antibody protein in the art, comprising 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 fully human full-length antibody protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3, or IgG4.

[0186] The antibodies of the present invention (anti-BDCA2 antibodies) can be full-length proteins (such as IgG1, IgG2a, IgG2b or IgG2c) or protein fragments containing the antigen-antibody binding domain (such as Fab, F(ab'), sdAb, ScFv fragments).

[0187] The antibody of the present invention (anti-BDCA2 antibody) can be a wild-type protein or a mutant protein that has undergone specific mutations to achieve a specific effect, for example, mutations that eliminate the effector function of the antibody.

[0188] 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, more preferably humanized antibodies, human-animal chimeric antibodies, and even more preferably fully humanized antibodies.

[0189] The antibody derivatives of the present invention can be 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 of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0190] The single-chain antibody is a conventional single-chain antibody in the art, which includes a heavy chain variable region, a light chain variable region and a short peptide of 15 to 20 amino acids.

[0191] The animal is preferably a mammal, such as a mouse.

[0192] As used herein, "Fc variant" or "variant Fc" means a protein comprising modifications in the Fc domain. The Fc variants of the present invention are defined according to the amino acid modifications that constitute them. Thus, for example, N434S or 434S is an Fc variant having a substituted serine at position 434 relative to the parent Fc polypeptide, wherein numbering is according to the EU index. Similarly, M428L / N434S defines an Fc variant having substitutions M428L and N434S. Relative to the parent Fc polypeptide. The identity of the wild-type amino acids may be non-specific, in which case the variant is referred to as 428L / 434S. Note that the order of substitutions is provided arbitrarily, that is, for example, 428L / 434S is the same Fc variant as M428L / N434S, and so on. For all positions discussed herein, numbering is according to the EU index. The EU index or EU index as in Kabat or EU numbering scheme refers to the EU numbering of antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, which is incorporated herein by reference in its entirety). Modifications can be additions, deletions, or substitutions. Substitutions can include naturally occurring amino acids and non-naturally occurring amino acids. Variants can include non-natural amino acids. Examples include US 6,586,207; WO 98 / 48032; WO 03 / 073238; US 2004-0214988 Al; WO 05 / 35727 A2; WO 05 / 74524 A2; JW Chin et al., (2002), Journal of the American Chemical Society 124:9026-9027; JW Chin and P.G. Schultz, (2002), Chem Bio Chem 11:1135-1137; JW Chin, et al., (2002), PIA S United States of America 99:11020-11024; and L. Wang and P.G. Schultz, (2002), Chem. 1-10, all of which are incorporated by reference in their entirety.

[0193] The antibody of the present invention may be a chimeric antibody, a humanized antibody, a CDR-grafted and / or modified antibody targeting BDCA2 (eg, human BDCA2).

[0194] Furthermore, the CDR regions of the mouse antibody were transplanted onto the selected humanized template, replacing the CDR regions of the human template. Chimeric antibodies were generated by recombining the heavy chain variable region with the human IgG1 constant region, and the light chain variable region with the human kappa chain constant region. Based on the three-dimensional structure of this antibody, back mutations were performed on buried residues, residues that directly interact with the CDR regions, and residues that have a significant impact on the antibody's VL and VH conformations, resulting in multiple humanized antibodies.

[0195] In the above content of 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%, more preferably 15-20%.

[0196] In the above content of the present invention, more preferably, the number of amino acids added, deleted, modified and / or substituted may be 1-7, more preferably 1-5, more preferably 1-3, more preferably 1-2.

[0197] Preferably, the humanized antibody comprises a heavy chain variable region and a light chain variable region, and relative to the murine antibody, the heavy chain variable region comprises a heavy chain complementary determining region having the following amino acid sequence:

[0198] H-CDR1 with the amino acid sequence SYX1IH, wherein X1 is I or L; and / or

[0199] H-CDR2 with an amino acid sequence of TIYPGX2GDTSYNQKFKG, wherein X2 is selected from the group consisting of N, G, A, S, R (preferably G); and / or

[0200] The light chain variable region includes a light chain complementarity determining region having the following amino acid sequence:

[0201] L-CDR2 with an amino acid sequence as shown in DAETLAX3, wherein X3 is selected from the group consisting of amino acids D, I, or E;

[0202] Here, X1, X2, and X3 are each independently not I, N, or D at the same time.

[0203] In another preferred embodiment, the heavy chain variable region and light chain variable region of the humanized antibody comprise a heavy chain complementary determining region and a light chain complementary determining region selected from the following group:

[0204] (1) H-CDR1 is selected from the following group:

[0205] SYIIH (SEQ ID NO: 6)

[0206] SYLIH (SEQ ID NO: 14)

[0207] (2) H-CDR2 is selected from the following group:

[0208] TIYPGNGDTSYNQKFKG(SEQ ID NO:7)

[0209] TIYPGGGDTSYNQKFKG(SEQ ID NO: 15)

[0210] TIYPGAGDTSYNQKFKG(SEQ ID NO: 16)

[0211] TIYPGSGDTSYNQKFKG(SEQ ID NO: 17)

[0212] TIYPGRGDTSYNQKFKG(SEQ ID NO: 18)

[0213] (3) H-CDR3 is MGDNEYFDY (SEQ ID NO: 8)

[0214] (4) L-CDR1 is RASGNIHNYLA (SEQ ID NO: 9)

[0215] (5) L-CDR2 is selected from the following group:

[0216] DAETLAD (SEQ ID NO: 10)

[0217] DAETLAI (SEQ ID NO: 19)

[0218] DAETLAE (SEQ ID NO: 20);

[0219] (6) L-CDR3 is QHFWSTPYT (SEQ ID NO: 11).

[0220] In another preferred example, the amino acid sequence of the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO. 3, 12, 21, 22, 23, 24 or 25, or has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity compared thereto.

[0221] In another preferred embodiment, the amino acid sequence of the light chain variable region contains 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 compared thereto.

[0222] In another preferred embodiment, the amino acid sequence of the heavy chain variable region (VH) and / or the amino acid sequence of the light chain variable region (VL) of the antibody targeting BDCA2 is as shown in Table B below:

[0223] Table B

[0224] recombinant protein

[0225] The present invention also provides a recombinant protein comprising the heavy chain CDR1 (H-CDR1), heavy chain CDR2 (H-CDR2), and heavy chain CDR3 (H-CDR3) of the BDCA2 antibody according to the 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.

[0226] The recombinant protein further comprises an antibody heavy chain constant region and / or an antibody light chain constant region. The antibody heavy chain constant region is conventional in the art, preferably a rat antibody heavy chain constant region or a human antibody heavy chain constant region, more preferably a human antibody heavy chain constant region. The antibody light chain constant region is conventional in the art, preferably a rat antibody light chain constant region or a human antibody light chain constant region, more preferably a human antibody light chain constant region.

[0227] The recombinant protein is a conventional protein in the art, 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 antibody or polyclonal antibody prepared from the above antibodies. The monoclonal antibody can be developed by a variety of approaches and technologies, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. The mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice through hybridoma technology.

[0228] The full-length antibody protein is a conventional full-length antibody protein in the art, comprising 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 fully human full-length antibody protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3, or IgG4.

[0229] The single-chain antibody is a conventional single-chain antibody in the art, which includes a heavy chain variable region, a light chain variable region and a short peptide of 15 to 20 amino acids.

[0230] The antigen-antibody binding domain protein fragment is a conventional antigen-antibody binding domain protein fragment in the art, which includes a light chain variable region, a light chain constant region, and an Fd segment of a heavy chain constant region. Preferably, the antigen-antibody binding domain protein fragment is Fab and F(ab').

[0231] The single-domain antibody is a conventional single-domain antibody in the art, which includes a heavy chain variable region and a heavy chain constant region.

[0232] The single-domain antibody is a conventional single-domain antibody in the art, which only includes the heavy chain variable region.

[0233] The recombinant protein preparation method is a conventional preparation method in the art. The preparation method is preferably: isolating and obtaining from a transformant expressing the recombinant protein or obtaining by artificially synthesizing the protein sequence. The isolation and obtaining from a transformant expressing the recombinant protein is preferably performed by cloning a nucleic acid molecule encoding the protein and carrying a point mutation into a recombinant vector, transforming the resulting recombinant vector into a transformant to obtain a recombinant expression transformant, and culturing the resulting recombinant expression transformant to isolate and purify the recombinant protein.

[0234] Nucleic Acids

[0235] The present invention also provides a nucleic acid encoding the heavy chain variable region or light chain variable region of the above-mentioned antibody (eg, anti-BDCA2 antibody) or recombinant protein or anti-BDCA2 antibody.

[0236] The method for preparing the nucleic acid is a conventional method in the art, and preferably comprises the following steps: obtaining a nucleic acid molecule encoding the above protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above protein by artificial full sequence synthesis.

[0237] Those skilled in the art will appreciate that the base sequence encoding the amino acid sequence of the aforementioned protein can be appropriately substituted, deleted, altered, inserted, or added to provide a polynucleotide homolog. The polynucleotide homologs 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.

[0238] carrier

[0239] The present invention also provides a recombinant expression vector comprising the nucleic acid.

[0240] The recombinant expression vector can be obtained by conventional methods in the art, namely, by ligating the nucleic acid molecule of the present invention to various expression vectors. The expression vector can be any conventional vector in the art, as long as it can carry the nucleic acid molecule. The vector preferably includes various plasmids, cosmids, phages, or viral vectors.

[0241] The present invention also provides a recombinant expression transformant comprising the above recombinant expression vector.

[0242] Wherein, the preparation method of the recombinant expression transformant is a conventional preparation method in the art, preferably: the above-mentioned recombinant expression vector is transformed into a host cell to obtain it. The host cell is various host cells conventional in the art, as long as it can satisfy the conditions for the above-mentioned recombinant expression vector to stably replicate on its own and the nucleic acid carried can be effectively expressed. Preferably, the host cell is E. coli TG1 or E. coli BL21 cell (expressing single-chain antibody or Fab antibody), or HEK293 or CHO cell (expressing full-length IgG antibody). The aforementioned recombinant expression plasmid is transformed into a host cell to obtain the preferred recombinant expression transformant of the present invention. The transformation method wherein is a conventional transformation method in the art, preferably a chemical transformation method, a heat shock method or an electroporation method.

[0243] Antibody preparation

[0244] The sequence of the DNA molecule of the antibody of the present invention or its fragment can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences of the light chain and heavy chain can be fused together to form a single-chain antibody.

[0245] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0246] In addition, the method of artificial synthesis can also be used to synthesize the relevant sequence, especially when the fragment length is shorter. Usually, by synthesizing multiple small fragments first and then connecting them, a very long fragment of sequence can be obtained.

[0247] Currently, DNA sequences encoding the antibodies (or fragments thereof, or derivatives thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the present invention through chemical synthesis.

[0248] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0249] The host cell can be a prokaryotic cell, such as a bacterial cell, 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.

[0250] Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present invention. The antibodies of the present invention are then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, among other conventional separation and purification methods well known to those skilled in the art.

[0251] The resulting monoclonal antibodies can be characterized using conventional methods. For example, the binding specificity of the monoclonal antibodies can be determined using immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, using the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0252] The antibodies of the present invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0253] Antibody-drug conjugates (ADCs)

[0254] The present invention also provides an antibody-drug conjugate (ADC) based on the antibody of the present invention.

[0255] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a therapeutically active drug. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

[0256] The antibody of the present invention and the effector molecule can be coupled via a coupling agent. Examples of the coupling agent may include any one or more of a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, and a coupling agent utilizing a disulfide bond. The non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde. The coupling agent utilizing a carboxyl group may include any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (where the coupling site is an acylhydrazone).

[0257] Certain residues on antibodies (such as Cys or Lys, etc.) are used to connect to a variety of functional groups, including imaging agents (such as chromophores and fluorescent groups), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers) and therapeutic agents. Antibodies can be coupled to functional agents to form antibody-functional agent conjugates. Functional agents (such as drugs, detection reagents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly connected to antibodies through linkers.

[0258] Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, ADCs contain a linker positioned between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers typically readily degrade in the intracellular environment, for example, at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including linkers containing peptidyl groups that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronide-containing linkers that can be degraded by glucuronidases. Peptide linkers can include, for example, 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 at a pH below 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug when the antibody is hydrolyzed by proteases.

[0259] Prior to attachment to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and attachment is achieved via the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimides, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethylketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, where the counter ion is acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. Linkers may include, for example, maleimides attached to the antibody via thiosuccinimide.

[0260] The drug can be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, a linker connects the antibody and the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group capable of forming a bond with the linker. In cases where the drug is directly attached to the linker, the drug has a reactive group prior to attachment to the antibody.

[0261] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, 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, and oxazolidinobenzodiazepines), and vinca alkaloids.

[0262] In the present invention, drug-linkers can be used to form ADCs in a single step. In other embodiments, bifunctional linker compounds can be used to form ADCs in a two-step or multi-step process. For example, a cysteine ​​residue is reacted with a reactive moiety of a linker in a first step, and in a subsequent step, the functional group on the linker reacts with the drug to form an ADC.

[0263] Typically, the functional group on the linker is selected to facilitate specific reaction with an appropriate reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Technology, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that, when a complementary pair of reactive functional groups is selected for selective reaction between the drug moiety and the linker, each member of the complementary pair can be used for both the linker and the drug.

[0264] The present invention also provides a method for preparing an ADC, which may further comprise: combining an antibody and a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

[0265] In certain embodiments, the methods of the present invention comprise conjugating an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the present invention further comprise conjugating the antibody-linker conjugate to a drug moiety under conditions sufficient to covalently attach the drug moiety to the antibody via the linker.

[0266] In some embodiments, the antibody drug conjugate ADC has the following molecular formula:

[0267] in:

[0268] Ab is an antibody.

[0269] LU is the connector;

[0270] D is for medicine;

[0271] And the subscript p is a value selected from 1 to 8.

[0272] Pharmaceutical composition

[0273] The present invention also provides a composition. In preferred embodiments, the composition is a pharmaceutical composition comprising the above-mentioned antibody, active fragment thereof, fusion protein thereof, ADC, or corresponding immune cell, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the condition to be treated.

[0274] The prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration. Typically, the route of administration of the pharmaceutical composition of the present invention is preferably injection or oral administration. The injection preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in the form of solid, semi-solid or liquid, and can be an aqueous solution, non-aqueous solution or suspension, more preferably tablets, capsules, granules, injections or infusions, etc.

[0275] The antibody of the present invention can also be expressed in cells by nucleotide sequences for cell therapy, for example, the antibody is used in chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

[0276] The pharmaceutical composition of the present invention is a pharmaceutical composition for preventing and / or treating diseases associated with abnormal BDCA2 expression or function.

[0277] The pharmaceutical composition of the present invention can be directly used to bind to BDCA2 protein molecules, and thus can be used to prevent and treat diseases such as tumors.

[0278] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned monoclonal antibody of the present invention (or its conjugate) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 μg / kg body weight to about 5 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used in conjunction with other therapeutic agents.

[0279] In the present invention, preferably, the pharmaceutical composition of the present invention further comprises one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutical carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, and preferably includes a pharmaceutically acceptable excipient, filler or diluent, etc. More preferably, the pharmaceutical composition comprises 0.01 to 99.99% of the above-mentioned protein and 0.01 to 99.99% of a pharmaceutical carrier, and the percentages are the mass percentages of the pharmaceutical composition.

[0280] In the present invention, the pharmaceutical composition is preferably administered in an effective amount, which is an amount capable of alleviating or delaying the progression of a disease, degenerative, or damaging condition. The effective amount can be determined on an individual basis and will be based in part on considerations of the symptoms to be treated and the desired outcome. Those skilled in the art can determine the effective amount by utilizing the above factors on an individual basis and using no more than routine experimentation.

[0281] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to about 20 mg / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0282] The present invention provides the use of the above-mentioned pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease associated with abnormal BDCA2 expression or function. Preferably, the disease associated with abnormal BDCA2 expression or function is an inflammatory disease or an autoimmune disease.

[0283] application

[0284] The present invention also provides uses of the antibodies, antibody conjugates ADCs, recombinant proteins, and / or immune cells of the present invention, for example, for preparing diagnostic preparations or preparing drugs.

[0285] Preferably, the drug is a drug for preventing and / or treating diseases associated with abnormal BDCA2 expression or function.

[0286] In the present invention, the disease associated with abnormal BDCA2 expression or function is a conventional disease associated with abnormal BDCA2 expression or function in the art. Preferably, the disease associated with abnormal BDCA2 expression or function is an inflammatory disease or autoimmune disease, including but not limited to systemic lupus erythematosus, cutaneous lupus erythematosus, discoid lupus, lupus nephritis, cutaneous lupus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type I diabetes, dermatomyositis, polymyositis, or a combination thereof.

[0287] The main advantages of the present invention include

[0288] (1) The mouse anti-BDCA2 antibody of the present invention has excellent affinity, which is superior to the positive control antibody litifilimab; the humanized anti-BDCA2 antibody also has higher affinity.

[0289] (2) The BDCA2 antibody of the present invention can effectively inhibit CpG-A from stimulating PBMC to secrete IFNa and IgM, and is expected to be used as a drug for treating autoimmune diseases.

[0290] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which detailed conditions are not specified, were generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0291] Example 1 Antigen Immunization of Animals and Preparation and Screening of Hybridomas

[0292] 1.1 Preparation of antigen protein and positive control antibody

[0293] The human BDCA2 extracellular domain (BDCA2-ECD) sequence, used as an antigen, was obtained from the UniProt database (entry: Q8WTT0), with the amino acid sequence shown in SEQ ID NO: 1. A 6×His tag was added to its C-terminus and constructed into the pcDNA 3.4 expression vector. The protein was transfected into HEK-293F cells. After 5 days of expression, the cell culture supernatant was collected and purified to obtain the BDCA2-his protein. Similarly, the 6×His tag was replaced with the human IgG1 Fc sequence and transfected into HEK-293F cells. The BDCA2-Fc protein was expressed and purified.

[0294] The heavy and light chain sequences of the positive control antibody litifilimab (BIIB059, 24F4A) were derived from patent CN111961134A. The heavy and light chain variable regions of this antibody were synthesized and recombined with the human heavy chain IgG1 and light chain kappa constant regions, respectively. These regions were then constructed into the pcDNA 3.4 expression vector, expressed using the HEK-293F system, and purified using Protein A affinity chromatography.

[0295] 1.2 Immunization of mice

[0296] Balb / c mice were routinely immunized with human BDCA2-His protein (prepared in-house, purity >95%) expressed in mammalian cell line 293F. On day 1, soluble human BDCA2-His protein was emulsified with Freund's complete adjuvant and injected subcutaneously at multiple sites into Balb / c mice (human BDCA2-His 100 μg / mouse / 0.5 mL). On day 14, soluble human BDCA2-His protein was emulsified with Freund's incomplete adjuvant and injected subcutaneously into Balb / c mice (human BDCA2-His 50 μg / mouse / 0.5 mL). On day 28, soluble human BDCA2-His protein was emulsified with Freund's incomplete adjuvant and injected subcutaneously into Balb / c mice (human BDCA2-His 50 μg / mouse / 0.5 mL). Three weeks later, soluble human BDCA2-His protein (50 μg / mouse / 0.2 mL) was injected intraperitoneally for challenge. 3-4 days later, the spleens of the mice were harvested for fusion experiments.

[0297] 1.3 Hybridoma cell preparation and screening

[0298] Three to four days after the last immunization of mice, mouse spleen cells were fused with mouse myeloma SP2 / 0 cells using a conventional hybridoma technology protocol. The fused cells were evenly suspended in complete culture medium consisting of RPMI1640-GLUMAX supplemented with 1% penicillin-streptomycin, 20% FBS (fetal bovine serum), and 1% HAT. The fused cells were plated at 3 x 10 4 10 cells / 200 μL / well were plated and cultured in 60 96-well plates. After 7-12 days, the supernatant was harvested and the hybridoma wells positive for human BDCA2 binding activity were screened by ELISA.

[0299] The ELISA method for screening hybridoma wells with positive human BDCA2 binding activity is as follows: BDCA2-Fc is diluted to 1 μg / mL in PBS buffer, 100 μL / well is added to the plate, and incubated overnight at 4°C; the next day, the supernatant is discarded, and the plate is blocked with 5% skim milk powder at 37°C for 1 hour. The plate is washed three times with PBST and set aside for use; the collected hybridoma supernatant is sequentially added to the blocked plate at 100 μL / well and incubated at 37°C for 1 hour; the plate is washed three times with PBST, and HRP-labeled goat anti-mouse IgG secondary antibody is added and incubated at 37°C for 30 minutes; after washing the plate three times with PBST, any remaining droplets are patted dry on absorbent paper as much as possible, 100 μL of TMB is added to each well, and the plate is incubated at room temperature (20±5°C) in the dark for 5 minutes; 50 μL of 2M H2SO4 stop solution is added to each well to terminate the substrate reaction, and the OD value is read at 450 nm on a microplate reader to analyze the binding ability of the test antibody to the target antigen BDCA2. The hybridoma cell lines obtained by amplification and screening in serum-containing complete medium were centrifuged and replaced with serum-free medium Hybridoma-SFM medium to a cell density of 1-2 × 10 6 / mL, cultured at 8% CO2 and 37°C for 1 week, the culture supernatant was obtained by centrifugation, and purified by Protein G affinity chromatography to obtain anti-human BDCA2 monoclonal antibody protein. After screening, a total of 30 hybridoma cell lines were obtained.

[0300] 1.4 Binding ability of mouse antibodies to human BDCA2-Fc protein

[0301] The binding ability of mouse antibodies to human BDCA2-Fc protein was determined by enzyme-linked immunosorbent assay (ELISA). The specific method is as follows:

[0302] BDCA2-Fc protein was diluted to 1 μg / mL with PBS buffer, 100 μL / well was added to the plate, and incubated at 4°C overnight; then blocked with 5% skim milk powder and incubated at 37°C for 1 hour; after washing the plate 3 times with PBST, the anti-human BDCA2 mouse antibody prepared in the laboratory was diluted 3-fold from 10 μg / mL in 11 gradients with 1% BSA-PBS buffer, 1% BSA-PBS was used as a blank control, and 100 μL / well was added to the pre-coated BDCA2-Fc plate and incubated at 37°C for 1 hour; the plate was washed 3 times with PBST, HRP-labeled goat anti-mouse IgG secondary antibody was added, and the plate was placed at 37°C for 3 minutes; after washing the plate 3 times with PBST, the residual droplets were patted dry on absorbent paper as much as possible, 100 μL of TMB was added to each well, and the plate was placed in the dark at room temperature (20±5°C) for 5 minutes, and 50 μL of 2M The substrate reaction was terminated with H2SO4 stop solution, and the OD value was read at 450 nm on a microplate reader to analyze the binding ability of the test antibody to the antigen human BDCA2-Fc. The obtained data were fitted and analyzed by GraphPad Prism 9 software, and the results are shown in Figures 1A-1B.

[0303] As shown in Figures 1A and 1B , most of the mouse antibodies have good binding activity to the target antigen BDCA2-Fc, and the EC50 values ​​are shown in Tables 1 and 2 .

[0304] Table 1:

[0305] Table 2:

[0306] 1.5 Determination of the binding affinity of mouse antibodies to the stably transfected 293FT-BDCA2 cell line

[0307] In this example, the binding affinity of the mouse antibody to the target cell 293FT-BDCA2 was determined by flow cytometry fluorescence activated cell sorting (FACS).

[0308] In this experiment, 293FT-BDCA2 (an engineered cell line that highly expresses human BDCA2 using lentivirus in the laboratory) was used as the target cell. The cells were counted and washed once with 1% BSA-PBS. 2×10 5 / well, 100 μL / well was plated, the supernatant was removed by centrifugation, and the anti-human BDCA2 mouse antibody was serially diluted 3-fold from 10 μg / mL in 1% BSA-PBS for 11 steps. 1% BSA-PBS was used as a blank control. 100 μL of each was resuspended in 293FT-BDCA2 cell wells and incubated at 4°C for 1 hour. The cells were washed twice with PBS to remove unbound mouse antibody. The cells were then resuspended in 100 μL / well and 1:500 diluted PE-conjugated anti-mouse secondary antibody (purchased from Jackson, cat. no. 715116150) and incubated at 4°C for 1 hour. The cells were washed twice with PBS to remove unbound secondary antibody. Finally, the cells were resuspended in 200 μL PBS. The binding affinity of the mouse antibody to the cells was determined by flow cytometry. The data were fitted and analyzed by GraphPad Prism 9 software. The results are shown in Figures 2A-2B.

[0309] The results showed that most of the mouse antibodies could specifically bind to BDCA2 on the surface of 293FT cells, and the EC50 values ​​are shown in Tables 3 and 4.

[0310] Table 3:

[0311] Table 4:

[0312] Example 2 Humanization of murine anti-human BDCA2 monoclonal antibody

[0313] 2.1 Determination of the variable region sequence of the mouse anti-human BDCA2 monoclonal antibody

[0314] Hybridoma clone 23C10A6B5 was selected as a candidate antibody. 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). PCR was performed using a primer combination reported in the literature ("Antibody Engineering," Volume 1, Edited by Roland Kontermann and Stefan Dübel, primer combination sequence from page 323). The resulting PCR product was sequenced and analyzed using the Kabat database, confirming that the obtained sequence was the variable region sequence of a murine antibody. The sequence information is as follows:

[0315] 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.

[0316] The light chain variable region gene sequence is 321 bp in length, encoding 107 amino acid residues. The nucleotide sequence is shown in SEQ ID NO: 4, and the amino acid sequence is shown in SEQ ID NO: 5.

[0317] 2.2 Construction of human-mouse chimeric antibodies and verification of their activity

[0318] The mouse heavy chain variable region and light chain variable region sequences obtained in Example 2.1 (SEQ ID NO: 3 and SEQ ID NO: 5) were linked to the human IgG1 heavy chain constant region and the kappa light chain constant region, respectively, to construct the human-mouse chimeric antibody C-23C10, and its binding activity to the antigen BDCA2 and its inhibitory effect on CpG-A-induced IFNa secretion by PBMC were verified.

[0319] The binding activity was detected as in Example 1.4, except that the coating antigen BDCA2-Fc was replaced with BDCA2-his, the coating concentration was 1 μg / mL, and the secondary antibody was replaced with anti-human Fc HRP (SinoBiological, Cat. No. SSA001) at a working concentration of 1:3000.

[0320] As shown in FIG3 , the experimental results show that the binding activity of C-23C10 to BDCA2 antigen is better than that of the positive control 24F4A.

[0321] CpG-A (CpG ODNs Class A) is an oligodeoxynucleotide with a CpG palindromic sequence that can stimulate pDCs to secrete a large amount of IFNα. In this example, PBMCs were used to detect the inhibitory effect of candidate antibodies on CpG-A-induced IFNα secretion by PBMCs.

[0322] CpG-A was diluted to 8 μg / mL (final concentration of 2 μg / mL) in RPMI 1640 + 3% FBS medium and added to a flat-bottom 96-well plate at 50 μL per well. The antibody was diluted to 16 nM in RPMI 1640 + 3% FBS medium, diluted 5-fold (final concentration of 4 nM, diluted 5-fold), and added to the CpG-A wells at 50 μL per well. CpG-A and antibody were co-incubated for 1 hour in a 37°C, 5% CO2 incubator. PBMC cells were centrifuged and counted, and the cell density was adjusted to seed the above 96-well plates at 500,000 cells per well. 100 μL per well was added and incubated overnight in a 37°C, 5% CO2 incubator. The cell supernatant was centrifuged and the H-IFNa content in the supernatant was assayed.

[0323] The experimental results are shown in Figure 4. C-23C10 can effectively inhibit the secretion of IFNa by PBMC stimulated by CpG-A, and its activity is better than that of the positive control 24F4.

[0324] 2.3 Humanization of mouse anti-human BDCA2 monoclonal antibody

[0325] The amino acid sequences of the heavy and light chain variable regions of 23C10A6B5 were analyzed and three antigen complementarity determining regions (CDRs) and four framework regions (FRs) were determined according to the Kabat rules.

[0326] HCDR1: SYIIH (SEQ ID NO: 6),

[0327] HCDR2: TIYPGNGDTSYNQKFKG (SEQ ID NO: 7) and

[0328] HCDR3: MGDNEYFDY (SEQ ID NO: 8), the amino acid sequence of the light chain complementary determining region is

[0329] LCDR1:RASGNIHNYLA(SEQ ID NO:9),

[0330] LCDR2: DAETLAD (SEQ ID NO: 10) and

[0331] LCDR3: QHFWSTPYT (SEQ ID NO: 11).

[0332] By performing homology comparison with human IgG germline sequences in NCBI IgBlast, IGHV1-46*01 was selected as the heavy chain CDR grafting template, and IGKV1-NL1*01 was selected as the light chain CDR grafting template. The CDR regions of the 23C10A6B5 antibody were transplanted onto the selected humanized templates, replacing the CDR regions of the humanized templates. The heavy chain variable region was then recombined with the human IgG1 constant region, and the light chain variable region was recombined with the human kappa chain constant region. Based on the three-dimensional structure of the antibody, back mutations were performed on buried residues, residues directly interacting with the CDR regions, and residues that have a significant impact on the VL and VH conformations of the antibody. Multiple humanized antibodies were obtained, and the heavy chain variable region sequences (SEQ ID NO: 12) and light chain variable region sequences (SEQ ID NO: 13) of the humanized antibody 230 were determined through affinity screening.

[0333] Example 3 Affinity maturation of humanized antibody 230

[0334] Using AlphaFold2 software, we predicted the complex structure of the 230 antibody and BDCA2-his antigen and analyzed the interaction interface. We then constructed a mutant library by introducing single-point saturation mutations at key interaction sites. Through ELISA affinity screening, we identified mutations that improved the affinity of the 230 antibody. The experimental method was similar to that in Example 1.4, except that the coating antigen, BDCA2-Fc, was replaced with BDCA2-his. The coating concentration was 0.1 μg / mL, or 10 ng per well. The secondary antibody was anti-human Fc HRP (SinoBiological, Cat. No. SSA001), at a working concentration of 1:3000.

[0335] The results are shown in Figure 5. The experimental results show that

[0336] Heavy chain CDR1: (SEQ ID NO: 6) Mutated to L to obtain SYLIH (SEQ ID NO: 14), and the heavy chain CDR2: (SEQ ID NO: 7) Mutated to G, A, S, R respectively to obtain

[0337] TIYPGGGDTSYNQKFKG (SEQ ID NO: 15),

[0338] TIYPGAGDTSYNQKFKG (SEQ ID NO: 16),

[0339] TIYPGSGDTSYNQKFKG (SEQ ID NO: 17),

[0340] The TIYPGRGDTSYNQKFKG (SEQ ID NO: 18) mutations (corresponding to antibodies 230-I33L, 230-N55G, 230-N55A, 230-N55S and 230-N55R) can effectively improve the affinity of the 230 antibody.

[0341] In addition, the light chain CDR2: (SEQ ID NO: 10) Mutations to I or E, respectively, gave DAETLAI (SEQ ID NO: 19) and DAETLAE (SEQ ID NO: 20). The mutants maintained an affinity comparable to that of 230, while the expression levels increased by an average of more than 30%. The remaining saturated mutants had comparable affinity and expression levels to the parent 230.

[0342] The mutated selected parts of the above CDR regions were combined and connected with the IgG1 subtype constant region 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. Among them, the heavy chains of 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 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 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 230NG-DI and 230NG-DE antibodies are the same, and the heavy chain variable region sequence is shown in SEQ ID NO: 24; and the heavy chains of 230NR-DI and 230NR-DE antibodies are the same, and the heavy chain variable region sequence is shown in SEQ ID NO: 25.

[0343] The light chains of 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 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.

[0344] According to the above 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 BDCA2-his antigen protein was investigated.

[0345] The results are shown in Figure 6. The experimental results show that the affinity of the above 10 mutants to the antigen protein is higher than that of the parent monoclonal antibody 230.

[0346] Example 4 Determination of affinity dissociation constant KD value

[0347] The heavy chain FC regions of the 230IL-NG-DE and 230-NG-DE (also referred to as 230NG-DE) antibodies in Example 3 were simultaneously introduced with M428L / N434S and G236A / S239D / I332E mutations according to patents CN200880123009.4 and CN200480018985.5 to construct the 237011 antibody (heavy chain sequence such as SEQ ID NO: 28, light chain variable region sequence such as SEQ ID NO: 27) and the 2391 antibody (heavy chain sequence such as SEQ ID NO: 29, light chain variable region sequence such as SEQ ID NO: 27).

[0348] The kinetic parameters of binding and dissociation between the candidate antibody protein and the antigen BDCA2-his were determined using a capture method using a Biacore 8K molecular interaction analyzer. The antibody was diluted to 1 μg / mL in 1*HBS-N, pH 7.0 buffer containing 5 mM CaCl2, 0.02% Tween 20, and the antibody was captured using a Protein A chip. The antigen was diluted in 1*HBS-N, pH 7.0 buffer containing 5 mM CaCl2, 0.02% Tween 20, and bound to the antibody over six concentration gradients with a maximum concentration of 12.5 nM. The antibody then dissociated in 1*HBS-N, pH 7.0 buffer containing 5 mM CaCl2, 0.02% Tween 20.

[0349] The experimental results are shown in Table 5. The affinity of candidate antibodies 237011 and 2391 to the antigen BDCA2-his is higher than that of the positive control antibody 24F4A.

[0350] Table 5: Affinity dissociation constants

[0351] Note: KD is the affinity constant; ka is the association rate constant; kd is the dissociation rate constant.

[0352] Example 5 Inhibitory Effect of CpG-A on IFNα Secretion by PBMC

[0353] The inhibitory effect of the humanized candidate antibody on CpG-A-induced IFNa secretion by PBMC was verified, and the experimental method was referred to Example 2.2.

[0354] The experimental results are shown in Figure 7. The candidate antibodies 237011, 2391, 230IL-NG-DE and 230-NG-DE can effectively inhibit the secretion of IFNa by PBMC stimulated by CpG-A, and their activity is significantly better than that of the positive control antibody 24F4A.

[0355] Example 6 Inhibitory Effect of CpG-A on IgM Secretion by PBMC

[0356] To verify the inhibitory effect of humanized candidate antibodies on CpG-A-induced IgM secretion by PBMC, the experimental method was as follows: PBMC cells were resuspended in RPMI 1640 + 10% FBS medium and the cell density was adjusted to 5×10 6 / ml, 100 μL per well, added to a 3599 plate. Add 50 μL of CPG-A (CpG2216) to each well to a final concentration of 2 μg / mL; dilute the antibody in culture medium, adding 50 μL per well to a final concentration of 3000 nM. First, incubate 100 μL of cells and 50 μL of antibody at 37°C, 5% CO2 for 1 hour. Then, add 50 μL of CPG-A and place in a 37°C, 5% CO2 incubator. After 7 days of incubation, centrifuge and collect the cell supernatant, and assay the IgM content in the supernatant.

[0357] The experimental results are shown in Figure 8. The candidate antibodies 237011, 2391, 230IL-NG-DE and 230-NG-DE can all effectively inhibit CpG-A-stimulated PBMC secretion of IgM, and their activity is significantly better than the positive control antibody 24F4A.

[0358] Example 7 Inhibitory Effects on IFNa in Mice

[0359] To verify the inhibitory effect of the humanized candidate antibody on IFNa secretion in mice, since the candidate antibody does not cross-react with the BDCA2 homologous protein in mice, in vivo animal experiments were performed using reconstructed humanized immune system mice (HuHSC-NCG immune system humanized mice, purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.) established by injecting human huCD34+ embryonic stem cells.

[0360] The experimental plan was as follows: On Day 0, mice were weighed and randomly divided into three groups based on body weight: a model group (PBS-treated), a 24F4A-treated group, and a 237011-treated group. That afternoon, each group received dosing according to the schedule in Table 6 via intraperitoneal injection. Eighteen hours after dosing (Day 1), mice in all three groups received a tail vein injection of CpG-A at a dose of 500 μg / 250 μL per mouse. Twenty-four hours after CpG-A injection, blood was collected from the retroorbital venous plexus, coagulated, and centrifuged to obtain serum. Serum human IFNα concentrations were measured by ELISA.

[0361] Table 6: Group dosing regimen

[0362] The experimental results are shown in FIG9 . The 237011-administered group inhibited IFNa activity in mice significantly better than the 24F4A-administered group.

[0363] The sequence of the present invention is as follows:

[0364] Human BDCA2 extracellular domain (BDCA2-ECD) SEQ ID NO: 1

[0365] 23C10A6B5 heavy chain variable region nucleotide sequence SEQ ID NO: 2

[0366] 23C10A6B5 heavy chain variable region amino acid sequence SEQ ID NO: 3

[0367] 23C10A6B5 light chain variable region nucleotide sequence SEQ ID NO: 4

[0368] 23C10A6B5 light chain variable region amino acid sequence SEQ ID NO: 5

[0369] HCDR1 of 23C10A6B5: SYIIH (SEQ ID NO: 6)

[0370] HCDR2 of 23C10A6B5: TIYPGNGDTSYNQKFKG (SEQ ID NO: 7)

[0371] HCDR3 of 23C10A6B5: MGDNEYFDY (SEQ ID NO: 8)

[0372] LCDR1 of 23C10A6B5: RASGNIHNYLA (SEQ ID NO: 9)

[0373] LCDR2 of 23C10A6B5: DAETLAD (SEQ ID NO: 10)

[0374] LCDR3 of 23C10A6B5: QHFWSTPYT (SEQ ID NO: 11)

[0375] Heavy chain variable region of humanized antibody 230 SEQ ID NO: 12

[0376] Light chain variable region of humanized antibody 230 SEQ ID NO: 13

[0377] 230 Antibody-IL Heavy Chain CDR1 SEQ ID NO: 14 SYLIH

[0378] 230 Antibody-NG Heavy Chain CDR2 SEQ ID NO: 15 TIYPGGGDTSYNQKFKG

[0379] 230 antibody-NA heavy chain CDR2 SEQ ID NO: 16 TIYPGAGDTSYNQKFKG

[0380] 230 Antibody-NS Heavy Chain CDR2 SEQ ID NO: 17 TIYPGSGDTSYNQKFKG

[0381] 230 antibody-NR heavy chain CDR2 SEQ ID NO: 18 TIYPGRGDTSYNQKFKG

[0382] 230 Antibody-DI Light Chain CDR2 SEQ ID NO: 19 DAETLAI

[0383] 230 Antibody-DE Light Chain CDR2 SEQ ID NO: 20 DAETLAE

[0384] 230IL-DI heavy chain variable region SEQ ID NO: 21

[0385] 230IL-NG-DI heavy chain variable region SEQ ID NO: 22

[0386] 230IL-NR-DI heavy chain variable region SEQ ID NO: 23

[0387] 230NG-DI heavy chain variable region SEQ ID NO: 24

[0388] 230NR-DI heavy chain variable region SEQ ID NO: 25

[0389] 230IL-DI light chain variable region SEQ ID NO: 26

[0390] 230IL-DE light chain variable region SEQ ID NO: 27

[0391] 237011 antibody heavy chain SEQ ID NO: 28

[0392] 2391 antibody heavy chain SEQ ID NO: 29

[0393] HCDR1SYX1IH of the anti-BDCA2 antibody, X1 is I or L (SEQ ID NO: 30)

[0394] HCDR2 of the anti-BDCA2 antibody is TIYPGX2GDTSYNQKFKG, where X2 is N, G, A, S, or R (SEQ ID NO: 31)

[0395] Antibody against BDCA2 LCDR2DAETLAX3, X3 is I or E (SEQ ID NO: 32)

[0396] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An antibody or antigen-binding fragment thereof that binds to BDCA2, characterized in that: It comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain complementary determining regions H-CDR1, H-CDR2, and H-CDR3, wherein The amino acid sequence of H-CDR1 is shown as 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 as MGDNEYFDY (SEQ ID NO: 8), and The light chain variable region includes light chain complementary determining regions L-CDR1, L-CDR2, and L-CDR3; wherein, The amino acid sequence of L-CDR1 is shown as RASGNIHNYLA (SEQ ID NO: 9), The amino acid sequence of L-CDR2 is shown as DAETLAX3 (SEQ ID NO: 32). The amino acid sequence of L-CDR3 is shown as QHFWSTPYT (SEQ ID NO: 11); wherein X1 is I or L; X2 is an amino acid selected from the group consisting of N, G, A, S, R; and / or X3 is an amino acid selected from the group consisting of D, I or E.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein X1, X2, X3 are selected from the following group: 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, and X3 is I; X1 is I, X2 is G, and X3 is E; X1 is I, X2 is R, and X3 is I; or X1 is I, X2 is R, and X3 is E.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain variable region includes heavy chain complementary determining regions H-CDR1, H-CDR2, and H-CDR3, wherein The amino acid sequence of H-CDR1 is shown in SYIIH (SEQ ID NO: 6). The amino acid sequence of H-CDR2 is shown as TIYPGNGDTSYNQKFKG (SEQ ID NO: 7), The amino acid sequence of H-CDR3 is shown as MGDNEYFDY (SEQ ID NO: 8), and The light chain variable region includes light chain complementary determining regions L-CDR1, L-CDR2, and L-CDR3; wherein, The amino acid sequence of L-CDR1 is shown as RASGNIHNYLA (SEQ ID NO: 9), The amino acid sequence of L-CDR2 is shown as DAETLAD (SEQ ID NO: 10). The amino acid sequence of L-CDR3 is shown as QHFWSTPYT (SEQ ID NO: 11); Wherein, any of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain the BDCA2 binding affinity.

4. The antibody or antigen-binding fragment thereof that binds to BDCA2 according to claim 1, wherein: The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof that binds to BDCA2 is as shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 5; or the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof that binds to BDCA2 is as shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 13; or the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof 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 antigen-binding fragment thereof according to claim 1, wherein: The antibody is selected from the group consisting of an animal-derived antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or a combination thereof.

6. A recombinant protein, characterized in that The recombinant protein comprises: (i) an antibody or antigen-binding fragment thereof that binds to BDCA2 according to any one of claims 1 to 5; and (ii) optionally a tag sequence to facilitate expression and / or purification.

7. A nucleotide molecule, characterized in that The nucleotide molecule encodes the antibody or antigen-binding fragment thereof that binds to BDCA2 according to any one of claims 1 to 5.

8. An expression vector, characterized in that The expression vector contains the nucleotide molecule according to claim 7.

9. A host cell, characterized in that The host cell contains the expression vector according to claim 8.

10. A method for preparing an antibody or an antigen-binding fragment thereof that binds to BDCA2 according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: a) culturing the host cell of claim 9 under expression conditions, thereby expressing the antibody or antigen-binding fragment thereof that binds to BDCA2; b) isolating and purifying the antibody or antigen-binding fragment thereof that binds to BDCA2 described in a).

11. A composition, characterized in that The composition contains the antibody or antigen-binding fragment thereof that binds to BDCA2 according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

12. Use of the antibody or antigen-binding fragment thereof binding 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 inflammatory diseases or autoimmune diseases.

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

14. An antibody-drug conjugate, characterized in that: The antibody-drug conjugate contains: (a) an antibody portion, the antibody portion comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; and (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

15. A CAR construct, characterized in that: The scFv segment of the monoclonal antibody antigen binding region of the CAR construct is a binding region that specifically binds to BDCA2, and the heavy chain variable region of the scFv includes: The heavy chain complementary determining regions H-CDR1, H-CDR2, H-CDR3, the amino acid sequence of H-CDR1 is as shown in SYIIH (SEQ The amino acid sequence of H-CDR2 is shown as TIYPGNGDTSYNQKFKG (SEQ ID NO: 7), the amino acid sequence of H-CDR3 is shown as MGDNEYFDY (SEQ ID NO: 8), and The light chain variable region of the scFv comprises: Light chain complementary determining regions L-CDR1, L-CDR2, and L-CDR3, wherein the amino acid sequence of L-CDR1 is shown as RASGNIHNYLA (SEQ ID NO: 9), the amino acid sequence of L-CDR2 is shown as DAETLAD (SEQ ID NO: 10), and the amino acid sequence of L-CDR3 is shown as QHFWSTPYT (SEQ ID NO: 11); Wherein, any of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain the BDCA2 binding affinity.

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