CN118638234A - Anti-BDCA2 antibody and its preparation method and application - Google Patents

Abstract

The present disclosure relates to anti-BDCA2 antibodies and preparation methods and applications thereof. Specifically, the present disclosure relates to a rabbit-derived antibody that specifically binds to the extracellular region of human BDCA2 and a humanized antibody thereof. The antibodies disclosed herein have potential therapeutic effects in autoimmune diseases (such as systemic lupus erythematosus).

Description

Anti-BDCA2 antibody and its preparation method and application

Technical Field

The present disclosure relates to the fields of biology and medicine. Specifically, the present disclosure relates to an antibody that specifically binds to human BDCA2, especially a rabbit antibody that specifically binds to human BDCA2 and a humanized antibody thereof.

Background Art

BDCA2 (Blood dendritic cell antigen 2), also known as CLEC4C or CD303, is the fourth member of the C-type lectin family and a new type II transmembrane glycoprotein containing 213 amino acids. BDCA2 is mainly expressed on the surface of plasmacytoid dendritic cells (pDCs), and its natural ligand has not yet been identified.

The extracellular part of BDCA2 contains a C-type carbohydrate recognition domain that can bind to mannose, glucose or N-acetylglucosamine, effectively inhibiting the secretion of type I interferon (IFN-I) in pDCs through a mechanism that depends on calcium mobilization and protein tyrosine. Antibodies targeting BDCA2 can rapidly internalize BDCA2 from the surface of pDCs and reduce the production of inflammatory cytokines including type I interferon, which plays an important role in autoimmune diseases such as systemic lupus erythematosus (SLE).

Summary of the invention

Antibodies that specifically bind to human BDCA2

In one aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds to human BDCA2, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region and the light chain variable region are selected from any one of the following groups:

The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 33; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 34; or

The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 37; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 38; or

The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 31; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 32; or

The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 35; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 36; or

The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 39; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 40;

In some embodiments, the HCDRs and the LCDRs are identified according to the Kabat, Chothia, AbM or IMTG numbering systems.

In some embodiments, the HCDRs and the LCDRs are identified according to the Kabat numbering system.

In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 as described above comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region and the light chain variable region are selected from any one of the following groups:

1) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, and HCDR3 shown in SEQ ID NO: 3; and

The light chain variable region comprises LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO: 6; or

2) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 7, HCDR2 shown in SEQ ID NO: 8, and HCDR3 shown in SEQ ID NO: 9; and

The light chain variable region comprises LCDR1 shown in SEQ ID NO: 10, LCDR2 shown in SEQ ID NO: 11, and LCDR3 shown in SEQ ID NO: 12; or

3) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 13, HCDR2 shown in SEQ ID NO: 14, and HCDR3 shown in SEQ ID NO: 15; and

The light chain variable region comprises LCDR1 shown in SEQ ID NO: 16, LCDR2 shown in SEQ ID NO: 17, and LCDR3 shown in SEQ ID NO: 18; or

4) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 19, HCDR2 shown in SEQ ID NO: 20, and HCDR3 shown in SEQ ID NO: 21; and

The light chain variable region comprises LCDR1 shown in SEQ ID NO: 22, LCDR2 shown in SEQ ID NO: 23, and LCDR3 shown in SEQ ID NO: 24; or

5) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 25, HCDR2 shown in SEQ ID NO: 26, and HCDR3 shown in SEQ ID NO: 27; and

The light chain variable region comprises LCDR1 shown in SEQ ID NO: 28, LCDR2 shown in SEQ ID NO: 29, and LCDR3 shown in SEQ ID NO: 30;

The HCDRs and the LCDRs are identified according to the Kabat numbering system.

In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 as described above, wherein the heavy chain variable region and the light chain variable region are selected from any one of the following groups:

1) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 33, and

the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:34, or comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:34;

2) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 37 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 37, and

the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:38, or comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:38;

3) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 31 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 31, and

the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:32, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:32;

4) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 35 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 35, and

the light chain variable region comprises the amino acid sequence of SEQ ID NO:36, or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:36;

5) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 39 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 39, and

the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:40, or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:40;

In this context, "at least 85%" means at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a range between any two of the foregoing values, which can be an integer or a decimal.

In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 as described above comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region are selected from any one of the following groups:

1) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 32;

2) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 34;

3) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 36;

4) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 38;

5) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 39, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 40.

In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 as described in any of the preceding items is a rabbit-derived antibody, a chimeric antibody, or a humanized antibody.

In some embodiments, the antigen-binding fragment of any of the aforementioned antibodies that specifically bind to human BDCA2 is selected from any one of the following: Fab, scFv, Fv, Fab', F(ab') ₂ , single domain antibody, scFab, linear antibody, and multispecific antibody.

In some embodiments, any of the aforementioned antibodies or antigen-binding fragments thereof that specifically bind to human BDCA2 further comprises a heavy chain constant region and a light chain constant region.

In some specific embodiments, the antibodies or antigen-binding fragments thereof that specifically bind to human BDCA2 of the present disclosure are of rabbit origin.

In other specific embodiments, the antibodies or antigen-binding fragments thereof that specifically bind to human BDCA2 of the present disclosure are chimeric.

In other specific embodiments, the antibodies or antigen-binding fragments thereof that specifically bind to human BDCA2 of the present disclosure are humanized.

In some specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 of the present disclosure comprises a heavy chain variable region and a light chain variable region, wherein:

1) the heavy chain variable region comprises an amino acid sequence selected from any one of SEQ ID NOs: 43 to 47, or an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises an amino acid sequence selected from any one of SEQ ID NOs: 48 to 49, or an amino acid sequence having at least 85% sequence identity thereto; or

2) the heavy chain variable region comprises an amino acid sequence selected from any one of SEQ ID NOs: 50 to 52, or an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises an amino acid sequence selected from any one of SEQ ID NOs: 53 to 54, or an amino acid sequence having at least 85% sequence identity thereto.

In some specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 of the present disclosure comprises a heavy chain variable region and a light chain variable region, wherein:

1) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence having at least 85% sequence identity thereto; or

2) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence having at least 85% sequence identity thereto; or

3) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence having at least 85% sequence identity thereto; or

4) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence having at least 85% sequence identity thereto; or

5) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence having at least 85% sequence identity thereto; or

6) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence having at least 85% sequence identity thereto; or

7) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence having at least 85% sequence identity thereto; or

8) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence having at least 85% sequence identity thereto; or

9) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 46 or an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 49 or an amino acid sequence having at least 85% sequence identity thereto; or

10) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 47 or an amino acid sequence having at least 85% sequence identity thereto, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 49 or an amino acid sequence having at least 85% sequence identity thereto.

In some specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 of the present disclosure comprises a heavy chain variable region and a light chain variable region, wherein:

1) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 48; or

2) the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 48; or

3) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 45, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 48; or

4) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 48; or

5) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 47, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 48; or

6) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 49; or

7) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 49; or

8) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 45, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 49; or

9) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 49; or

10) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 47, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 49.

In some specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 of the present disclosure comprises a heavy chain variable region and a light chain variable region, wherein:

1) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 50, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 53; or

2) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 51, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 53; or

3) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 52, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 53; or

4) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 50, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 54; or

5) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 51, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 54; or

6) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 52, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 54.

In some specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 of the present disclosure comprises a heavy chain variable region and a light chain variable region, wherein:

1) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 50, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 53; or

2) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 51, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 53; or

3) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 52, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 53; or

4) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 50, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 54; or

5) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 51, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 54; or

6) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 52, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 54.

In some embodiments, the heavy chain constant region is selected from the constant regions of human IgG1, human IgG2, human IgG3 and human IgG4 or mutants thereof, wherein the amino acid at position 252 is Y, the amino acid at position 254 is T and the amino acid at position 256 is E, or the amino acid at position 428 is L and the amino acid at position 434 is S mutated, and the amino acid positions are determined by EU numbering.

In some embodiments, the light chain constant region is selected from a lambda and a kappa light chain constant region.

In some embodiments, the heavy chain constant region is a human IgG1 heavy chain constant region, and the light chain constant region is a kappa light chain constant region.

In some embodiments, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:41, 60 or 61, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:42.

In some specific embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:41, and the light chain constant region comprises the amino acid sequence of SEQ ID NO:42.

In some specific embodiments, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:41, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:42.

In other specific embodiments, the heavy chain constant region is a variant of the amino acid sequence shown in SEQ ID NO: 41, and preferably the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 60 or 61.

In some specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 of the present disclosure comprises a heavy chain and a light chain, and the heavy chain and the light chain are selected from any one of the following groups:

1) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 55, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 56;

2) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 57, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 58;

3) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 62, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 63;

4) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 64, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 65;

5) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 66, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 67; and

6) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 68, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 69.

In some specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 of the present disclosure comprises a heavy chain and a light chain, and the heavy chain and the light chain are selected from any one of the following groups:

1) the heavy chain comprises the amino acid sequence of SEQ ID NO: 55 or an amino acid sequence having at least 85% sequence identity thereto, and the light chain comprises the amino acid sequence of SEQ ID NO: 56 or an amino acid sequence having at least 85% sequence identity thereto;

2) the heavy chain comprises the amino acid sequence of SEQ ID NO: 57, or an amino acid sequence having at least 85% sequence identity thereto, and the light chain comprises the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence having at least 85% sequence identity thereto;

3) the heavy chain comprises the amino acid sequence of SEQ ID NO: 62 or an amino acid sequence having at least 85% sequence identity thereto, and the light chain comprises the amino acid sequence of SEQ ID NO: 63 or an amino acid sequence having at least 85% sequence identity thereto;

4) the heavy chain comprises the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence having at least 85% sequence identity thereto, and the light chain comprises the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence having at least 85% sequence identity thereto;

5) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 66, or an amino acid sequence having at least 85% sequence identity thereto, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 67, or an amino acid sequence having at least 85% sequence identity thereto; and

6) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 68 or an amino acid sequence having at least 85% sequence identity thereto, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 69 or an amino acid sequence having at least 85% sequence identity thereto.

In some specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 of the present disclosure comprises a heavy chain and a light chain, and the heavy chain and the light chain are selected from any one of the following groups:

1) the amino acid sequence of the heavy chain is shown in SEQ ID NO: 55, and the amino acid sequence of the light chain is shown in SEQ ID NO: 56;

2) the amino acid sequence of the heavy chain is shown in SEQ ID NO: 57, and the amino acid sequence of the light chain is shown in SEQ ID NO: 58;

3) the amino acid sequence of the heavy chain is shown in SEQ ID NO: 62, and the amino acid sequence of the light chain is shown in SEQ ID NO: 63;

4) the amino acid sequence of the heavy chain is shown in SEQ ID NO: 64, and the amino acid sequence of the light chain is shown in SEQ ID NO: 65;

5) the amino acid sequence of the heavy chain is shown in SEQ ID NO: 66, and the amino acid sequence of the light chain is shown in SEQ ID NO: 67; and

6) The amino acid sequence of the heavy chain is shown in SEQ ID NO: 68, and the amino acid sequence of the light chain is shown in SEQ ID NO: 69.

In some specific embodiments, the humanized antibodies or antigen-binding fragments thereof of the present disclosure bind to human BDCA2 or its extracellular region with a KD value of no higher than 4E-09, 3E-09, 2E-09, 1E-09, 9E-10, 8E-10, 7E-10, 6E-10, 5E-10, 4E-10, 3E-10 (M).

In some specific embodiments, the humanized antibodies or antigen-binding fragments thereof of the present disclosure bind to monkey BDCA2 or its extracellular region with a KD value of no higher than 7E-09, 6E-09, 5E-09, 4E-09, 3E-09, 2E-09, 1E-09, 9E-10, 8E-10 (M).

In other specific embodiments, the chimeric antibodies or antigen-binding fragments thereof of the present disclosure bind to human BDCA2 or its extracellular region with an _EC50 of no higher than 0.04, 0.03, 0.02, 0.019, 0.018, 0.017, 0.016, 0.015, 0.014, 0.013, 0.012, 0.011 (μg/mL).

In other specific embodiments, the chimeric antibodies or antigen-binding fragments thereof of the present disclosure bind to monkey BDCA2 or its extracellular region with an _EC50 of no higher than 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 (μg/mL).

In some specific embodiments, the chimeric and humanized antibodies or antigen-binding fragments thereof of the present disclosure bind to cells overexpressing BDCA2.

The antibodies or antigen-binding fragments thereof specifically binding to human BDCA2 provided by the present disclosure have any one or more of the following functional characteristics:

1) Binds to human or cynomolgus monkey BDCA2 with high binding affinity;

2) Effectively inhibit TLR9-induced IFN-α in human PBMCs;

3) effectively inhibit TLR7-induced IFN-α in human PBMCs; and

4) The killing of target cells is mediated by ADCC.

In a specific embodiment, the EC50 value of the antibody or antigen-binding fragment thereof specifically binding to human BDCA2 provided by the present disclosure for inhibiting the secretion of IFN-α induced by TLR9 of human PBMC is 0.0005546 (μg/mL). For specific detection methods, please refer to Example 7.

In a specific embodiment, the antibody or antigen-binding fragment thereof specifically binding to human BDCA2 provided by the present disclosure has an EC5 value of 0.002841 (μg/mL) for killing HEK-hBDCA2 cells through ADCC activity, and the maximum killing rate is 38.55%. For specific detection schemes, please refer to Example 9.

Pharmaceutical composition

The antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 according to the present disclosure can be formulated into a pharmaceutical composition that is administered to a subject (e.g., to prevent or treat a disease according to the present disclosure). Therefore, the present disclosure provides a pharmaceutical composition that contains a preventive or therapeutically effective amount of any of the above-described antibodies or antigen-binding fragments that specifically bind to human BDCA2, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

Typically, a pharmaceutical composition includes a pharmaceutically acceptable carrier, diluent, buffer or excipient.

In some embodiments, a "pharmaceutical composition" refers to a mixture containing one or more antibodies or antigen-binding fragments thereof described herein and other components, such as physiologically/pharmaceutically acceptable carriers and excipients. One of the purposes of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

In some embodiments, the antibody or antigen-binding fragment thereof described herein is an antibody or antigen-binding fragment thereof that specifically binds to human BDCA2.

The formulation of drugs is a well-known technique, as further described, for example, in Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins (2000); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed., Lippincott Williams & Wilkins Publishers (1999).

Pharmaceutical compositions can also be in a variety of forms, including, for example, liquid, semisolid, and solid dosage forms. For example, liquid solutions (e.g., injectable and infusible solutions), suspensions, tablets, pills, powders, liposomes, suppositories. The preferred form may depend on the mode of administration and therapeutic application. Typically, the pharmaceutical compositions described herein are in the form of injectable or infusible solutions.

In one embodiment, an antibody that specifically binds to human BDCA2 according to the present disclosure is provided at a suitable concentration in a buffer and stored at 2-8°C.

The pharmaceutical composition can be administered parenterally (e.g., intravenous, subcutaneous, intraperitoneal or intramuscular injection). As used herein, "parenteral administration" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular

, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. In some specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 is administered intravenously.

In some embodiments, antibodies or antigen-binding fragments thereof that specifically bind to human BDCA2 can be prepared together with a controlled release carrier (eg, a controlled release formulation, an implant, a microencapsulated delivery system). Methods for preparing such formulations are generally known.

Isolated nucleic acids

The present disclosure also provides an isolated nucleic acid encoding any of the aforementioned antibodies or antigen-binding fragments thereof that specifically bind to human BDCA2.

It should be understood that even if the present application provides specific nucleotide sequence examples, the nucleotide sequences capable of "encoding the antibodies or antigen-binding fragments thereof that specifically bind to human BDCA2 of the present disclosure" are not limited to these specific sequences, because equivalent nucleotide sequences can be used to encode the same amino acid sequence according to the codon preference and codon degeneracy of the expression host.

Expression vector

The present disclosure also provides an expression vector comprising the isolated nucleic acid according to the present disclosure.

Nucleic acids encoding the desired antibodies or fragments thereof can be constructed, introduced into expression vectors, and expressed in appropriate host cells.

The expression vector can be a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus or a retrovirus.

Host cells

The present disclosure also provides a host cell, which comprises the aforementioned nucleic acid or expression vector for expressing the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 of the present disclosure.

Host cells include prokaryotic and eukaryotic host cells. Eukaryotic host cells include, but are not limited to, mammalian cells, insect cells, plant cells, and fungal cells. Mammalian cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells. Exemplary host cells include, but are not limited to, CHO, NSO, COS, SP2 cells, HeLa cells, BHK cells, human hepatocellular carcinoma cells, A549 cells, 3T3 cells, and HEK-293 cells. Fungal cells include yeast, such as, but not limited to, Pichia, Saccharomyces, Hansenula polymorpha, and Kluyveromyces.

In the present disclosure, a host cell is incapable of developing into a complete animal or plant individual.

Methods of producing antibodies

Antibodies can be produced in bacteria or eukaryotic cells. Antibodies can be produced in bacterial cells or in eukaryotic cells (e.g., CHO, 293E). To produce the desired antibody, a nucleic acid encoding the antibody is constructed, introduced into an expression vector, and then expressed in a suitable host cell. Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture host cells, and recover the antibody.

If the antibody is expressed in an animal cell (such as CHO), the expression vector includes a promoter required for expression, such as an SV40 promoter, an EF1α promoter, or a CMV promoter. In addition to the nucleic acid sequence encoding the antibody or its domain, the recombinant expression vector can carry additional sequences, such as a selection marker. The selection marker facilitates the selection of the host cell into which the vector is introduced. For example, the resistance of the selection marker to a drug (such as G418, hygromycin, or methotrexate) is usually selected.

In some embodiments, the antibody is produced in a mammalian cell. Exemplary mammalian host cells for expressing the antibody include Chinese hamster ovary (CHO cells), human embryonic kidney 293 cells, COS cells, NIH3T3 cells, NS0 myeloma cells, SP2 cells.

The antibodies of the present disclosure can be separated from host cells and purified to substantially pure antibodies. Separation and purification methods commonly used for antibody purification can be used for separation and purification of antibodies, such as chromatography, filtration, ultrafiltration, salting out, precipitation, extraction, distillation, electrophoresis, isoelectric focusing, dialysis, recrystallization. Chromatography includes, for example, affinity, ion exchange, hydrophobic, reverse phase and adsorption chromatography, etc. Columns for affinity chromatography, such as protein A columns and protein G columns.

In some embodiments, the antibody is produced by chemical synthesis, which includes the steps of synthesizing the amino acid sequence according to the antibody or antigen-binding fragment thereof.

In some embodiments, the antibodies that specifically bind to human BDCA2 in the present invention include murine antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.

Prevention or treatment methods

The antibodies or antigen-binding fragments thereof that specifically bind to human BDCA2 described in the present disclosure can be used to treat or prevent a variety of immune diseases, such as inflammatory and autoimmune diseases. In some embodiments, the disease is associated with abnormal expression or abnormal activity of BDCA2 on pDCs.

The present disclosure provides a method for preventing or treating a disease, the method comprising administering to a subject a preventive or therapeutically effective amount of an antibody or antigen-binding fragment thereof, a nucleic acid or a pharmaceutical composition that specifically binds to human BDCA2. In some embodiments, the disease is an inflammatory disease or an autoimmune disease; more preferably, the inflammatory disease or autoimmune disease is selected from: systemic lupus erythematosus, discoid lupus, lupus nephritis, epidermal lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis, progressive systemic sclerosis (scleroderma), psoriasis, psoriatic arthritis, type I diabetes, fibrosis (e.g., skin fibrosis, pulmonary fibrosis), pemphigus vulgaris, Graves' disease, morphea (localized scleroderma), Sjögren's disease, Hashimoto's disease, dermatomyositis, polymyositis, asthma, Behcet's disease, Crohn's disease, autoimmune glomerulonephritis, membranous glomerulopathy, juvenile rheumatoid arthritis, mixed connective tissue disease, multiple sclerosis, nephrotic syndrome, panniculitis, pemphigoid, pemphigus, pemphigus erythematosus, pemphigus foliaceus, pemphigus vulgaris, polymyalgia rheumatica, Raynaud's phenomenon/syndrome, Sjögren's syndrome, and ulcerative colitis.

In specific embodiments, the inflammatory or autoimmune disease is systemic lupus erythematosus.

In specific embodiments, the inflammatory or autoimmune disease is discoid lupus.

In specific embodiments, the inflammatory or autoimmune disease is epidermal lupus erythematosus.

In specific embodiments, the disease or disorder is a BDCA2-mediated disease.

Systemic lupus erythematosus is a chronic autoimmune disease in which multiple organs are damaged by immune complexes and tissue-bound autoantibodies. Systemic sclerosis (or systemic scleroderma) is a systemic connective tissue disease. Psoriasis is an autoimmune disease that affects the skin. Psoriasis occurs when the immune system mistakes skin cells for pathogens and sends erroneous signals that accelerate the growth cycle of skin cells. Rheumatoid arthritis is a chronic inflammatory disease that affects many tissues and organs, but mainly attacks active joints. Inflammatory bowel disease (IBD) is a group of inflammatory diseases of the colon and small intestine. The main types of IBD are Crohn's disease and ulcerative colitis (UC). Dermatomyositis (DM) is a class of autoimmune connective tissue diseases associated with polymyositis (PM), characterized by inflammation of muscles and skin. Type I diabetes is a form of diabetes that results from autoimmune destruction of the insulin-producing β cells of the pancreas, with subsequent insulin deficiency leading to increased blood and urine glucose.

Examples of other diseases suitable for prevention or treatment with the antibodies or antigen-binding fragments thereof that specifically bind to human BDCA2 of the present disclosure include asthma, Behcet's disease, CREST syndrome, pulmonary fibrosis, autoimmune glomerulonephritis, membranous glomerulopathy, juvenile rheumatoid arthritis, mixed connective tissue disease, multiple sclerosis, nephrotic syndrome, panniculitis, pemphigoid, pemphigus, pemphigus erythematosus, pemphigus foliaceus, and pemphigus vulgaris.

An antibody or an antigen-binding fragment thereof that specifically binds to human BDCA2 is administered to a subject at risk for, diagnosed with, or having these diseases in an amount for a period of time to provide a therapeutic effect. An antibody or an antigen-binding fragment thereof that specifically binds to human BDCA2 can be administered alone (monotherapy) or in combination with a second therapeutic agent (combination therapy).

In specific embodiments, the subject is an individual susceptible to, suspected of having, or already having a disease.

"Treatment" means providing an antibody or antigen-binding fragment thereof that specifically binds to human BDCA2, or a pharmaceutical composition thereof, to a subject. The subject has one or more symptoms of a disease. Typically, an antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 is administered in an amount effective to alleviate the symptoms of the disease in the treated subject or subject population.

In specific embodiments, the selection of an effective amount can be determined by those skilled in the art based on consideration of a variety of factors (e.g., through clinical trials), including the disease to be treated, the symptoms involved, the route of administration, the severity of the disease, the subject's weight, the subject's immune status, and other factors known to those skilled in the art.

The effective amount in the specific embodiment can be obtained from the dose-response curve derived from the animal model test system, and it is allowed to be determined according to the doctor's judgment and the situation of each subject. As an example, the amount of the drug required for the subject's one-time administration can be conveniently obtained by calculating the product of the subject's body weight and the unit body weight dose required for the subject's one-time medication. For example, in the process of preparing the drug, it is generally believed that the adult body weight is 50-70kg, and the dosage can be initially determined by the equivalent dose conversion relationship between the unit body weight dose of the experimental animal and the human. For example, it can be determined according to the guidance opinions proposed by drug administration agencies such as SFDA and FDA. In some embodiments, the dosage of humans and mice can be converted using the body surface area conversion coefficient of 0.0026 for humans and mice.

The active ingredient herein (e.g., an antibody or antigen-binding fragment thereof that specifically binds to human BDCA2) can be administered at once, or can be divided into a number of smaller unit doses and administered at certain time intervals. It should be understood that the dosage, duration, and interval of treatment are a function of the disease being treated, and can be determined by inference using animal or clinical trial data. The administration may include a single administration, or two or more administrations separated by appropriate time intervals. Two adjacent administrations may be spaced 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, one and a half days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months apart.

The present disclosure provides a method for reducing the production of inflammatory cytokines or chemokines by plasmacytoid dendritic cells in a subject, the method comprising contacting plasmacytoid dendritic cells expressing BDCA2 with a certain amount of the anti-BDCA2 antibody or antigen-binding fragment thereof described in the present disclosure, or the pharmaceutical composition described in the present disclosure.

In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2, or the pharmaceutical composition disclosed herein is used in the preparation of a drug for inducing death of plasmacytoid dendritic cells.

In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 described in the present disclosure, or the pharmaceutical composition described in the present disclosure is used in the preparation of a medicament for reducing the production of inflammatory cytokines or chemokines by plasmacytoid dendritic cells.

In some embodiments, the antibody or antigen-binding fragment thereof specifically binding to human BDCA2 described in the present disclosure, or the pharmaceutical composition described in the present disclosure can be used as a drug for inducing death of plasmacytoid dendritic cells, or reducing the production of inflammatory cytokines or chemokines by plasmacytoid dendritic cells.

use

In another aspect, the present disclosure also provides use of an antibody or an antigen-binding fragment thereof, a nucleic acid or a pharmaceutical composition that specifically binds to human BDCA2 in the preparation of a drug for treating or preventing a disease.

In some embodiments, the disease is an inflammatory disease or an autoimmune disease.

In some embodiments, the inflammatory disease or autoimmune disease is selected from the group consisting of systemic lupus erythematosus, discoid lupus, lupus nephritis, epidermal lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis, progressive systemic sclerosis (scleroderma), psoriasis, psoriatic arthritis, type I diabetes, fibrosis (e.g., skin fibrosis, pulmonary fibrosis), pemphigus vulgaris, Graves' disease, morphea (localized scleroderma), Sjogren's disease, Hashimoto's disease, dermatomyositis, polymyositis, asthma, Behcet's disease, Crohn's disease, autoimmune glomerulonephritis, membranous glomerulopathy, juvenile rheumatoid arthritis, mixed connective tissue disease, multiple sclerosis, nephrotic syndrome, panniculitis, pemphigoid, pemphigus, pemphigus erythematosus, pemphigus foliaceus, pemphigus vulgaris, polymyalgia rheumatica, Raynaud's phenomenon/syndrome, Sjogren's syndrome, and ulcerative colitis.

The present disclosure also provides antibodies or antigen-binding fragments thereof, nucleic acids or pharmaceutical compositions that specifically bind to human BDCA2 for use as drugs. In some embodiments, the present disclosure also provides antibodies or antigen-binding fragments thereof, nucleic acids or pharmaceutical compositions that specifically bind to human BDCA2 for use as drugs for treating diseases.

In some embodiments, the aforementioned antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 is provided for use in treating a disease, wherein the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 is administered in combination with a second therapeutic agent.

In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 and the second therapeutic agent are in the same or different containers.

Detection Methods

The antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 of the present disclosure is used for immunoassay. The immunoassay includes contacting the biological sample with the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 of the present disclosure, and detecting the expression level of BDCA2 in the biological sample from the subject. Typically, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 of the present disclosure can be coupled to a fluorescent label or other label.

Reagents/Kits

The present disclosure also provides a drug kit comprising at least one container, wherein each container independently comprises: any one of the aforementioned antibodies or antigen-binding fragments thereof that specifically bind to human BDCA2, nucleic acids, or pharmaceutical compositions.

In diagnostic or research applications, the kits include one or more of the following components: analytical reagents, buffers, and the antibodies or antigen-binding fragments thereof of the present disclosure. In addition, these reagents/kits may include instructions.

The invention discloses a rabbit-derived antibody, a chimeric antibody and a humanized antibody specifically binding to the extracellular region of human BDCA2, which are found to have high affinity with BDCA2 protein and have potential therapeutic effects in autoimmune diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Activity of antibodies of the present disclosure in inhibiting TLR9-induced IFN-α secretion.

Figure 2. Activity of antibodies of the present disclosure in inhibiting TLR7-induced IFN-α secretion.

Figure 3. ADCC activity of antibodies of the present disclosure.

DETAILED DESCRIPTION

the term

The terminology used herein is for the purpose of describing the embodiments only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.

Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising", "having", "including", etc. should be understood as meaning "including but not limited to". Unless otherwise specified, "comprising" includes "consisting of", for example, for VH comprising HCDR1 as shown in SEQ ID NO: 31, it clearly covers the HCDR1 with an amino acid sequence as shown in SEQ ID NO: 31.

It should be understood that the ordinal numbers "first", "second", "Formula 1", "Formula 2", "1)", "2)", etc. in the present disclosure are only used to distinguish different technical features, elements, components, and steps, and are not intended to limit the level, sequence, or quantity.

The target refers to the object to which the therapeutic active ingredient (antibody or antigen-binding fragment thereof that specifically binds to human BDCA2) of the present disclosure is directed. The target can be a nucleic acid (gene, mRNA, etc.) or a protein (precursor, mature protein, isoform, modified form, free, surface expressed). In the present disclosure, the target particularly refers to a protein. As an example, an antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 targets BDCA2.

The nucleotide or amino acid information of BDCA2 as a target is well known in the art, and can be obtained, for example but not limited to, from literature or databases.

In the context of the present disclosure, BDCA2 should be understood broadly, including various forms of BDCA2 molecules at various stages, such as, but not limited to, molecules generated during amplification, replication, transcription, splicing, processing, translation, and modification of the BDCA2 gene, such as cDNA, mRNA, precursor protein, mature protein, and fragments thereof. In specific embodiments, BDCA2 refers to human BDCA2. In some specific embodiments, human BDCA2 refers to mature BDCA2 protein, especially BDCA2 expressed on the cell surface.

In the field of antibodies, targets usually exist in the form of antigens. "Antigen" refers to a molecule or part thereof that can be selectively recognized or bound by an antigen-binding molecule (e.g., an antibody or antigen-binding fragment thereof that specifically binds to human BDCA2). An antigen may have one or more epitopes.

"Epitope" refers to a region on an antigen that can specifically bind to an antibody or its antigen-binding fragment. An epitope can be formed by continuous amino acids (linear epitope); or comprise non-continuous amino acids (conformational epitope). An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids. Routine methods in the art can be used to screen for antibodies or their antigen-binding fragments that bind to a specific epitope, such as but not limited to alanine scanning, peptide cleavage analysis, epitope extraction, and chemical modification of the antigen.

"Antibody" is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and full-length antibodies, as long as they exhibit the desired antigen-binding activity. Typically, natural IgG antibodies are heterotetrameric proteins consisting of two light chains and two heavy chains bound by disulfide bonds. From N to C-terminus, each heavy chain has a variable region (VH) and three constant domains (CH1, CH2, and CH3). From N to C-terminus, each light chain has a variable region (VL) and a constant light domain (CL). Depending on the context, a technician can determine the specific meaning of "antibody".

The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. Antibodies are divided into five main classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

"Specific binding" means that the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 of the present disclosure binds to the targeted antigen or epitope thereof with a higher affinity than other antigens or epitopes thereof. Generally, an equilibrium dissociation constant (KD) of about 1×10 ^-7 M or less is considered to be "specific" binding. KD can be measured using known methods, such as FACS or surface plasmon resonance. "Specific binding" does not exclude cross-reactivity to homologous antigens of other species (such as cynomolgus monkeys (Macaca fascicularis), chimpanzees (Pan troglodytes) or marmosets (Callithrix jacchus). In view of this, "specifically binds to human BDCA2...", "specifically binds to BDCA2..." means that the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 is able to specifically recognize or bind to BDCA2 (or its epitope).

"Affinity" is used to describe the strength of the non-covalent interaction between an antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 and its antigen or epitope thereof. Affinity is usually expressed by the equilibrium dissociation constant (KD). "Kassoc" or "ka" refers to the association rate of a particular antibody-antigen interaction, and "kdis" or "kd" is intended to refer to the dissociation rate of a particular antibody-antigen interaction. "KD" is obtained from the ratio of kd to ka (i.e., kd/ka), expressed as a molar concentration.

A "chimeric antibody" is an antibody formed by fusing the variable region of an antibody from one species (such as rabbit) with the constant region of an antibody from another species (such as human).

A "humanized antibody" refers to an antibody produced by transplanting non-human CDR sequences (such as rabbit-derived CDRs) into the framework of a human antibody variable region.

"Antibody fragments" or "antigen-binding fragments" are molecules other than intact antibodies, which contain a portion of an intact antibody that retains the antigen-binding ability of the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ , single-domain antibodies, single-chain Fab (scFab), diabodies, linear antibodies, single-chain antibodies (e.g., scFv); and multispecific antibodies formed from antibody fragments.

"Fab" consists of a light chain and the CH1 and variable region of a heavy chain.

"Fab' fragment", which is a Fab fragment having one or more cysteine residues at the C-terminus of the CH1 domain.

"F(ab') ₂ fragment" is a bivalent fragment formed by two Fab' fragments linked by a disulfide bridge at the hinge region.

"Fab'-SH" refers to Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.

"Single-chain Fab fragment" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein the antibody domain and the linker have one of the following orders from N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL; and wherein the linker is a peptide linker. In some embodiments, the peptide linker is a polypeptide of at least 30 amino acids, preferably a polypeptide of 32-50 amino acids.

"Diabodies" have two antigen-binding sites and include a heavy chain variable domain (VH) and a light chain variable domain (VL) connected to each other in the same polypeptide chain.

A "linear antibody" comprises a pair of tandem Fd segments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen binding regions.

A "Fv fragment" has the VL and VH domains of one arm of an antibody.

A "single domain antibody" is an antibody fragment that comprises all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody.

A single-chain antibody fragment (scFv) is an antibody composed of a heavy chain variable region and a light chain variable region connected by a linker.

"Fc" contains two heavy chain fragments including CH2 and CH3 domains. The two heavy chain fragments are held together by disulfide bonds and the hydrophobic interaction of the CH3 domain. As a supplement, in the anti-BDCA2 antibody or antigen-binding fragment thereof of the present application, although the C-terminus of the Fc region is a complete C-terminus ending with PGK, it can still be a truncated C-terminus. Exemplarily, one or two C-terminal residues are removed in the truncated C-terminus (e.g., a truncated C-terminus ending with PG). In the composition containing antibodies, antibodies with complete C-termini and antibodies with truncated C-termini can be included.

"Variable region" refers to the domain of an antibody or antigen-binding fragment that binds antigen.

"Complementarity determining region" or "CDR" refers to the region within the variable region that primarily contributes to antigen binding. VH and VL each contain four framework regions (FR) and three complementarity determining regions (CDR). VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL has the following sequence from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

"Framework" or "FR" refers to the variable domain residues outside the hypervariable region (CDR) residues. The FR of the variable domain is usually composed of four FR domains: FR1, FR2, FR3 and FR4. Therefore, HVR and FR sequences usually appear in the following order in VH (or VL): FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

The heavy chain variable region of the antibody that specifically binds to BDCA2 is represented as VH, and the light chain variable region is represented as VL. The three CDR regions in VH are represented as HCDR1, HCDR2, and HCDR3, respectively; the three CDR regions in VL are represented as LCDR1, LCDR2, and LCDR3, respectively.

The amino acid sequence boundaries of CDRs can be determined by various well-known schemes, such as: the Kabat numbering system (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD), the Chothia numbering system, and the ImMunoGenTics (IMGT) numbering system (Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9:2278).

The correspondence between various numbering systems is well known to those skilled in the art. In other words, when a CDR sequence under one numbering system and its position in an antibody are provided, the technician is capable of determining the corresponding CDR sequence under another numbering system and its position in an antibody. Technical solutions corresponding to different numbering systems will be regarded as equivalent technical solutions.

In one embodiment, the antibody CDRs of the present disclosure are positioned according to the Kabat numbering scheme.

The anti-BDCA2 antibodies disclosed herein may also include antibodies with "alternative CDRs". "Alternative" CDRs refer to CDRs (CDR1, CDR2, and CDR3) defined according to any one of Chothia (from AbYsis), AbM CDR, or ImMunoGeneTics database (IMGT). For example, these alternative CDRs can be obtained by using the AbYsis database (www.bioinf.org.uk/abysis/sequence_input/key_annotation/key_annotation.cg). Exemplarily, in the following table, the amino acid sequences of the "alternative" CDR1, CDR2, and CDR3 of the heavy chain variable region and the light chain variable region of 14D10-VH1VL1 are compared with the CDRs defined according to Kabat.

Table 1

Unless otherwise indicated, in this disclosure, when referring to residue positions in antibody variable regions and CDRs, reference is made to the numbered positions according to the Kabat numbering system.

In view of the above, the skilled person understands that when the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 of the present disclosure is described according to the Kabat numbering system, it also includes the corresponding sequence under the Chothia, AbM CDR or IMGT numbering system. As an example, the antibody or antigen-binding fragment thereof that specifically binds to human BDCA2 of the present disclosure comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region and the light chain variable region are selected from any one of the following groups:

The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 7, HCDR2 shown in SEQ ID NO: 8, and HCDR3 shown in SEQ ID NO: 9; and

The light chain variable region comprises LCDR1 shown in SEQ ID NO: 10, LCDR2 shown in SEQ ID NO: 11, and LCDR3 shown in SEQ ID NO: 12;

or

The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 70, HCDR2 shown in SEQ ID NO: 71, and HCDR3 shown in SEQ ID NO: 9; and

The light chain variable region comprises LCDR1 shown in SEQ ID NO: 10, LCDR2 shown in SEQ ID NO: 11, and LCDR3 shown in SEQ ID NO: 12;

or

The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 72, HCDR2 shown in SEQ ID NO: 73, and HCDR3 shown in SEQ ID NO: 9; and

The light chain variable region comprises LCDR1 shown in SEQ ID NO: 10, LCDR2 shown in SEQ ID NO: 11, and LCDR3 shown in SEQ ID NO: 12;

or

The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 74, HCDR2 shown in SEQ ID NO: 75, and HCDR3 shown in SEQ ID NO: 9; and

The light chain variable region comprises LCDR1 shown in SEQ ID NO:76, LCDR2 shown in SEQ ID NO:77, and LCDR3 shown in SEQ ID NO:12.

The term "percent (%) amino acid sequence identity" or simply "identity" is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the amino acid residues in a reference amino acid sequence, after the amino acid sequences are aligned (and gaps introduced where necessary) to obtain the maximum percent sequence identity, and any conservative substitutions are not considered part of the sequence identity. Sequence alignments can be performed using various methods in the art to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignments, including any algorithm required to achieve maximum alignment over the entire length of the compared sequences.

As an example, "the heavy chain variable region comprises an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 31" means that the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, and HCDR3 shown in SEQ ID NO: 3, and amino acid mutations are allowed to be introduced in regions outside the CDRs so as to have at least 85% sequence identity with SEQ ID NO: 31.

The term "humanization" refers to a humanized form of a non-human (e.g., mouse) antibody or fragment thereof (e.g., Fv, Fab, Fab', F(ab'), scFv or other antigen binding portion sequences of an antibody) comprising some portion of a sequence derived from a non-human antibody. Humanized antibodies include human immunoglobulins in which residues from a complementarity determining region (CDR) of a human immunoglobulin are replaced with residues from a CDR of a non-human species (e.g., mouse, rat or rabbit) having the desired binding specificity, affinity and capacity. In general, a humanized antibody will comprise at least one, and usually substantially all of two variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. Optimally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin; see, especially, Jones et al., Nature 321 (1986), 522-525, Presta, Curr. Op. Struct. Biol. 2 (1992), 593-596.

Methods for humanizing non-human antibodies are well known in the art. Typically, a humanized antibody has one or more amino acids introduced from a non-human source, while retaining the original binding activity of the antibody. Methods for humanizing antibodies/antibody molecules are also described in detail in Jones et al., Nature 321 (1986), 522-525; Reichmann et al., Nature 332 (1988), 323-327; and Verhoeyen et al., Science 239 (1988), 1534-1536. Popular methods for antibody humanization involve CDR grafting, in which a functional antigen binding site from a non-human "donor" antibody is grafted onto a human "acceptor" antibody. CDR grafting methods are known in the art and are described, for example, in US 5,225,539, US 5,693,761, and US 6,407,213. Another related approach is to generate humanized antibodies from transgenic animals that are genetically engineered to contain one or more humanized immunoglobulin loci capable of gene rearrangement and gene conversion (see, e.g., US 7,129,084).

"Amino acid mutation" includes amino acid substitution, deletion, insertion, modification or any combination thereof. Any combination of substitution, deletion, insertion and modification can be performed to achieve the final construct, as long as the final construct has the desired characteristics, such as amino acid substitutions that reduce homodimerization in the Fc region. Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for the desired activity, such as retained/improved antigen binding, reduced immunogenicity. Amino acid sequence deletions and insertions include deletions and insertions at the amino terminus and/or carboxyl terminus of the polypeptide chain. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, that is, an amino acid is replaced with another amino acid having different structural and/or chemical properties. Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc.

"Nucleic acid" is used interchangeably with "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. The nucleic acids are synthetic, naturally occurring, and non-naturally occurring. Unless otherwise indicated, a nucleic acid sequence also encompasses conservative variants thereof (e.g., degenerate substitutions) and complementary sequences as well as explicitly specified sequences. The term "nucleic acid molecule" includes a plurality of nucleic acid molecules.

"Vector" means a polynucleotide molecule capable of transporting another polynucleotide to which it is attached. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which an exogenous DNA segment is attached. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV), in which an exogenous DNA segment is attached to the viral genome. Some vectors are capable of autonomous replication in host cells, while others can be integrated into the host's genome, thereby replicating along with the host genome.

"Host cell", "cell line" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced and their progeny, regardless of the number of generations of the progeny. Progeny are not allowed to be completely identical to the parent cell in terms of nucleic acid, but may contain mutations, as long as the progeny with mutations have the same desired function or activity as the original cell. The term "BDCA2-mediated disease" refers to a disease in which BDCA2 is involved in the onset, progression or prolongation of the disease, such as involving the activation of BDCA2 (e.g., abnormal activation or overactivation), and the involvement of BDCA2 directly or indirectly produces at least one of the following effects: the onset/occurrence of the disease; the increase/deepening of the pathological changes of the disease; the expansion of the site/range affected by the disease; the aggravation of the body damage caused by the disease; the increase of the pain caused by the disease; the insensitivity/resistance of the disease to treatment measures; the decrease of the tendency of the disease to heal itself and the worsening of the prognosis of the disease. In some embodiments of the disclosure, the BDCA2-mediated disease is an inflammatory disease or an autoimmune disease; in some specific embodiments of the disclosure, the BDCA2-mediated disease is systemic lupus erythematosus, discoid lupus, lupus nephritis, epidermal lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type I diabetes, fibrosis (e.g., cutaneous fibrosis), pemphigus vulgaris, psoriasis, Graves' disease, morphea, Sjogren's disease, Hashimoto's disease, dermatomyositis, and polymyositis.

"Subject" or "individual" refers to an animal, preferably a mammal. According to a specific embodiment, the subject is a mammal, including, for example, camels, horses, cows, pigs, sheep, cats, dogs, rabbits, rats, guinea pigs, mice, primates (e.g., humans). In a specific embodiment, the subject is a human.

It should be understood that when a numerical range is disclosed, for example, "A to B" is a concise way of writing, although not every point value in the range is given, it is considered that the integers and decimals within the range have been explicitly disclosed.

“And/or”, for example, “A and/or B” should be understood to mean “A and B” or “A or B”.

"Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur. As used in the specification and claims, the singular forms "a", "an" include the corresponding plural referents unless the content clearly dictates otherwise.

The following are examples of the practice of the present disclosure and should not be construed as limiting the scope of the invention in any way.

Example

Example 1. Preparation of rabbit anti-BDCA2 antibody

1. Preparation of Immunogen

Entrust Bio-Bio to express the extracellular region protein of human BDCA2 with human IgG Fc (hFc) fragment:

NFMYSKTVKRLSKLREYQQYHPSLTCVMEGKDIEDWSCCPTPWTSFQSSCYFISTGMQSWTKSQKNCSVMGADLVVINTREEQDFIIQNLKRNSSYFLGLSDPGGRRHWQWVDQTPYNENVTFWHSGEPNNLDERCAIINFRSSEEWGWNDIHCHVPQKSICKMKKIYI (SEQ ID NO: 59); the immunogen hBDCA2-hFc was obtained with an expression level of 10.3 mg/L and a SDS-PAGE purity of >95%.

The full-length cDNA sequences encoding human or monkey BDCA2 (NM_130441.3; XM_005570023.1) and human or monkey FcεRIγ (NM_004106.1; NM_001265840.2) were cloned into the lentiviral vector pLVX-Puro (source: Clontech) and transfected by Lipofectamine 2000 (purchased from ThermoFisher; catalog number: 11668-019) was used to transfect 293T cells, and the viral supernatant was collected and used to infect the target cells. After 2 weeks of screening in a selective medium containing puromycin, subcloning was performed in a 96-well culture plate by limiting dilution. After 2 weeks, some monoclonal amplification was selected and screened by flow cytometry using the known anti-BDCA2 antibody 24F4 (produced according to the sequence disclosed in patent application WO2014093396, and the light/heavy chains correspond to the third and fourth sequences numbered in WO2014093396, respectively). Monoclonal cells with higher fluorescence intensity were selected for expansion culture and cryopreservation to obtain overexpression cell lines CHO-K1-HuBDCA2 and CHO-K1-CynoBDCA2 expressing human or monkey BDCA2.

2. Animal immunization and antibody screening

The immunized animals were female New Zealand white rabbits. The hBDCA2-hFc protein was used as an immunogen to immunize rabbits A and B. For the first immunization, Freund's complete adjuvant (purchased from Sigma; item number: F5881-10ML) was emulsified with an equal volume of antigen, with a dose of 300 μg/rabbit. For the booster immunization, Freund's incomplete adjuvant (purchased from Sigma, item number F5506-10ML) was emulsified with an equal volume of antigen, with a dose of 150 μg/rabbit. After successful emulsification, New Zealand white rabbits were injected subcutaneously at multiple points on the back. The first immunization was separated by 21 days, and each subsequent immunization was separated by 14 days. Blood was collected one week after each immunization to detect the antibody titer in the serum. After the third immunization, the ELISA method was used to detect whether the serum of rabbits A and B could specifically bind to the human BDCA2 protein, and the results showed that the titer was above 1:256000.

The stable strain of CHO-K1-HuBDCA2 was selected as the immunogen to immunize rabbits C and D. Cells in the logarithmic growth phase were collected and diluted with PBS. Intraperitoneal immunization was performed at 5×10 ⁷ cells/time/rabbit. The first immunization was separated by 10 days, and the subsequent immunizations were separated by one week. After four immunizations, hBDCA2-hFc was used for booster immunization. After the immunization, the ELISA method was used to detect the binding of the sera of rabbits C and D to human BDCA2. The results showed that the titers of the sera of both rabbits were greater than 1:256000.

After immunization, animal B cells were taken, and the target B cells were specifically screened using immunomagnetic beads. B cell complete culture medium was added, and the cells were plated into 96-well cell culture plates by limiting dilution method. ELISA and FACS detection were performed after culturing for 7-10 days. A total of 355 positive B cell supernatants were screened, and 42 positive clones were selected for sequencing and expression.

Example 2. Sequencing of light/heavy chain variable regions and expression of chimeric antibodies

The positive B cells cultured in Example 1 were collected, RNA was extracted and reverse transcribed, and the heavy chain and light chain variable region sequences of the positive clones were obtained after sequencing, and rabbit antibodies 4C12, 14D10, 14G5, 16C6, and 17H5 were obtained. The CDR amino acid sequences were determined according to the database of Kabat (Kabat, E.A. et al. 1991).

The heavy chain and light chain variable regions obtained by sequencing were connected and cloned into the expression vector pcDNA3.4 with the heavy chain IgG1 constant region of the human antibody and the light chain kappa constant region of the human antibody, respectively, and transiently transfected into CHO-K1 cells. After culturing at 37°C, 8% CO ₂ , and 110rpm for 3 to 7 days, the cell supernatant was collected and purified by protein A affinity to obtain chimeric antibodies ch-4C12, ch-14D10, ch-14G5, ch-16C6, and ch-17H5. The purified antibodies were subjected to protein concentration, purity, endotoxin detection analysis, and various characterization tests, and finally 5 antibodies were obtained, and their CDR and heavy and light chain variable region amino acid sequences are shown in the table below.

Table 2. CDR sequences of rabbit antibodies

Note: The CDRs in the table are obtained according to the Kabat numbering convention.

The variable regions of the rabbit antibody are as follows:

4C12-VH:

QSLEESGGRLVTPGTPLTLTCTVSGFSLN NYAVS WVRQAPGKGLEWIG LISNSGNTYYASWAKG RFTISKTSTTVDLKGTSPTTEDTATYFCAR YGYDDYGYYWLDL WGQGTLVTVSS(SEQ ID NO: 31)

4C12-VL:

IKMTQTPASVSAAVGGTVSINC QASEDIESYLA WYQQKPGQPPKLLIY DADDLAS GVPSRFKGSGSGTQFTLTISGVQCDDAATYYC QHGHYTSSSDNA FGGGTEVVVK(SEQ ID NO: 32)

14D10-VH:

QSVEESGGRLVTPGTPLTLTCTVSGIDLS TYGMG WVRQAPGKGLEYIG IISNTGGTYSASWAKG RFTISKTSTTVDLKMTSLTTEDTATYFCAR GLYDDYGTYYFDL WGQGTLVTVSS (SEQ ID NO: 33)

14D10-VL:

IKMTQTPASVSAAVGGRVSINC QASEDIESYLA WYQQKPGQPPKLLIY DASDLAS GVPSRFKGSGSGKQFTLTISGVQCDDAATYYC QHGFYTSDSDNA FGGGTEVVVE(SEQ ID NO: 34)

14G5-VH:

QSVEESGGRLVTPGTPLTLTCTVSGFSLS EYSMG WVRQAPGKGLEYIG IISNTGNTYYASWAKG RFTISKTSTTVDLRTTSPTTEDTATYFCAR SLYDDYGAYYFDL WGQGTLVTVSS(SEQ ID NO: 35)

14G5-VL:

IKLTQTPASVSAAVGGTVTINC QASEDIETYLA WYQQKPGQPPNLLIY DASDLAS GVPSRFKGSGSGKQFTLTISGVQCDDAATYYC QHGYYTSTSDNA FGGGTAVVVK(SEQ ID NO: 36)

16C6-VH:

QSVEESGGRLVTPGTPLTLTCTVSGFSLS SYAMS WVRQAPGKGLEWIG IISSSGNTYYASWAKG RFAISKTSTKVDLRITSPTTEDTATYFCAR GYSYDDYGDFFDAFDP WGPGTLVTVSS(SEQ ID NO: 37)

16C6-VL:

ADIVMTQTPASVEAAVGGTVTIKC QASQSIVGYLA WYQQKPGQPPKLLIY RASTLPS GVPSRFKGSGSGAQFTLTISDLECADAATYYC QSWHYDTSSTYGWA FGGGTEVVVK(SEQ ID NO: 38)

17H5-VH:

QSVEESGGRLVTPGTPLTLTCTVSGFSLS SYAMS WVRQAPGKGLEWIG IISTSGRTYDASWAKG RFTISKTSTTVDLKITSPTTEDTATYFCAR ALYDDYGDYWGYFNL WGQGTLVTVSS(SEQ ID NO: 39)

17H5-VL:

IKMTQTPASVSAAVGGTVSINC QASEDIETFLA WYQQKPGQPPKLLIY DASDLAS GVPSRFKGSGSGKQFTLTISGVQCDDAATYYC QHGYYTSSSENA FGGGTEVVVK (SEQ ID NO: 40);

Human IgG1 constant region:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 41)

Human light chain kappa constant region:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC (SEQ ID NO: 42).

Example 3. Humanization of antibodies

Through sequence comparison, the germline gene sequence with the highest homology to the heavy chain variable region and light chain variable region of the rabbit antibody was selected as the variable region transplantation skeleton.

14D10 selected IGHV3-64, IGHV3-23 and IGHV1-46 as the heavy chain variable region grafting framework, and selected IGKV1-5 and IGKV3-7 as the light chain variable region grafting framework. 16C6 selected IGHV3-64 as the heavy chain variable region grafting framework, and selected IGKV1-27 and IGKV1-12 as the light chain variable region grafting framework. Through homology modeling, the grafted framework region was back mutated, and the humanized antibodies of 14D10 and 16C6 were designed and expressed.

The variable region sequences of the humanized antibody are as follows:

14D10-VH1:

QVQLVESGGGLVQPGGSLRLSCSASGIDLSTYGMGWVRQAPGKGLEYIGII SNTGGTYSASWAKGRFTISKDSSKNTVYLQMNSLRAEDTAVYYCARGLYDDY GTYYFDLWGRGTLVTVSS (SEQ ID NO: 43)

14D10-VH2:

QVQLVESGGGLVQPGGSLRLSCSASGIDLSTYGMGWVRQAPGKGLEYIGII SNTGGTYSASWAKGRFTISKDSSKNTLYLQMNSLRAEDTAVYYCARGLYDDY GTYYFDLWGRGTLVTVSS (SEQ ID NO: 44)

14D10-VH3:

QVQLVESGGGLVQPGGSLRLSCSASGIDLSTYGMGWVRQAPGKGLEYIGII SNTGGTYSASWAKGRFTISKDNSKNTLYLQMNSLRAEDTAVYYCARGLYDDY GTYYFDLWGRGTLVTVSS (SEQ ID NO: 45)

14D10-VH7:

EVQLLESGGGLVQPGGSLRLSCAASGIDLSTYGMGWVRQAPGKGLEYIGII SNTGGTYSASWAKGRFTISKDSSKNTVYLQMNSLRAEDTAVYYCARGLYDDY GTYYFDLWGRGTLVTVSS (SEQ ID NO: 46)

14D10-VH8:

QVQLVQSGAEVKKPGASVKVSCKASGIDLSTYGMGWVRQAPGQGLEYIGI ISNTGGTYSASWAKGRFTITKDSSTSTVYMELSSLRSEDTAVYYCARGLYDDYG TYYFDLWGRGTLVTVSS(SEQ ID NO: 47)

14D10-VL1:

DIQMTQSPSSVSASVGDRVTITCQASEDIESYLAWYQQKPGKAPKLLIYDA SDLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHGFYTSDSDNAFGGGTK VEIK (SEQ ID NO: 48)

14D10-VL2:

EIVMTQSPPTLSLSPGERVTLSCQASEDIESYLAWYQQKPGQAPRLLIYDAS DLASGIPARFSGSGSGKDFTLTISSLQPEDFAVYYCQHGFYTSDSDNAFGGGTK VEIK (SEQ ID NO: 49)

16C6-VH1:

QVQLVESGGGLVQPGGSLRLSCSASGFSLSSYAMSWVRQAPGKGLEWISII SSSGNTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYSYDD YGDFFDAFDPWGQGTMVTVSS (SEQ ID NO: 50)

16C6-VH2:

QVQLVESGGGLVQPGGSLRLSCSASGFSLSSYAMSWVRQAPGKGLEWIGII SSSGNTYYASWAKGRFTISKDSSKNTVYLQMNSLRAEDTAVYYCARGYSYDD YGDFFDAFDPWGQGTMVTVSS(SEQ ID NO: 51)

16C6-VH3:

QVQLVESGGGLVQPGGSLRLSCSASGFSLSSYAMSWVRQAPGKGLEWIGII SSSGNTYYASWAKGRFTISKDSSKNTVYLQMNSLRAEDTAVYYCARGYSYDD YGDFFDAFEPWGQGTMVTVSS(SEQ ID NO: 52)

16C6-VL1:

DIQMTQSPSSSLSASVGDRVTITCQASQSIVGYLAWYQQKPGKVPKLLIYRA STLPSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSWHYDTSSTYGWAFGQ GTKVEIK(SEQ ID NO: 53)

16C6-VL2:

DIQMTQSPSSVSASVGDRVTITCQASQSIVGYLAWYQQKPGKAPKLLIYRA STLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSWHYDTSSTYGWAFGQ GTKVEIK (SEQ ID NO: 54).

According to the method of Example 2, the heavy chain and light chain variable regions of the humanized antibody were connected to the human antibody heavy chain IgG1 constant region and the human antibody light chain kappa constant region, respectively, and cloned into the expression vector pcDNA3.4 for transient expression to obtain the humanized antibody.

Table 3. Combinations of variable region sequences of humanized antibodies

The sequences of exemplary humanized antibodies are as follows:

14D10-VH1VL1 HC:

QVQLVESGGGLVQPGGSLRLSCSASGIDLSTYGMGWVRQAPGKGLEYIGIISNTGGTYSASWAKGRFT ISKDSSKNTVYLQMNSLRAEDTAVYYCARGLYDDYGTYYFDLWGRGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 55)

14D10-VH1VL1 LC:

DIQMTQSPSSVSASVGDRVTITCQASEDIESYLAWYQQKPGKAPKLLIYDASDLASGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQHGFYTSDSDNAFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC(SEQID NO:56)

16C6-VH1VL1 HC:

QVQLVESGGGLVQPGGSLRLSCSASGFSLSSYAMSWVRQAPGKGLEWISIISSSGNTYYASWAKGRFT ISRDNSKNTLYLQMNSLRAEDTAVYYCARGYSYDDYGDFFDAFDPWGQGTMVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 57)

16C6-VH1VL1 LC:

DIQMTQSPSSSLSASVGDRVTITCQASQSIVGYLAWYQQKPGKVPKLLIYRASTLPSGVPSRFSGSGSG TDFTLTISSLQPEDVATYYCQSWHYDTSSTYGWAFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 58).

Underlining indicates variable regions.

Example 4. Fc mutation of humanized antibody

The heavy chain constant region of the humanized antibody screened in Example 3 was introduced with M252Y, S254T and T256E (YTE) mutations, or M428L and N434S (LS) mutations to increase the affinity of the antibody to FcRn. The amino acid positions are determined by EU numbering.

Fc-YTE:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 60) The mutation sites are in bold;

Fc-LS:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK (SEQ ID NO: 61) The mutation sites are in bold.

The sequences of exemplary mutant antibodies are as follows:

Heavy chain sequence of 14D10-VH1VL1(YTE):

QVQLVESGGGLVQPGGSLRLSCSASGIDLSTYGMGWVRQAPGKGLEYIGIISNTGGTYSASWAKGRFT ISKDSSKNTVYLQMNSLRAEDTAVYYCARGLYDDYGTYYFDLWGRGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 62)

Light chain sequence of 14D10-VH1VL1(YTE):

DIQMTQSPSSVSASVGDRVTITCQASEDIESYLAWYQQKPGKAPKLLIYDASDLASGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQHGFYTSDSDNAFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC (SEQID NO: 63; same as 14D10-VH1VL1 LC)

Heavy chain sequence of 14D10-VH1VL1(LS):

QVQLVESGGGLVQPGGSLRLSCSASGIDLSTYGMGWVRQAPGKGLEYIGIISNTGGTYSASWAKGRFT ISKDSSKNTVYLQMNSLRAEDTAVYYCARGLYDDYGTYYFDLWGRGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK(SEQ ID NO: 64)

Light chain sequence of 14D10-VH1VL1(LS):

DIQMTQSPSSVSASVGDRVTITCQASEDIESYLAWYQQKPGKAPKLLIYDASDLASGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQHGFYTSDSDNAFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC (SEQID NO: 65; same as 14D10-VH1VL1 LC)

Heavy chain sequence of 16C6-VH1VL1(YTE):

QVQLVESGGGLVQPGGSLRLSCSASGFSLSSYAMSWVRQAPGKGLEWISIISSSGNTYYASWAKGRFT ISRDNSKNTLYLQMNSLRAEDTAVYYCARGYSYDDYGDFFDAFDPWGQGTMVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 66)

Light chain sequence of 16C6-VH1VL1(YTE):

DIQMTQSPSSSLSASVGDRVTITCQASQSIVGYLAWYQQKPGKVPKLLIYRASTLPSGVPSRFSGSGSG TDFTLTISSLQPEDVATYYCQSWHYDTSSTYGWAFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 67, same as 16C6-VH1VL1 LC)

Heavy chain sequence of 16C6-VH1VL1(LS):

QVQLVESGGGLVQPGGSLRLSCSASGFSLSSYAMSWVRQAPGKGLEWISIISSSGNTYYASWAKGRFT ISRDNSKNTLYLQMNSLRAEDTAVYYCARGYSYDDYGDFFDAFDPWGQGTMVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK(SEQ ID NO: 68)

Light chain sequence of 16C6-VH1VL1(LS):

DIQMTQSPSSSLSASVGDRVTITCQASQSIVGYLAWYQQKPGKVPKLLIYRASTLPSGVPSRFSGSGSG TDFTLTISSLQPEDVATYYCQSWHYDTSSTYGWAFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 69, same as 16C6-VH1VL1 LC);

The mutation sites are in bold and the variable regions are underlined.

Example 5. Antibody Binding Activity Detection

1. Antibody affinity testing

Octet R8 was used to detect the affinity of the antibody to BDCA2 protein.

Sample treatment: Use running buffer SD buffer (0.1% BSA-PBST) to dilute the test antibody to 5 μg/mL, dilute the analyte human BDCA2 protein (purchased from ACRO; Catalog No.: CLC-H5245) or monkey BDCA2 (purchased from KACTUS; Catalog No.: BCA-CM102) to 5 μg/mL, and then dilute it by 2 times, for a total of 5 concentrations. Use ProA biosensor (purchased from Sartorius) to capture the antibody, and then immerse the sensor in the analyte.

This example runs five steps: 1. Baseline (60 seconds), 2. Loading (capture antibody) (100 seconds, 1.0 nm), 3. Baseline (60 seconds), 4. Association (45 seconds), 5. Dissociation (200 seconds). After the experiment, the Octet Analysis Studio 12.2 software was used for fitting. The results are shown in the table below.

The results showed that the screened chimeric antibodies all had high affinity to BDCA2, and the chimeric antibodies and humanized antibodies of 14D10 and 16C6 had high affinity to both human and cynomolgus monkey BDCA2.

Table 4. Affinity of chimeric antibodies to hBDCA2

Table 5. Affinity of 14D10 humanized antibody to BDCA2 from different species

Table 6. Affinity of 16C6 humanized antibody to BDCA2 from different species

2. ELISA to detect the binding of antibodies to BDCA2 protein

The binding of antibodies to human or monkey BDCA2 protein was detected by ELISA.

Human BDCA2 (purchased from Kactus; Catalog No.: BCA-HM102) or monkey BDCA2 (purchased from Kactus; Catalog No.: BCA-CM102) was diluted into PBS at a concentration of 1 μg/mL and coated on a 96-well plate at 4°C overnight. Blocked with 5% BSA-PBST blocking solution at 37°C for 1 hour, washed, and then added with gradient dilutions of antibodies (concentration of 20 μg/mL in the first well, then 6-fold gradient dilutions), and incubated at 37°C for 1 hour. After washing, HRP-labeled goat anti-human IgG (purchased from Sigma; Catalog No.: 129825) was added, reacted at 37°C for 30 minutes, washed, added TMB, protected from light for 10 minutes, and then 2N H ₂ SO ₄ was added to terminate the reaction. The absorbance was detected at a wavelength of 450nm on an enzyme reader (TECAN SPARK).

The results showed that the chimeric antibody had good binding activity to both human and monkey BDCA2, and the binding activity of 14D10 and 16C6 humanized antibodies to hBDCA2 protein was comparable to that of the chimeric antibody.

Table 7. Binding activity of chimeric antibodies to hBDCA2 and cynoBDCA2

Table 8. Binding activity of antibodies to hBDCA2

3. Detection of the binding activity of antibodies to cells expressing BDCA2

Flow cytometry (FACS) was used to detect the binding of humanized antibodies to cells overexpressing BDCA2.

The 293T-hBDCA2 cells in Example 1 were collected, washed twice with PBS containing 2% BSA, and added to a 96-well cell culture plate at 3×10 ⁵ cells per well. Antibodies of different dilution concentrations were added (the first well concentration was 80 μg/mL, 4-fold gradient dilution), and incubated on ice for 30 minutes. After washing, PE-labeled goat anti-human IgG (purchased from Invitrogen; catalog number: 12-4998-82) was added, incubated at 4°C in the dark for 30 minutes, and detected by flow cytometer (BD Accuri C6 Plus).

The results showed that the binding activities of humanized antibodies and chimeric antibodies of 14D10 and 16C6 to 293T-hBDCA2 cells were comparable.

Table 9. Binding activity of antibodies to 293T-hBDCA2 cells

Example 6. Detection of antibody endocytosis ability

The HEK293 cells expressing human BDCA2 in Example 1 were collected, inoculated into 96-well white microplates at 5×10 ³ cells/well, and cultured in an incubator overnight. The antibody was mixed with DT3C (purchased from Huamei Biotechnology; catalog number: CSB-EP360556CQR1) at a molar ratio of 1:4, incubated at room temperature for 30 minutes, and the antibody and DT3 mixture was diluted to 3 μg/mL in the first well, and then added to the cells after 2.5-fold gradient dilution. Cultivated in a 37°C, 5% CO ₂ incubator for 3 days, and CTG (purchased from Promega; catalog number: G7572) was added for detection.

The results showed that the chimeric antibodies all had good endocytosis activity; the binding activities of 14D10 and 16C6 humanized antibodies were comparable to those of the chimeric antibodies.

Table 10. Endocytic activity of chimeric antibodies

Example 7. Antibodies inhibit TLR9-induced IFN-α secretion

Resuscitate human PBMC cells, resuspend in complete medium (RPMI 1640 + 10% FBS + 1 × NEAA + 1 × sodium pyruvate + 1 × GlutaMax), adjust the cell density to 5 × 10 ⁶ cells / mL, and inoculate into 96-well cell culture plates at 100 μL / well; dilute the antibody to 40 μg / mL, dilute 20 times to 2 concentration points, dilute 10 times to 1 concentration point, and then dilute 3 times to 4 concentration points, and add to the plate at 50 μL / well; dilute ODN2216 (manufacturer: Invivogen; catalog number: tlrl-2216-1) to 2 μM, add to the plate at 50 μL / well, and culture at 37 ° C for 18 hours. Collect the supernatant and detect the IFN-α concentration in the supernatant using an IFN-α ELISA kit (purchased from Dayou; catalog number: 1110013). The results are shown in Table 11 and Figure 1 . The humanized antibody can significantly inhibit the secretion of IFN-α induced by TLR9.

Table 11. Antibody inhibition of IFN-α secretion activity

Antibody EC50(μg/mL) 14D10-VH1VL1 0.0005546

Example 8. Antibodies inhibit TLR7-induced IFN-α secretion

Resuscitate human PBMC cells, resuspend in complete medium (RPMI 1640 + 10% FBS + 1 × NEAA + 1 × sodium pyruvate + 1 × GlutaMax), adjust the cell density to 5 × 10 ⁶ / mL, and inoculate into 96-well cell culture plates at 100 μL / well; dilute the antibody to 40 μg / mL, dilute 20 times to 2 concentration points, dilute 10 times to 1 concentration point, and then dilute 3 times to 4 concentration points, and add to the plate at 50 μL / well; add 50 μL of 4 μg / ml concentration of R837 (manufacturer: Invivogen; item number: tlrl-imqs), and culture at 37 ° C for 18 hours. Collect the supernatant and use IFN-α ELISA kit (purchased from Dayou; item number: 1110013) to detect the concentration of IFN-α in the supernatant. The results are shown in Table 12 and Figure 2. The humanized antibody has a significant inhibitory effect on the secretion of IFN-α induced by TLR7.

Table 12. Antibody inhibition of IFN-α secretion activity

Antibody EC50(μg/mL) 14D10-VH1VL1 0.0005933

Example 9. Antibody ADCC activity detection

PBMC was used to detect the ADCC activity of humanized antibodies. PBMC cells were revived and inoculated into 96-well cell culture plates at 2×10 ⁵ /well; HEK-hBDCA2 cells were collected, the cell density was adjusted, and 1×10 ⁴ /well was added to the plate; the antibody was diluted to a concentration of 2 μg/mL in the first well, and then added to the plate after 7-fold gradient dilution, and incubated at 37°C for 6 h; the LDH kit (purchased from Roche; manufacturer: 04744934001) was used for color development, and the plate was placed on an ELISA reader for reading.

The results are shown in Table 13 and Figure 3. 14D10-VH1VL1 mediated the killing of target cells through ADCC.

Table 13. Antibody ADCC activity

Antibody EC50(μg/mL) Maximum killing rate (%) 24F4 0.004632 30.15 14D10-VH1VL1 0.002841 38.55

Claims (11)

1. An antibody or antigen-binding fragment thereof that specifically binds human BDCA2, comprising a heavy chain variable region and a light chain variable region; wherein:

the heavy chain variable region comprises SEQ ID NO:33, HCDR1, HCDR2, HCDR3 in the sequence shown in seq id no; the light chain variable region comprises SEQ ID NO:34, LCDR1, LCDR2, LCDR3 in the sequence shown in seq id no; or (b)

The heavy chain variable region comprises SEQ ID NO:37, HCDR1, HCDR2, HCDR3 in the sequence shown in seq id no; the light chain variable region comprises SEQ ID NO:38 LCDR1, LCDR2, LCDR3 in the sequence shown in seq id no; or (b)

The heavy chain variable region comprises SEQ ID NO:31, HCDR1, HCDR2, HCDR3 in the sequence shown in seq id no; the light chain variable region comprises SEQ ID NO:32, LCDR1, LCDR2, LCDR3 in the sequence shown in seq id no; or (b)

The heavy chain variable region comprises SEQ ID NO:35, HCDR1, HCDR2, HCDR3 in the sequence shown in seq id no; the light chain variable region comprises SEQ ID NO:36 LCDR1, LCDR2, LCDR3 in the sequence shown in seq id no; or (b)

The heavy chain variable region comprises SEQ ID NO:39, HCDR1, HCDR2, HCDR3 in the sequence shown in seq id no; the light chain variable region comprises SEQ ID NO:40, LCDR1, LCDR2, LCDR3 in the sequence shown in seq id no;

Preferably, the amino acid sequences of said HCDR and said LCDR are determined according to the numbering system of Kabat, chothia, abM or IMTG.

2. An antibody or antigen-binding fragment thereof that specifically binds human BDCA2 according to claim 1, comprising a heavy chain variable region and a light chain variable region; wherein:

1) The heavy chain variable region comprises SEQ ID NO:7, HCDR1, SEQ ID NO:8, HCDR2, SEQ ID NO: HCDR3 as shown in 9; and

The light chain variable region comprises SEQ ID NO:10, LCDR1, SEQ ID NO:11, LCDR2, SEQ ID NO: LCDR3 as shown in 12; or (b)

2) The heavy chain variable region comprises SEQ ID NO:19, HCDR1, SEQ ID NO:20, HCDR2, SEQ ID NO: HCDR3 as shown in 21; and

The light chain variable region comprises SEQ ID NO:22, LCDR1, SEQ ID NO:23, LCDR2, SEQ ID NO: LCDR3 as shown at 24; or (b)

3) The heavy chain variable region comprises SEQ ID NO:1, HCDR1, SEQ ID NO:2, HCDR2, SEQ ID NO: HCDR3 as shown in 3; and

The light chain variable region comprises SEQ ID NO:4, LCDR1, SEQ ID NO:5, LCDR2, SEQ ID NO: LCDR3 as shown in 6; or (b)

4) The heavy chain variable region comprises SEQ ID NO:13, HCDR1, SEQ ID NO:14, HCDR2, SEQ ID NO: HCDR3 as shown at 15; and

The light chain variable region comprises SEQ ID NO:16, LCDR1, SEQ ID NO:17, LCDR2, SEQ ID NO: LCDR3 as shown at 18; or (b)

5) The heavy chain variable region comprises SEQ ID NO:25, HCDR1, SEQ ID NO:26, HCDR2, SEQ ID NO: HCDR3 as shown in 27; and

The light chain variable region comprises SEQ ID NO:28, LCDR1, SEQ ID NO:29, LCDR2, SEQ ID NO:30, LCDR3.

3. An antibody or antigen-binding fragment thereof that specifically binds human BDCA2 according to claim 1 or 2, which is a rabbit-derived antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.

4. An antibody or antigen-binding fragment thereof that specifically binds human BDCA2 according to any one of claims 1 to 3, wherein the heavy chain variable region and the light chain variable region are selected from any one of the following groups:

1) The heavy chain variable region comprises SEQ ID NO:33 or comprises an amino acid sequence as set forth in SEQ ID NO:33, and an amino acid sequence having at least 85% sequence identity, and

The light chain variable region comprises SEQ ID NO:34 or comprises an amino acid sequence as set forth in SEQ ID NO:34 having an amino acid sequence with at least 85% sequence identity;

2) The heavy chain variable region comprises SEQ ID NO:37 or comprises an amino acid sequence as set forth in SEQ ID NO:37, and having at least 85% sequence identity, and

The light chain variable region comprises SEQ ID NO:38 or comprises an amino acid sequence as set forth in SEQ ID NO:38 having an amino acid sequence having at least 85% sequence identity;

3) The heavy chain variable region comprises SEQ ID NO:31 or comprises an amino acid sequence as set forth in SEQ ID NO:31, and having at least 85% sequence identity, and

The light chain variable region comprises SEQ ID NO:32 or comprises an amino acid sequence as set forth in SEQ ID NO:32 having at least 85% sequence identity;

4) The heavy chain variable region comprises SEQ ID NO:35 or comprises an amino acid sequence as set forth in SEQ ID NO:35, and an amino acid sequence having at least 85% sequence identity, and

The light chain variable region comprises SEQ ID NO:36 or comprises an amino acid sequence as set forth in SEQ ID NO:36 having an amino acid sequence with at least 85% sequence identity;

5) The heavy chain variable region comprises SEQ ID NO:39 or comprises an amino acid sequence as set forth in SEQ ID NO:39, and an amino acid sequence having at least 85% sequence identity, and

The light chain variable region comprises SEQ ID NO:40 or comprises an amino acid sequence as set forth in SEQ ID NO:40 has an amino acid sequence having at least 85% sequence identity.

5. An antibody or antigen-binding fragment thereof that specifically binds human BDCA2 according to any one of claims 1 to 4, wherein the antigen-binding fragment is selected from any one of the following: fab, scFv, fv, fab ', F (ab') ₂, single domain antibodies, scFab, linear antibodies and multispecific antibodies.

6. An antibody or antigen-binding fragment thereof that specifically binds human BDCA2 according to any one of claims 1 to 5, which is a humanized antibody; preferably, the method comprises the steps of,

1) The heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 43 to 47 or an amino acid sequence comprising at least 85% sequence identity thereto, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 to 49 or an amino acid sequence comprising at least 85% sequence identity thereto; or (b)

2) The heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 50 to 52 or an amino acid sequence comprising at least 85% sequence identity thereto, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 54 or an amino acid sequence comprising at least 85% sequence identity thereto.

7. An antibody or antigen-binding fragment thereof that specifically binds human BDCA2 according to any one of claims 1 to 6, further comprising a heavy chain constant region and a light chain constant region; preferably, the method comprises the steps of,

The heavy chain constant region is selected from a constant region of human IgG1, human IgG2, human IgG3 and human IgG4 or a mutant thereof, wherein the amino acid of the mutant at position 252 is Y, the amino acid at position 254 is T and the amino acid at position 256 is E, or the amino acid at position 428 is an amino acid S mutation at positions L and 434, and the amino acid positions are determined by EU numbering; the light chain constant region is selected from the group consisting of a lambda light chain constant region and a kappa light chain constant region; most preferably, the heavy chain constant region comprises SEQ ID NO: 41. 60 or 61, and the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 42.

8. An antibody or antigen-binding fragment thereof that specifically binds human BDCA2 according to any one of claims 1 to 7, comprising a heavy chain and a light chain selected from any one of the following groups:

1) The heavy chain comprises SEQ ID NO:55 or an amino acid sequence comprising at least 85% sequence identity thereto, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:56 or an amino acid sequence comprising at least 85% sequence identity thereto;

2) The heavy chain comprises SEQ ID NO:57 or an amino acid sequence comprising at least 85% sequence identity thereto, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:58 or an amino acid sequence comprising at least 85% sequence identity thereto;

3) The heavy chain comprises SEQ ID NO:62 or an amino acid sequence comprising at least 85% sequence identity thereto, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:63 or an amino acid sequence comprising at least 85% sequence identity thereto;

4) The heavy chain comprises SEQ ID NO:64 or an amino acid sequence comprising at least 85% sequence identity thereto, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:65 or an amino acid sequence comprising at least 85% sequence identity thereto;

5) The heavy chain comprises SEQ ID NO:66 or an amino acid sequence comprising at least 85% sequence identity thereto, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:67 or an amino acid sequence comprising at least 85% sequence identity thereto; and

6) The heavy chain comprises SEQ ID NO:68 or an amino acid sequence comprising at least 85% sequence identity thereto, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:69 or an amino acid sequence comprising at least 85% sequence identity thereto.

9. A pharmaceutical composition comprising:

1) A therapeutically effective amount of an antibody or antigen-binding fragment thereof of any one of claims 1 to 8 that specifically binds human BDCA2, and

2) One or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

10. A biological material, which is:

1) A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of any one of claims 1 to 8 that specifically binds human BDCA 2;

2) A vector comprising the nucleic acid molecule of 1) above;

3) A cell comprising the nucleic acid molecule of 1) above or the vector of 2) above.

11. A method of treating and/or preventing a disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof that specifically binds human BDCA2 of any one of claims 1 to 8, or a therapeutically effective amount of the pharmaceutical composition of claim 9; preferably, the method comprises the steps of,

The disease is an inflammatory disease or an autoimmune disease; more preferably, the inflammatory or autoimmune disease is selected from: systemic lupus erythematosus, discoid lupus, lupus nephritis, epidermoid lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis, progressive systemic sclerosis (scleroderma), psoriasis, psoriatic arthritis, type I diabetes, fibrosis (e.g., skin fibrosis, pulmonary fibrosis), pemphigus vulgaris, graves 'disease, scleroderma (localized scleroderma), xerosis, hashimoto's disease, dermatomyositis, polymyositis, asthma, behcet's disease, crohn's disease, autoimmune glomerulonephritis, membranous glomerulopathy, juvenile rheumatoid arthritis, mixed connective tissue disease, multiple sclerosis, nephrotic syndrome, panama, pemphigus, erythroid pemphigus, leaf-type pemphigus, pemphigus vulgaris, polymyositis rheumatica, raynaud's phenomenon/syndrome, sjogren's syndrome, and ulcerative colitis.