Anti-BDCA2 antibodies

Abstract

The present invention provides an anti-BDCA2 antibody and a preparation method thereof. The anti-BDCA2 antibody disclosed by the invention can be combined with a BDCA2 antigen with high specificity, has very high affinity, can effectively inhibit CpG-A from stimulating PBMC to secrete IFNa, and is expected to be used as a medicine for treating autoimmune diseases.

Description

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 mainly produced by plasmacytoid dendritic cells (pDC) and is a component of the innate immune system. Type I IFN genes are strictly regulated. Under normal circumstances, the formation of IFN-α is almost undetectable in healthy individuals. However, many patients with systemic autoimmune diseases have signs of continued production of type I IFN, and the expression of IFN-α regulatory genes is increased. IFN-α activates the innate and adaptive immune systems. When there is a genetic setting that leads to strong IFN-α production and / or a significant IFN-α response, the individual's tolerance is destroyed and antibodies against self-antigens are produced. These antibodies form interfering ICs together with self-antigens, stimulate pDC to synthesize IFN-α, and stimulate B cells to promote the production of autoantibodies, leading to a vicious cycle of continued production of IFN-α and autoimmune reactions.

[0003] Blood dendritic cell antigen 2 (BDCA2), also named CLEC4C or CD303, is specifically expressed only on pDCs. After BDCA2 is triggered, it activates 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 start from intervening in the function of pDC cells, allowing BDCA2 to be quickly internalized from the surface of pDC, and indirectly inhibiting the release of IFN-I, pro-inflammatory cytokines, etc. from the upstream. In addition, the signal transduction of BDCA-2 is not affected by other induction stimuli of IFN-I, so targeting BDCA2 is a new option for treating autoimmune diseases. However, BDCA2 targeted drugs in the prior art still have many shortcomings, and the field still needs to provide new anti-BDCA2 antibodies. Summary of the invention

[0005] The object of the present invention is to provide an anti-BDCA2 antibody.

[0006] In the first aspect of the present invention, an antibody or antigen-binding fragment thereof that binds to BDCA2 is provided, 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

[0007] The amino acid sequence of H-CDR1 is shown as SYX1IH (SEQ ID NO: 30),

[0008] The amino acid sequence of H-CDR2 is shown as TIYPGX2GDTSYNQKFKG (SEQ ID NO: 31),

[0009] The amino acid sequence of H-CDR3 is shown as MGDNEYFDY (SEQ ID NO: 8), and

[0010] The light chain variable region includes light chain complementary determining regions L-CDR1, L-CDR2, and L-CDR3; wherein,

[0011] The amino acid sequence of L-CDR1 is shown as RASGNIHNYLA (SEQ ID NO: 9),

[0012] The amino acid sequence of L-CDR2 is shown as DAETLAX3 (SEQ ID NO: 32).

[0013] The amino acid sequence of L-CDR3 is shown as QHFWSTPYT (SEQ ID NO: 11);

[0014] 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.

[0015] In another preferred embodiment, X1, X2, and X3 are selected from the following groups:

[0016] X1 is I, X2 is N, and X3 is D;

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

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

[0019] X1 is L, X2 is G, and X3 is I;

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

[0021] X1 is L, X2 is R, and X3 is I;

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

[0023] X1 is I, X2 is G, and X3 is I;

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

[0025] X1 is I, X2 is R, and X3 is I; or

[0026] X1 is I, X2 is R, and X3 is E.

[0027] In another preferred embodiment, the heavy chain variable region includes heavy chain complementary determining regions H-CDR1, H-CDR2, and H-CDR3, wherein

[0028] The amino acid sequence of H-CDR1 is shown in SYIIH (SEQ ID NO: 6).

[0029] The amino acid sequence of H-CDR2 is shown as TIYPGNGDTSYNQKFKG (SEQ ID NO: 7),

[0030] The amino acid sequence of H-CDR3 is shown as MGDNEYFDY (SEQ ID NO: 8), and

[0031] The light chain variable region includes light chain complementary determining regions L-CDR1, L-CDR2, and L-CDR3; wherein,

[0032] The amino acid sequence of L-CDR1 is shown as RASGNIHNYLA (SEQ ID NO: 9),

[0033] The amino acid sequence of L-CDR2 is shown as DAETLAD (SEQ ID NO: 10).

[0034] The amino acid sequence of L-CDR3 is shown as QHFWSTPYT (SEQ ID NO: 11);

[0035] 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.

[0036] In another preferred embodiment, the ratio of the binding affinity F1 of the derivatized antibody to BDCA2 to the binding affinity F0 of the corresponding non-derivatized antibody to BDCA2 (F1 / F0) is 0.5-4, preferably 0.7-1.5, and more preferably 0.8-1.2.

[0037] 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).

[0038] 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 complementary determining region having the following amino acid sequence:

[0039] H-CDR1 with the amino acid sequence SYX1IH, wherein X1 is I or L; and / or

[0040] H-CDR2 with an amino acid sequence as shown in TIYPGX2GDTSYNQKFKG, wherein X2 is selected from the group consisting of N, G, A, S, R (preferably G); and / or

[0041] The light chain variable region includes a light chain complementarity determining region having the following amino acid sequence:

[0042] The amino acid sequence of L-CDR2 is shown as DAETLAX3, wherein X3 is selected from the following amino acids: D, I or E; wherein X1, X2, and X3 are each independently not I, N, or D at the same time.

[0043] In another preferred embodiment, in the derivative sequence, X1, X2, and X3 are selected from the following groups:

[0044] X1 is L, X2 is N, and X3 is I;

[0045] X1 is L, X2 is N, and X3 is E;

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

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

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

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

[0050] X1 is I, X2 is G, and X3 is I;

[0051] X1 is I, X2 is G, and X3 is E;

[0052] X1 is I, X2 is R, and X3 is I; or

[0053] X1 is I, X2 is R, and X3 is E.

[0054] In another preferred embodiment, in the derived sequence, the heavy chain variable region comprises

[0055] The amino acid sequence of H-CDR1 is shown in SYLIH (SEQ ID NO: 14),

[0056] H-CDR2 with an amino acid sequence as shown in TIYPGGGDTSYNQKFKG (SEQ ID NO: 15), H-CDR3 with an amino acid sequence as shown in MGDNEYFDY (SEQ ID NO: 8), and

[0057] The light chain variable region comprises

[0058] The amino acid sequence of L-CDR1 is as shown in RASGNIHNYLA (SEQ ID NO: 9), and the amino acid sequence of L-CDR2 is as shown in DAETLAE (SEQ ID NO: 20),

[0059] The amino acid sequence of L-CDR3 is shown as QHFWSTPYT (SEQ ID NO: 11).

[0060] In another preferred embodiment, in the derived sequence, the heavy chain variable region comprises

[0061] The amino acid sequence of H-CDR1 is shown in SYIIH (SEQ ID NO: 6),

[0062] H-CDR2 with an amino acid sequence as shown in TIYPGGGDTSYNQKFKG (SEQ ID NO: 15), H-CDR3 with an amino acid sequence as shown in MGDNEYFDY (SEQ ID NO: 8), and

[0063] The light chain variable region comprises

[0064] The amino acid sequence of L-CDR1 is as shown in RASGNIHNYLA (SEQ ID NO: 9), and the amino acid sequence of L-CDR2 is as shown in DAETLAE (SEQ ID NO: 20),

[0065] The amino acid sequence of L-CDR3 is shown as QHFWSTPYT (SEQ ID NO: 11).

[0066] In another preferred embodiment, in the derived sequence, the heavy chain variable region comprises

[0067] The amino acid sequence of H-CDR1 is shown in SYLIH (SEQ ID NO: 14),

[0068] H-CDR2 with an amino acid sequence as shown in TIYPGNGDTSYNQKFKG (SEQ ID NO: 7), H-CDR3 with an amino acid sequence as shown in MGDNEYFDY (SEQ ID NO: 8), and

[0069] The light chain variable region comprises

[0070] The amino acid sequence of L-CDR1 is as shown in RASGNIHNYLA (SEQ ID NO: 9), and the amino acid sequence of L-CDR2 is as 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, the antibody is a murine antibody, a chimeric antibody or a humanized antibody.

[0073] 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.

[0074] 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.

[0075] In another preferred embodiment, the heavy chain constant region is of human origin, and / or the light chain constant region is of human origin.

[0076] 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.

[0077] In another preferred embodiment, the heavy chain constant region comprises an Fc segment.

[0078] In another preferred embodiment, the Fc segment is an Fc variant, and the modified Fc variant comprises the following mutations: M428L / N434S; G236A / S239D / I332E; S239D / I332E / A330L; S239D / I332E / G236A; S298A; A330L; I332E; E333A; and / or K334A;

[0079] The numbering of the residues in the Fc variant is that of the EU index as in Kabat.

[0080] In another preferred embodiment, the antibody is selected from the following group: animal-derived antibody, chimeric antibody, humanized antibody, fully human antibody, or a combination thereof.

[0081] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0082] In another preferred embodiment, the antibody is a full-length antibody protein or an antigen-binding fragment.

[0083] In another preferred embodiment, the antibody is a bispecific antibody or a multispecific antibody.

[0084] In another preferred embodiment, the antibody is in the form of a drug conjugate.

[0085] In another preferred embodiment, the affinity of the antibody to the antigen BDCA2 is ≤1E-9M; preferably ≤1E-10M; more preferably ≤1E-11M.

[0086] In another preferred embodiment, the BDCA2 is human BDCA2; preferably, it is the extracellular domain of human BDCA2.

[0087] In another preferred embodiment, the sequence of the human BDCA2 extracellular domain is shown as SEQ ID NO: 1.

[0088] In another preferred embodiment, the antigen-binding fragment includes a Fab fragment, a F(ab')2 fragment, a Fv fragment, and a scFv.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] In a second aspect of the present invention, a recombinant protein is provided, characterized in that the recombinant protein comprises:

[0095] (i) an antibody or antigen-binding fragment thereof that binds to BDCA2 according to the first aspect of the present invention; and

[0096] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0097] In another preferred embodiment, the tag sequence includes a 6His tag.

[0098] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0099] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0100] In the third aspect of the present invention, a nucleotide molecule is provided, wherein the nucleotide molecule encodes the antibody or antigen-binding fragment thereof that binds to BDCA2 as described in the first aspect of the present invention.

[0101] 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.

[0102] In the fourth aspect of the present invention, an expression vector is provided, characterized in that the expression vector contains the nucleotide molecule as described in the third aspect of the present invention.

[0103] In the fifth aspect of the present invention, a host cell is provided, characterized in that the host cell contains the expression vector as described in the fourth aspect of the present invention.

[0104] In a sixth aspect of the present invention, a method for preparing an antibody or an antigen-binding fragment thereof that binds to BDCA2 according to the first aspect of the present invention is provided, wherein the method comprises the following steps:

[0105] a) culturing the host cell according to the fifth aspect of the present invention under expression conditions, thereby expressing the antibody or antigen-binding fragment thereof that binds to BDCA2;

[0106] b) isolating and purifying the antibody or antigen-binding fragment thereof that binds to BDCA2 described in a).

[0107] In the seventh aspect of the present invention, a composition is provided, characterized in that the composition contains the antibody or antigen-binding fragment thereof binding to BDCA2 as described in the first aspect of the present invention and a pharmaceutically acceptable carrier.

[0108] In the eighth aspect of the present invention, there is provided use of the antibody or antigen-binding fragment thereof binding 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 drug for treating inflammatory diseases or autoimmune diseases.

[0109] In another preferred embodiment, the inflammatory disease or autoimmune disease is selected from the group consisting of systemic lupus erythematosus, discoid lupus, lupus nephritis, cutaneous lupus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type I diabetes, dermatomyositis and polymyositis.

[0110] In the ninth aspect of the present invention, a CAR construct is provided, 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 comprises:

[0111] Heavy chain complementary determining regions H-CDR1, H-CDR2, H-CDR3, 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), the amino acid sequence of H-CDR3 is shown as MGDNEYFDY (SEQ ID NO: 8), and

[0112] The light chain variable region of the scFv comprises:

[0113] 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 one of the above amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified, and / or substituted with at least one amino acid and can retain BDCA2 binding affinity.

[0114] 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 complementary determining region having the following amino acid sequence:

[0115] H-CDR1 with the amino acid sequence SYX1IH, wherein X1 is I or L; and / or

[0116] H-CDR2 with an amino acid sequence as shown in TIYPGX2GDTSYNQKFKG, wherein X2 is selected from the group consisting of N, G, A, S, R (preferably G); and / or

[0117] The light chain variable region includes a light chain complementarity determining region having the following amino acid sequence:

[0118] 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;

[0119] Among them, X1, X2, and X3 are each independently I, N, and D at the same time.

[0120] In the tenth aspect of the present invention, a recombinant immune cell is provided, characterized in that the immune cell expresses an exogenous CAR construct as described in the ninth aspect of the present invention.

[0121] In the eleventh aspect of the present invention, an antibody-drug conjugate is provided, characterized in that the antibody-drug conjugate contains:

[0122] (a) an antibody portion, the antibody portion comprising the antibody or antigen-binding fragment thereof according to the first aspect of the invention; and

[0123] (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.

[0124] In the twelfth aspect of the present invention, a method for detecting BDCA2 protein in a sample in vitro is provided, characterized in that the method comprises the steps of:

[0125] (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;

[0126] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of BDCA2 protein in the sample.

[0127] In the thirteenth aspect of the present invention, a method for preventing and / or treating a BDCA2-related disease is provided, the method 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.

[0128] In another preferred embodiment, the BDCA2-related disease is selected from inflammatory diseases or autoimmune diseases, including but not limited to systemic 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.

[0129] 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 specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0130] Figure 1A-1B The results show that the mouse antibody has the ability to bind to the human BDCA2-Fc protein.

[0131] Figure 2A-2B The binding affinity determination of the murine antibody to the stably transfected strain 293FT-BDCA2 is shown.

[0132] Figure 3 The results show that the human-mouse chimeric antibody has the ability to bind to human BDCA2-his protein.

[0133] Figure 4 The results showed that the human-mouse chimeric antibody could inhibit the secretion of IFNa by PBMC induced by CpG-A.

[0134] Figure 5 The ELISA screening of the effects of different mutation sites on the affinity of the 230 antibody is shown.

[0135] Figure 6 The ELISA test of the affinity of anti-BDCA2 antibody mutants to BDCA2-his antigen protein is shown.

[0136] Figure 7 The inhibitory effect of candidate antibodies on CpG-A-induced IFNa secretion by PBMCs was shown using PBMCs. DETAILED DESCRIPTION

[0137] After extensive and in-depth research and a large number of screenings, the inventors unexpectedly discovered a series of mouse antibodies that have good binding activity to the target antigen BDCA2-Fc. Candidate antibodies were further selected, and the affinity and expression of the obtained humanized antibodies were equivalent to or better than the parent antibodies through humanization and antibody CDR modification. The antibodies of the present invention can effectively inhibit CpG-A from stimulating PBMC to secrete IFNa, and the activity is much better than the positive control antibody Litifilimab. The present invention was completed on this basis.

[0138] the term

[0139] 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.

[0140] 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.

[0141] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0142] Antibody

[0143] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 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 between the heavy chains of different immunoglobulin isotypes varies. 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 to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0144] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms 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 fragments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative part of the variable region is called the framework region (FR). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0145] The "light chains" of vertebrate antibodies (immunoglobulins) can be classified into 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 can be 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 structure and three-dimensional configuration of the different classes of immunoglobulins are well known in the art.

[0146] Generally, the antigen binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs). This segment is divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs in between are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chains constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

[0147] 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.

[0148] 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 from different animal species, such as a chimeric antibody having a variable region from a monoclonal antibody from a mouse, and a constant region from a human immunoglobulin (see, for example, U.S. Pat. No. 4,816,567 and U.S. Pat. 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. Pat. 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.

[0149] In the present invention, the antibodies may be monospecific, bispecific, trispecific, or more multispecific.

[0150] In the present invention, the antibody of the present invention also includes its conservative variants, which refers to a polypeptide formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids of similar or similar properties compared to the amino acid sequence of the antibody of the present invention. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.

[0151] Table A

[0152]

[0153]

[0154] Anti-BDCA2 antibodies

[0155] In the present invention, the antibody is an anti-BDCA2 antibody. The present invention provides a high-specificity 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.

[0156] Wherein, the heavy chain variable region includes heavy chain complementary determining regions H-CDR1, H-CDR2, and H-CDR3,

[0157] The amino acid sequence of H-CDR1 is shown as SYX1IH (SEQ ID NO: 30),

[0158] The amino acid sequence of H-CDR2 is shown as TIYPGX2GDTSYNQKFKG (SEQ ID NO: 31),

[0159] The amino acid sequence of H-CDR3 is shown as MGDNEYFDY (SEQ ID NO: 8), and

[0160] The light chain variable region includes light chain complementary determining regions L-CDR1, L-CDR2, and L-CDR3; wherein,

[0161] The amino acid sequence of L-CDR1 is shown as RASGNIHNYLA (SEQ ID NO: 9),

[0162] The amino acid sequence of L-CDR2 is shown as DAETLAX3 (SEQ ID NO: 32).

[0163] The amino acid sequence of L-CDR3 is shown as QHFWSTPYT (SEQ ID NO: 11);

[0164] 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.

[0165] Preferably,

[0166] The amino acid sequence of H-CDR1 is shown in SYIIH (SEQ ID NO: 6).

[0167] The amino acid sequence of H-CDR2 is shown as TIYPGNGDTSYNQKFKG (SEQ ID NO: 7),

[0168] The amino acid sequence of H-CDR3 is shown as MGDNEYFDY (SEQ ID NO: 8), and

[0169] The light chain variable region includes light chain complementary determining regions L-CDR1, L-CDR2, and L-CDR3; wherein,

[0170] The amino acid sequence of L-CDR1 is shown as RASGNIHNYLA (SEQ ID NO: 9),

[0171] The amino acid sequence of L-CDR2 is shown as DAETLAD (SEQ ID NO: 10).

[0172] The amino acid sequence of L-CDR3 is shown as QHFWSTPYT (SEQ ID NO: 11);

[0173] 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.

[0174] 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%.

[0175] Methods for determining sequence homology or identity 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, 1996. 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 the 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.

[0176] 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 (scFv), a single-domain antibody (sdAb), and a single-domain antibody (Signle-domain antibody), as well as a monoclonal antibody or a polyclonal antibody prepared from the above antibodies. The monoclonal antibody can be prepared by a variety of approaches and techniques, 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 by hybridoma technology.

[0177] The full-length antibody protein is a conventional full-length antibody protein in the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region and a light chain constant region. The heavy chain variable region and light chain variable region of the protein and the human heavy chain constant region and the human light chain constant region constitute a full-length human antibody protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3 or IgG4.

[0178] The antibody (anti-BDCA2 antibody) of the present invention may be a full-length protein (such as IgG1, IgG2a, IgG2b or IgG2c), or a protein fragment containing the antigen-antibody binding domain (such as Fab, F(ab'), sdAb, ScFv fragment).

[0179] The antibody (anti-BDCA2 antibody) of the present invention may be a wild-type protein, or a mutant protein that has undergone specific mutations to achieve a specific effect, for example, mutations are used to eliminate the effector function of the antibody.

[0180] 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 more preferably fully humanized antibodies.

[0181] The antibody derivatives of the present invention can be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other known antibody derivatives in the field, as well as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0182] 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.

[0183] Wherein, the animal is preferably a mammal, such as a mouse.

[0184] As used herein, "Fc variant" or "variant Fc" means a protein comprising a modification 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 performed 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 acid 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 performed according to the EU index. The EU index or the EU index or EU numbering scheme as in Kabat refers to the numbering of EU antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, which is incorporated herein by reference in its entirety). Modifications may be additions, deletions, or substitutions. Substitutions may include naturally occurring amino acids and non-naturally occurring amino acids. Variants may include non-natural amino acids. Examples include US 6,586,207; WO 98 / 48032; WO 03 / 073238; US 2004-0214988 A1; 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 PG Schultz, (2002), Chem Bio Chem 11:1135-1137; JW Chin, et al., (2002), PICAS United States of America 99:11020-11024; and L. Wang and PG Schultz, (2002), Chem. 1-10, all of which are incorporated by reference in their entirety.

[0185] 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).

[0186] Furthermore, the CDR region of the mouse antibody was transplanted to the selected humanized template to replace the CDR region of the human template. The heavy chain variable region was recombined with the human IgG1 constant region, and the light chain variable region was recombined with the human kappa chain constant region to obtain a chimeric antibody. Based on the three-dimensional structure of the antibody, the embedded residues, the residues that directly interacted with the CDR region, and the residues that had an important influence on the conformation of the antibody's VL and VH were back-mutated to obtain multiple humanized antibodies.

[0187] 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%.

[0188] In the above content of the present invention, more preferably, the number of the added, deleted, modified and / or substituted amino acids may be 1-7, more preferably 1-5, more preferably 1-3, more preferably 1-2.

[0189] 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:

[0190] H-CDR1 with the amino acid sequence SYX1IH, wherein X1 is I or L; and / or

[0191] H-CDR2 with an amino acid sequence as shown in TIYPGX2GDTSYNQKFKG, wherein X2 is selected from the group consisting of N, G, A, S, R (preferably G); and / or

[0192] The light chain variable region includes a light chain complementarity determining region having the following amino acid sequence:

[0193] 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;

[0194] Among them, X1, X2, and X3 are each independently I, N, and D at the same time.

[0195] In another preferred embodiment, the heavy chain variable region and the 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:

[0196] (1) H-CDR1 is selected from the following group:

[0197] SYIIH (SEQ ID NO: 6)

[0198] SYLIH (SEQ ID NO: 14)

[0199] (2) H-CDR2 is selected from the following group:

[0200] TIYPGNGDTSYNQKFKG(SEQ ID NO:7)

[0201] TIYPGGGDTSYNQKFKG(SEQ ID NO: 15)

[0202] TIYPGAGDTSYNQKFKG(SEQ ID NO: 16)

[0203] TIYPGSGDTSYNQKFKG(SEQ ID NO: 17)

[0204] TIYPGRGDTSYNQKFKG(SEQ ID NO: 18)

[0205] (3) H-CDR3 is MGDNEYFDY (SEQ ID NO: 8)

[0206] (4) L-CDR1 is RASGNIHNYLA (SEQ ID NO: 9)

[0207] (5) L-CDR2 is selected from the following group:

[0208] DAETLAD (SEQ ID NO: 10)

[0209] DAETLAI (SEQ ID NO: 19)

[0210] DAETLAE (SEQ ID NO: 20);

[0211] (6) L-CDR3 is QHFWSTPYT (SEQ ID NO: 11).

[0212] 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.

[0213] 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.

[0214] 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:

[0215] Table B

[0216]

[0217] Recombinant protein

[0218] 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 of 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.

[0219] The recombinant protein further comprises an antibody heavy chain constant region and / or an antibody light chain constant region, wherein the antibody heavy chain constant region is 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 light chain antibody constant region or a human antibody light chain constant region, more preferably a human antibody light chain constant region.

[0220] The recombinant protein is a conventional protein in the art, preferably, it 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 (scFv), a single-domain antibody (sdAb), and a single-domain antibody (Signle-domainantibody), 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 techniques, 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.

[0221] The full-length antibody protein is a conventional full-length antibody protein in the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region and a light chain constant region. The heavy chain variable region and light chain variable region of the protein and the human heavy chain constant region and the human light chain constant region constitute a full-length human antibody protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3 or IgG4.

[0222] 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.

[0223] 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').

[0224] 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.

[0225] The single-domain antibody is a conventional single-domain antibody in the art, which only includes the heavy chain variable region.

[0226] Wherein, the preparation method of the recombinant protein is a conventional preparation method in the art. The preparation method is preferably: obtaining by separation from an expression transformant that recombinantly expresses the protein or obtaining by artificially synthesizing a protein sequence. The separation from the expression transformant that recombinantly expresses the protein is preferably the following method: cloning a nucleic acid molecule encoding the protein and having a point mutation into a recombinant vector, transforming the obtained recombinant vector into a transformant, obtaining a recombinant expression transformant, and culturing the obtained recombinant expression transformant to obtain the recombinant protein.

[0227] Nucleic Acids

[0228] 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.

[0229] 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.

[0230] Those skilled in the art know that the base sequence encoding the amino acid sequence of the above-mentioned protein can be appropriately introduced with substitution, deletion, change, insertion or addition to provide a polynucleotide homologue. The polynucleotide homologue of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the protein sequence within the range of maintaining the antibody activity.

[0231] Carrier

[0232] The present invention also provides a recombinant expression vector comprising the nucleic acid.

[0233] The recombinant expression vector can be obtained by conventional methods in the art, that is, by connecting the nucleic acid molecule of the present invention to various expression vectors. The expression vector is any conventional vector in the art, as long as it can carry the aforementioned nucleic acid molecule. The vector preferably includes various plasmids, cosmids, phages or virus vectors, etc.

[0234] The present invention also provides a recombinant expression transformant comprising the recombinant expression vector.

[0235] 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 any conventional host cell in the art, as long as it can satisfy the above-mentioned recombinant expression vector to stably replicate itself, 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 a preferred recombinant expression transformant of the present invention. Wherein the transformation method is a conventional transformation method in the art, preferably a chemical transformation method, a heat shock method or an electroporation method.

[0236] Antibody preparation

[0237] 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 the heavy chain can be fused together to form a single-chain antibody.

[0238] 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.

[0239] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0240] At present, the DNA sequence encoding the antibody (or its fragment, or its derivative) of the present invention can be obtained completely by chemical synthesis. The 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 sequence of the present invention by chemical synthesis.

[0241] 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.

[0242] 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, HEK-293 cells.

[0243] Typically, the transformed host cells are cultured under conditions suitable for the expression of the antibodies of the present invention, and then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc., conventional separation and purification means well known to those skilled in the art to obtain the antibodies of the present invention.

[0244] The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0245] The antibodies of the present invention can be expressed in cells, on cell membranes, or secreted outside cells. If necessary, the recombinant protein can be separated and purified by various separation methods using 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 protein precipitants (salting out method), centrifugation, osmotic sterilization, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0246] Antibody-drug conjugates (ADCs)

[0247] The present invention also provides an antibody-drug conjugate (ADC) based on the antibody of the present invention.

[0248] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is coupled to the effector molecule, and preferably chemically coupled. The effector molecule is preferably a drug with therapeutic activity. In addition, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug or a radionuclide.

[0249] The antibody of the present invention and the effector molecule can be coupled via a coupling agent. Examples of the coupling agent can be 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 can be any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (the coupling site is an acylhydrazone).

[0250] 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 agents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly connected to antibodies through linkers.

[0251] Antibodies can be coupled to drugs to form antibody drug conjugates (ADCs). Typically, ADCs contain a linker between the drug and the antibody. The linker can be a degradable or non-degradable linker. Degradable linkers are typically easily degraded in the intracellular environment, such as degradation of the linker at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. 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 (such as linkers that hydrolyze when the pH is less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (such as disulfide linkers). Non-degradable linkers typically release drugs under conditions where the antibody is hydrolyzed by proteases.

[0252] 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 through the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimide compounds, halogenated amides (e.g., iodinated, brominated or chlorinated); halogenated esters (e.g., iodinated, brominated or chlorinated); halogenated methyl ketones (e.g., iodinated, brominated or chlorinated), benzyl halides (e.g., iodinated, brominated or chlorinated); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, maleimide attached to the antibody via thiosuccinimide.

[0253] The drug can be any cytotoxic, cytostatic or immunosuppressive drug. In an embodiment, a linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group that can form a bond with the linker. In the case where the drug is directly connected to the linker, the drug has a reactive group before being connected to the antibody.

[0254] 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.

[0255] In the present invention, drug-linkers can be used to form ADCs in one simple 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, a functional group on the linker reacts with a drug to form an ADC.

[0256] Typically, the functional groups on the linker are selected to react specifically with the appropriate reactive groups on the drug moiety. As a non-limiting example, an azide-based moiety can be used to react specifically with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker by a 1,3-dipolar cycloaddition between the azide and the alkynyl group. 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 attachment 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 of the drug moiety and the linker, each member of the complementary pair can be used for both the linker and the drug.

[0257] The present invention also provides a method for preparing ADC, which may further comprise: combining an antibody with a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

[0258] In certain embodiments, the methods of the invention include: combining the antibody with the bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the invention further include: combining the antibody-linker conjugate with the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via the linker.

[0259] In some embodiments, the antibody drug conjugate ADC has the following molecular formula:

[0260]

[0261] in:

[0262] Ab is antibody,

[0263] LU is the connector;

[0264] D is for medicine;

[0265] And the subscript p is a value selected from 1 to 8.

[0266] Pharmaceutical composition

[0267] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its ADC or corresponding immune cells, and a pharmaceutically acceptable carrier. Generally, these substances can be 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 disease to be treated.

[0268] 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.

[0269] The antibody of the present invention can also be expressed in cells by nucleotide sequences for cell therapy, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

[0270] The pharmaceutical composition of the present invention is a pharmaceutical composition for preventing and / or treating diseases associated with abnormal BDCA2 expression or function.

[0271] 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.

[0272] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned monoclonal antibody (or its conjugate) of the present invention 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, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. 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 microgram / kg body weight to about 5 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used with other therapeutic agents.

[0273] 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, preferably including 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 percentage is the mass percentage of the pharmaceutical composition.

[0274] In the present invention, preferably, the amount of the pharmaceutical composition administered is an effective amount, which is an amount that can alleviate or delay 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 results sought. Those skilled in the art can determine the effective amount by using the above factors on an individual basis and using experiments that do not exceed routine.

[0275] 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 usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 50 milligrams / kg body weight, preferably the dosage is about 10 micrograms / kg body weight to about 20 milligrams / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0276] The present invention provides the use of the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and / or treating a disease associated with abnormal expression or function of BDCA2. Preferably, the disease associated with abnormal expression or function of BDCA2 is an inflammatory disease or an autoimmune disease.

[0277] application

[0278] 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.

[0279] Preferably, the drug is a drug for preventing and / or treating diseases associated with abnormal BDCA2 expression or function.

[0280] In the present invention, the disease associated with abnormal expression or function of BDCA2 is a conventional disease associated with abnormal expression or function of BDCA2 in the art. Preferably, the disease associated with abnormal expression or function of BDCA2 is an inflammatory disease or an autoimmune disease, including but not limited to systemic 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.

[0281] The main advantages of the present invention include

[0282] (1) The mouse anti-BDCA2 antibody of the present invention has excellent affinity, which is better than the positive control antibody Litifilimab; the anti-BDCA2 antibody modified after humanization also has higher affinity.

[0283] (2) The BDCA2 antibody of the present invention can effectively inhibit the secretion of IFNa by PBMC stimulated by CpG-A, and is expected to be used as a drug for the treatment of autoimmune diseases.

[0284] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify detailed conditions are usually based on conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0285] Example 1 Antigen immunization of animals and preparation and screening of hybridomas

[0286] 1.1 Preparation of antigen protein and positive control antibody

[0287] The sequence of the human BDCA2 extracellular domain (BDCA2-ECD) used as an antigen was obtained from the UniProt database (Entry: Q8WTT0), and the amino acid sequence is shown in SEQ ID NO: 1. A 6×His tag was added to its C-terminus, constructed into the pcDNA 3.4 expression vector, and 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 above 6×His tag was replaced with the Fc sequence of human IgG1 and then transfected into HEK-293F cells. After expression and purification, the BDCA2-Fc protein was obtained.

[0288] The sequences of the heavy and light chains of the positive control antibody Litifilimab (BIIB059, 24F4A) are from patent CN111961134A. After the heavy chain variable region and light chain variable region genes of the antibody were synthesized, they were recombined with the human heavy chain IgG1 and light chain Kappa constant regions, respectively, and constructed into the pcDNA 3.4 expression vector, expressed using the HEK-293F system, and purified using Protein A affinity chromatography.

[0289] 1.2 Immunization of mice

[0290] Balb / c mice were routinely immunized with human BDCA2-His protein (homemade, purity>95%) expressed by mammalian cell line 293F. On the first day, the soluble human BDCA2-His protein was emulsified with Freund's complete adjuvant and injected subcutaneously at multiple points into Balb / c mice (human BDCA2-His 100 μg / mouse / 0.5 mL). On the 14th day, the 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 the 28th day, the 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 stimulation. After 3-4 days, the spleens of the mice were taken for fusion experiments.

[0291] 1.3 Hybridoma cell preparation and screening

[0292] 3-4 days after the last immunization of mice, mouse spleen cells were PEG-fused with mouse myeloma cells SP2 / 0 using conventional hybridoma technology. The fused cells were evenly suspended in complete culture medium, which was a culture medium composed of RPMI1640-GLUMAX with 1% Penicillin-streptomycin, 20% FBS, and 1*HAT. The fused cells were plated at 3*10 4 Cells / 200 μL / well were plated in 60 96-well culture plates. After 7-12 days, the supernatant was harvested and the hybridoma wells positive for human BDCA2 binding activity were screened by ELISA.

[0293] Among them, the method of screening the hybridoma wells with positive human BDCA2 binding activity by ELISA method is as follows: BDCA2-Fc is diluted to 1 μg / mL with PBS buffer, 100 μL / well is added to the plate, and cultured at 4°C overnight; the supernatant is discarded the next day, 5% skim milk powder is added to block at 37°C for 1 hour, and the plate is washed 3 times with PBST for standby use; the collected hybridoma supernatant is added to the blocked plate in sequence, 100 μL / well, and placed at 37°C for 1 hour; the plate is washed 3 times with PBST, HRP-labeled goat anti-mouse IgG secondary antibody is added, and it is placed at 37°C for 30 minutes; after washing the plate 3 times with PBST, the residual droplets are patted dry on absorbent paper as much as possible, 100 μL of TMB is added to each well, and it is placed at room temperature (20±5°C) away from light for 5 minutes; 50 μL of 2M H2SO4 stop solution is added to each well to stop the substrate reaction, and the OD value is read at 450nm by an enzyme reader to analyze the binding ability of the antibody to be tested with 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, purified by Protein G affinity chromatography, and anti-human BDCA2 monoclonal antibody protein was obtained. After screening, a total of 30 hybridoma cell lines were obtained.

[0294] 1.4 Binding ability of mouse antibodies to human BDCA2-Fc protein

[0295] The binding ability of mouse antibodies to human BDCA2-Fc protein was determined by enzyme-linked immunosorbent assay (ELISA).

[0296] The specific method is as follows:

[0297] BDCA2-Fc protein was diluted to 1 μg / mL with PBS buffer, 100 μL / well was added to the plate, and cultured 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, and HRP-labeled goat anti-mouse IgG secondary antibody was added, and 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 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 450nm by a microplate reader to analyze the binding ability of the test antibody with the antigen human BDCA2-Fc. The obtained data were fitted and analyzed by GraphPadPrism 9 software. The results are shown in Figure 1A-1B shown.

[0298] Depend on Figure 1A-1B It can be seen that most mouse antibodies have good binding activity with the target antigen BDCA2-Fc, and the EC50 is shown in Tables 1 and 2.

[0299] Table 1:

[0300] Antibody EC50(ng / mL) Antibody EC50(ng / mL) 2B7E5 14.69 17G7D3 107.2 2E3F6 18.85 20E10F5 32.65 2D12H3 21.86 21B10A3 22.51 11G11C12 16.86 21E1A3 9.28 27G9G9 24.19 21E5C8 25.41 39E3B2 23.59 26C3F1 28.01 44H3A7 16.44 35E12F1 31.31 52G1A3 28.15 36F2A1 63.8 52H10D11 28.2 39F4D1 15.79 106G8F8 25.34 55G10A9 26.19 24F4A 34.61

[0301] Table 2:

[0302] Antibody EC50(ng / mL) 16G1H3 21.34 23C10A6B5 20.14 36A6C5E2 138.3 39F6C9 24.55 40H1F3G8 49.29 48A5H1F10 19.54 52E5D3 26.54 136B4H2E3 26.51 144A4G1A1 83.4 146D6A9E5 25.33 24F4A 46.27

[0303] 1.5 Determination of the binding affinity of mouse antibodies to the stably transfected 293FT-BDCA2

[0304] In this example, the binding affinity of mouse antibodies to target cells 293FT-BDCA2 was determined by flow cytometry (Fluorescence activated Cell Sorting, FACS).

[0305] In this experiment, 293FT-BDCA2 (an engineered cell line that highly expresses human BDCA2 constructed by 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, and the supernatant was removed by centrifugation. The anti-human BDCA2 mouse antibody was diluted 3-fold from 10 μg / mL in 11 gradients with 1% BSA-PBS, and 1% BSA-PBS was used as a blank control. 100 μL of each was resuspended in 293FT-BDCA2 cell wells, incubated at 4°C for 1 hour, and the cells were washed twice with PBS to remove unbound mouse antibody. The cells were then resuspended in 100 μL / well, 1:500 diluted PE-labeled anti-mouse secondary antibody (purchased from Jackson, catalog number 715116150), incubated at 4°C for 1 hour, and the cells were washed twice with PBS to remove unbound secondary antibody. Finally, the cells were resuspended in 200 μL PBS, and the binding affinity of the mouse antibody to the cells was determined by flow cytometry. The data obtained were fitted and analyzed by GraphPadPrism 9 software. The results are shown in Figure 2. Figure 2A-2B shown.

[0306] The results showed that most mouse antibodies could specifically bind to BDCA2 on the surface of 293FT cells, and the EC50 values ​​were shown in Tables 3 and 4.

[0307] Table 3:

[0308] Antibody EC50(ng / mL) Antibody EC50(ng / mL) 2B7E5 72.08 17G7D3 5251 2E3F6 306.4 20E10F5 257.3 2D12H3 139.5 21B10A3 124.7 11G11C12 87.09 21E1A3 85.94 27G9G9 198.5 21E5C8 108.2 39E3B2 78.75 26C3F1 78.28 44H3A7 103.1 35E12F1 44.23 52G1A3 114.8 36F2A1 349.7 52H10D11 248.8 39F4D1 135.6 106G8F8 1348 55G10A9 92.82 24F4A 103.4

[0309] Table 4:

[0310] Antibody EC50(ng / mL) 16G1H3 72.7 23C10A6B5 83.53 36A6C5E2 4787 39F6C9 81.91 40H1F3G8 410.1 48A5H1F10 172.6 52E5D3 272.3 136B4H2E3 94.27 144A4G1A1 31337 146D6A9E5 17724 24F4A 124.2

[0311] Example 2 Humanization of mouse anti-human BDCA2 monoclonal antibody

[0312] 2.1 Determination of the variable region sequence of mouse anti-human BDCA2 monoclonal antibody

[0313] Hybridoma clone 23C10A6B5 was selected as a candidate antibody, and total RNA was extracted from the corresponding hybridoma monoclonal cell line using Trizol (purchased from Life technologies). The mRNA was reverse transcribed into cDNA using a reverse transcription kit (purchased from Takara). PCR was performed using a combination of primers reported in the literature (Antibody Engineering Volume 1, Edited by Roland Kontermann and Stefan Dübel, the sequence of the combination of primers is from page 323). The obtained PCR product was sequenced and analyzed using the Kabat database to determine that the obtained sequence was the variable region sequence of a mouse antibody. The sequence information is as follows:

[0314] 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;

[0315] 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.

[0316] 2.2 Construction of human-mouse chimeric antibodies and verification of their activity

[0317] 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 connected 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 with the antigen BDCA2 and its inhibitory effect on CpG-A-induced IFNa secretion by PBMC were verified.

[0318] The binding activity detection method is the same as in Example 1.4, except that the coating antigen BDCA2-Fc is replaced with BDCA2-his, the coating concentration is 1 μg / mL, and the secondary antibody is replaced with anti-human Fc HRP (SinoBiological, catalog number SSA001) with a working concentration of 1:3000.

[0319] The experimental results are as follows Figure 3 As shown, the binding activity of C-23C10 to BDCA2 antigen is better than that of the positive control 24F4A.

[0320] CpG-A (CpG ODNs Class A) is an oligodeoxynucleotide with a CpG palindromic sequence, which can stimulate pDC cells to secrete a large amount of IFNa. In this example, PBMC was used to detect the inhibitory effect of the candidate antibody on CpG-A-induced PBMC secretion of IFNa.

[0321] CpG-A was diluted to 8 μg / mL (final concentration was 2 μg / mL) with RPMI 1640+3% FBS medium and added to a flat-bottomed 96-well plate, 50 μL per well. Antibodies were diluted to 16 nM with RPMI 1640+3% FBS medium, 5-fold dilution (final concentration was 4 nM, 5-fold dilution), and added to CpG-A wells, 50 μL per well. CpG-A and antibodies 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 and inoculated into the above 96-well plate, 500,000 cells per well, 100 μL per well, and incubated overnight in a 37°C, 5% CO2 incubator. The cell supernatant was centrifuged and the H-IFNa content in the supernatant was detected.

[0322] The experimental results are as follows 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.

[0323] 2.3 Humanization of mouse anti-human BDCA2 monoclonal antibody

[0324] The amino acid sequences of the heavy and light chain variable regions of 23C10A6B5 were analyzed, and three antigen complementary determining regions (CDRs) and four framework regions (FRs) were determined according to the Kabat rule. Among them, the amino acid sequence of the heavy chain complementary determining region is

[0325] HCDR1: SYIIH (SEQ ID NO: 6),

[0326] HCDR2: TIYPGNGDTSYNQKFKG (SEQ ID NO: 7) and

[0327] HCDR3: MGDNEYFDY (SEQ ID NO: 8), the amino acid sequence of the light chain complementary determining region is

[0328] LCDR1:RASGNIHNYLA(SEQ ID NO:9),

[0329] LCDR2: DAETLAD (SEQ ID NO: 10) and

[0330] LCDR3: QHFWSTPYT (SEQ ID NO: 11).

[0331] By comparing homology with human IgG germline sequences in NCBI IgBlast, IGHV1-46*01 was selected as the heavy chain CDR transplantation template and IGKV1-NL1*01 was selected as the light chain CDR transplantation template. The CDR region of the 23C10A6B5 antibody was transplanted to the selected humanized template to replace the CDR region of the human template. 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. At the same time, based on the three-dimensional structure of the antibody, the buried residues, the residues that directly interacted with the CDR region, and the residues that had an important influence on the conformation of the antibody's VL and VH were back mutated to obtain multiple humanized antibodies. The heavy chain variable region sequence (SEQ ID NO: 12) and the light chain variable region sequence (SEQ ID NO: 13) of the humanized antibody 230 were determined through affinity screening.

[0332] Example 3 Affinity maturation of humanized antibody 230

[0333] The AlphaFold2 software was used to predict the complex structure and analyze the interaction interface of the 230 antibody and BDCA2-his antigen, and a mutant library was constructed by introducing single-point saturation mutations at the key sites of interaction. The ELISA affinity screening was used to identify mutation sites that can improve the affinity of the 230 antibody. The experimental method is as described in Example 1.4, except that the coating antigen BDCA2-Fc was replaced with BDCA2-his, the coating concentration was 0.1 μg / mL, i.e., 10 ng per well, and the secondary antibody was replaced with anti-human Fc HRP (SinoBiological, catalog number SSA001), with a working concentration of 1:3000.

[0334] The results are as follows Figure 5 The experimental results show that

[0335] 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

[0336] TIYPGGGDTSYNQKFKG (SEQ ID NO: 15),

[0337] TIYPGAGDTSYNQKFKG (SEQ ID NO: 16),

[0338] TIYPGSGDTSYNQKFKG (SEQ ID NO: 17),

[0339] The TIYPGRGDTSYNQKFKG (SEQ ID NO: 18) mutation (the corresponding antibodies are 230-I33L, 230-N55G, 230-N55A, 230-N55S and 230-N55R, respectively) can effectively improve the affinity of the 230 antibody.

[0340] In addition, the light chain CDR2: (SEQ ID NO: 10) Mutated to I or E respectively, and obtained DAETLAI (SEQ ID NO: 19) and DAETLAE (SEQ ID NO: 20). The mutants maintained the same affinity as 230, while the expression level increased by more than 30% on average. The remaining saturated mutants had the same affinity and expression level as the parent 230.

[0341] The mutated selected parts of the above-mentioned CDR regions were combined and connected to 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; 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.

[0342] 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.

[0343] According to the above-mentioned 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.

[0344] The results are as follows 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.

[0345] Example 4 Determination of affinity dissociation constant KD value

[0346] The heavy chain FC region of the 230IL-NG-DE and 230-NG-DE antibodies in Example 3 was 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).

[0347] The kinetic parameters of the binding and dissociation of the candidate antibody protein with the antigen BDCA2-his were determined by the capture method using a Biacore 8K molecular interaction analyzer. The antibody was diluted to 1 μg / mL with a 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 with a 1*HBS-N, pH 7.0 buffer containing 5 mM CaCl2, 0.02% Tween 20, and the antibody was bound to 6 concentration gradients with a maximum concentration of 12.5 nM, and dissociated in a 1*HBS-N, pH 7.0 buffer containing 5 mM CaCl2, 0.02% Tween 20.

[0348] 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.

[0349] Table 5: Affinity dissociation constants

[0350]

[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 IFNa secretion induced 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 as follows 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] The sequence of the present invention is as follows:

[0356] Human BDCA2 extracellular domain (BDCA2-ECD) SEQ ID NO: 1

[0357] NFMYSKTVKRLSKLREYQQYHPSLTCVMEGKDIEDWSCCPTPWTSFQSSCYFISTGMQSW

[0358] TKSQKNCSVMGADLVVINTREEQDFIIQNLKRNSSYFLGLSDPGGRRHWQWVDQTPYNENVTF

[0359] WHSGEPNNLDERCAIINFRSSEEWGWNDIHCHVPQKSICKMKKIYI

[0360] 23C10A6B5 heavy chain variable region nucleotide sequence SEQ ID NO: 2

[0361] CAGGTGCAACTACAGCAGCCTGGGGCTGACCTGGTGAAGCCTGGGGCCTCAGTGATGATGTCCTGCAAGGCTTCTGGATACACATTTACCAGTTACATTATTCACTGGGTAAAACAGACACCTGGACAGGGCCTGGAATGGATTGGAACTATTTATCCAGGAAATGGTGATACTTCC TACAATCAGAAATTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAATGGGGGATAACGAATACTTTGACTACTGGGGCCAAGGAACCACTCTCACAGTCTCCTCA

[0362] 23C10A6B5 heavy chain variable region amino acid sequence SEQ ID NO: 3

[0363] QVQLQQPGADLVKPGASVMMSCKASGYTFTSYIIHWVKQTPGQGLEWIGTIYPGNGDTSY NQKFKGKATLTADKSSSTAYMQLSSSLTSEDSAVYYCARMGDNEYFDYWGQGTTLTVSS

[0364] 23C10A6B5 light chain variable region nucleotide sequence SEQ ID NO: 4

[0365] GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGGAAATATTCACAATTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATGATGCAGAAACCT TAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCCGGAACACAATATTCTCTCAATATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTTTTACTGTCAACATTTTTGGAGTACTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAAATAAAA

[0366] 23C10A6B5 light chain variable region amino acid sequence SEQ ID NO: 5

[0367] DIQMTQSPASSLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYDAETLADGVPSR FSGSGSGTQYSLNINSLQPEDFGSFYCQHFWSTPYTFGGGTKLEIK

[0368] HCDR1 of 23C10A6B5: SYIIH (SEQ ID NO: 6)

[0369] HCDR2 of 23C10A6B5: TIYPGNGDTSYNQKFKG (SEQ ID NO: 7)

[0370] HCDR3 of 23C10A6B5: MGDNEYFDY (SEQ ID NO: 8)

[0371] LCDR1 of 23C10A6B5: RASGNIHNYLA (SEQ ID NO: 9)

[0372] LCDR2 of 23C10A6B5: DAETLAD (SEQ ID NO: 10)

[0373] LCDR3 of 23C10A6B5: QHFWSTPYT (SEQ ID NO: 11)

[0374] Heavy chain variable region of humanized antibody 230 SEQ ID NO: 12

[0375] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYIIHWVRQAPGQGLEWMGTIYPGNGDTS YNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARMGDNEYFDYWGQGTLVTVSS

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

[0377] DIQMTQSPSSSLSASVGDRVTITCRASGNIHNYLAWYQQKPGKAPKLLLYDAETLADGVPSR FSGSGSGTDYTLTISSLQPEDFATYYCQHFWSTPYTFGGGTKVEIK

[0378] 230 Antibody-IL Heavy Chain CDR1 SEQ ID NO: 14SYLIH

[0379] 230 Antibody-NG Heavy Chain CDR2 SEQ ID NO: 15TIYPGGGDTSYNQKFKG

[0380] 230 antibody-NA heavy chain CDR2 SEQ ID NO: 16TIYPGAGDTSYNQKFKG

[0381] 230 Antibody-NS Heavy Chain CDR2 SEQ ID NO: 17TIYPGSGDTSYNQKFKG

[0382] 230 Antibody-NR Heavy Chain CDR2 SEQ ID NO: 18TIYPGRGDTSYNQKFKG

[0383] 230 Antibody-DI Light Chain CDR2 SEQ ID NO: 19DAETLAI

[0384] 230 Antibody-DE Light Chain CDR2 SEQ ID NO: 20DAETLAE

[0385] 230IL-DI heavy chain variable region SEQ ID NO: 21

[0386] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYLIHWVRQAPGQGLEWMGTIYPGNGDTS YNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARMGDNEYFDYWGQGTLVTVSS

[0387] 230IL-NG-DI Heavy Chain Variable Region SEQ ID NO: 22

[0388] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYLIHWVRQAPGQGLEWMGTIYPGGGDTS YNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARMGDNEYFDYWGQGTLVTVSS

[0389] 230IL-NR-DI Heavy Chain Variable Region SEQ ID NO: 23

[0390] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYLIHWVRQAPGQGLEWMGTIYPGRGDTS YNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARMGDNEYFDYWGQGTLVTVSS

[0391] 230NG-DI Heavy Chain Variable Region SEQ ID NO: 24

[0392] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYIIHWVRQAPGQGLEWMGTIYPGGGDTS YNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARMGDNEYFDYWGQGTLVTVSS

[0393] 230NR-DI Heavy Chain Variable Region SEQ ID NO: 25

[0394] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYIIHWVRQAPGQGLEWMGTIYPGRGDTSY NQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARMGDNEYFDYWGQGTLVTVSS

[0395] 230IL-DI Light Chain Variable Region SEQ ID NO: 26

[0396] DIQMTQSPSSLSASVGDRVTITCRASGNIHNYLAWYQQKPGKAPKLLLYDAETLAIGVPSRF SGSGSGTDYTLTISSLQPEDFATYYCQHFWSTPYTFGGGTKVEIK

[0397] 230IL-DE Light Chain Variable Region SEQ ID NO: 27

[0398] DIQMTQSPSSLSASVGDRVTITCRASGNIHNYLAWYQQKPGKAPKLLLYDAETLAEGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWSTPYTFGGGTKVEIK

[0399] Heavy chain of antibody 237011, SEQ ID NO: 28

[0400] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYLIHWVRQAPGQGLEWMGTIYPGGGDTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARMGDNEYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK

[0401] Heavy chain of antibody 2391, SEQ ID NO: 29

[0402] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYIIHWVRQAPGQGLEWMGTIYPGGGDTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARMGDNEYFDYWGQGT LVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK

[0403] HCDR1 SYX1IH of the anti-BDCA2 antibody, X1 is I or L (SEQ ID NO: 30)

[0404] H-CDR2 of anti-BDCA2 antibody TIYPGX2GDTSYNQKFKG, X2 is N or G or A or S or R (SEQ ID NO: 31)

[0405] Antibody L-CDR2 DAETLAX3 of anti-BDCA2, X3 is I or E (SEQ ID NO: 32)

[0406] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

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 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.

3. The antibody or antigen-binding fragment thereof that binds to BDCA2 according to claim 1 or 2, 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.

4. 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 3; and (ii) optionally a tag sequence to facilitate expression and / or purification.

5. 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 3.

6. An expression vector, characterized in that: The expression vector contains the nucleotide molecule according to any one of claim 5.

7. A host cell, characterized in that The host cell contains the expression vector according to claim 6.

8. A method for preparing an antibody or antigen-binding fragment thereof that binds to BDCA2 according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: a) culturing the host cell of claim 7 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).

9. 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 3 and a pharmaceutically acceptable carrier.

10. Use of the antibody or antigen-binding fragment thereof binding to BDCA2 according to any one of claims 1 to 3 or the composition according to claim 9 in the preparation of a drug for treating inflammatory diseases or autoimmune diseases; preferably, The inflammatory disease or autoimmune disease is selected from the group consisting of systemic lupus erythematosus, discoid lupus, lupus nephritis, cutaneous lupus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type I diabetes, dermatomyositis and polymyositis.

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