WO2024010118A1 - Anticorps anti-bdca-2 et son utilisation - Google Patents

Anticorps anti-bdca-2 et son utilisation Download PDF

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WO2024010118A1
WO2024010118A1 PCT/KR2022/009887 KR2022009887W WO2024010118A1 WO 2024010118 A1 WO2024010118 A1 WO 2024010118A1 KR 2022009887 W KR2022009887 W KR 2022009887W WO 2024010118 A1 WO2024010118 A1 WO 2024010118A1
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bdca
seq
cells
antibody
present
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PCT/KR2022/009887
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Korean (ko)
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권병세
유미진
김나래
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주식회사 유틸렉스
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present invention relates to a novel antibody that specifically binds to BDCA-2 (Blood dendritic cell antigen 2), a chimeric antigen receptor based thereon, transformed T cells expressing the same, chimeric antibodies, and humanized antibodies.
  • BDCA-2 Bood dendritic cell antigen 2
  • a chimeric antigen receptor based thereon transformed T cells expressing the same, chimeric antibodies, and humanized antibodies.
  • BDCA-2 (Blood Dendritic Cell Antigen 2) is a single-transmembrane type membrane protein. BDCA-2 is expressed exclusively in human plasmacytoid dendritic cells (pDCs), and is known to be responsible for regulating the function of pDCs by transmitting signals within the cells of pDCs.
  • pDCs human plasmacytoid dendritic cells
  • BDCA-2 functions suppressively against activated immune responses. Although the details of the mechanism are still largely unclear, it has been reported that pDCs in an activated state can be inhibited by cross-linking BDCA-2 molecules using an antibody against BDCA-2, as described later.
  • pDCs cells that specifically express BDCA-2, show abnormal activation in autoimmune diseases such as systemic lupus erythematosus, systemic sclerosis, polymyositis and dermatomyositis, psoriasis, Sjögren's syndrome, rheumatoid arthritis, Grave's disease, and Hashimoto's disease.
  • autoimmune diseases such as systemic lupus erythematosus, systemic sclerosis, polymyositis and dermatomyositis, psoriasis, Sjögren's syndrome, rheumatoid arthritis, Grave's disease, and Hashimoto's disease.
  • IFN- ⁇ interferon- ⁇
  • systemic lupus erythematosus a type of autoimmune disease
  • genetic modification is performed on systemic lupus erythematosus condition model mice so as not to generate pDCs, the onset of systemic lupus erythematosus is suppressed, proving the direct involvement of pDCs in the systemic lupus erythematosus condition.
  • AC144 a mouse monoclonal antibody
  • AC144 can inhibit pDCs in an activated state by cross-linking BDCA-2 molecules.
  • TLR 9 Toll-like receptor 9
  • BDCA-2-specific chimeric antigen receptor (CAR), CAR-T cells expressing it, chimeric antibodies, and humanized antibodies were produced, and the activity of each was confirmed to complete the present invention.
  • the purpose of the present invention is to provide an antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 (Blood dendritic cell antigen 2).
  • Another object of the present invention is to provide a nucleic acid encoding an antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 according to the present invention.
  • Another object of the present invention is to provide a recombinant expression vector containing the nucleic acid according to the present invention.
  • Another object of the present invention is to provide host cells transfected with the recombinant expression vector according to the present invention.
  • Another object of the present invention is to provide a method for producing an antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 according to the present invention.
  • Another object of the present invention is to provide a BDCA-2 specific chimeric antigen receptor (CAR).
  • CAR BDCA-2 specific chimeric antigen receptor
  • Another object of the present invention is to provide a nucleic acid encoding the BDCA-2 specific CAR according to the present invention.
  • Another object of the present invention is to provide a recombinant expression vector containing a nucleic acid encoding the BDCA-2 specific CAR according to the present invention.
  • Another object of the present invention is to provide host cells transfected with a recombinant expression vector containing a nucleic acid encoding the BDCA-2 specific CAR according to the present invention.
  • Another object of the present invention is to provide transformed T cells expressing the BDCA-2 specific CAR according to the present invention.
  • Another object of the present invention is to provide a chimeric antibody against BDCA-2.
  • Another object of the present invention is to provide a humanized antibody against BDCA-2.
  • Another object of the present invention is a chimeric antibody against BDCA-2 according to the present invention; or providing a nucleic acid encoding a humanized antibody against BDCA-2.
  • Another object of the present invention is a chimeric antibody against BDCA-2 according to the present invention.
  • a recombinant expression vector containing a nucleic acid encoding a humanized antibody against BDCA-2 is provided.
  • Another object of the present invention is a chimeric antibody against BDCA-2 according to the present invention.
  • host cells transfected with a recombinant expression vector containing a nucleic acid encoding a humanized antibody against BDCA-2 are provided.
  • Another object of the present invention is to provide a method for producing a chimeric antibody against BDCA-2 or a humanized antibody against BDCA-2 according to the present invention.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating autoimmune diseases.
  • Another object of the present invention is to provide a method for treating autoimmune diseases.
  • the present invention provides a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 1; and an antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 (Blood dendritic cell antigen 2), including a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 2.
  • the present invention provides a nucleic acid encoding an antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 according to the present invention.
  • the present invention provides a recombinant expression vector containing a nucleic acid encoding an antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 according to the present invention.
  • the present invention provides host cells transfected with a recombinant expression vector containing a nucleic acid encoding an antibody that specifically binds to BDCA-2 or an antigen-binding fragment thereof according to the present invention.
  • the present invention includes the steps of culturing the host cell according to the present invention to produce an antibody; and isolating and purifying the produced antibody.
  • a method for producing an antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 is provided.
  • the present invention provides a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 1; and a single-chain variable fragment (scFv) containing a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 2. It provides a BDCA-2 specific chimeric antigen receptor (CAR).
  • a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 1; and a single-chain variable fragment (scFv) containing a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 2. It provides a BDCA-2 specific chimeric antigen receptor (CAR).
  • CAR BDCA-2 specific chimeric antigen receptor
  • the present invention provides a nucleic acid encoding the BDCA-2 specific CAR according to the present invention.
  • the present invention provides a recombinant expression vector containing a nucleic acid encoding the BDCA-2 specific CAR according to the present invention.
  • the present invention provides a host cell transfected with a recombinant expression vector containing a nucleic acid encoding the BDCA-2 specific CAR according to the present invention.
  • the present invention provides transformed T cells expressing the BDCA-2 specific CAR according to the present invention.
  • the present invention provides a light chain comprising the amino acid sequence represented by SEQ ID NO: 10; And a chimeric antibody against BDCA-2 comprising a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 11 is provided.
  • the present invention provides a light chain comprising the amino acid sequence shown in SEQ ID NO: 14; and a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 15.
  • the present invention provides a chimeric antibody against BDCA-2 according to the present invention.
  • nucleic acids encoding humanized antibodies against BDCA-2 are provided.
  • the present invention provides a chimeric antibody against BDCA-2 according to the present invention.
  • a recombinant expression vector containing a nucleic acid encoding a humanized antibody against BDCA-2 is provided.
  • the present invention provides a chimeric antibody against BDCA-2 according to the present invention.
  • host cells transfected with a recombinant expression vector containing a nucleic acid encoding a humanized antibody against BDCA-2 are provided.
  • the present invention provides a chimeric antibody against BDCA-2 according to the present invention; or producing an antibody by culturing a host cell transfected with a recombinant expression vector containing a nucleic acid encoding a humanized antibody against BDCA-2; It provides a method for producing a chimeric antibody against BDCA-2 or a humanized antibody against BDCA-2, comprising the step of isolating and purifying the produced antibody.
  • the present invention provides an antibody that specifically binds to BDCA-2 according to the present invention; BDCA-2 specific CAR; Transduced T cells expressing BDCA-2 specific CAR; Chimeric antibody against BDCA-2; It provides a pharmaceutical composition for preventing or treating autoimmune diseases, comprising at least one selected from the group consisting of humanized antibodies against BDCA-2.
  • the present invention provides an antibody that specifically binds to BDCA-2 according to the present invention; BDCA-2 specific CAR; Transduced T cells expressing BDCA-2 specific CAR; Chimeric antibody against BDCA-2; and administering to an individual at least one selected from the group consisting of humanized antibodies against BDCA-2.
  • the novel antibody that specifically binds to BDCA-2 according to the present invention recognizes BDCA-2 to a degree similar to known antibodies and binds to BDCA-2 from the surface of human plasmacytoid dendritic cells (pDC). It was confirmed that it was effective in suppressing the expression of type I interferon by mediating the internalization of 2, and that the effect was superior to that of known antibodies.
  • CAR produced based on an antibody that specifically binds to BDCA-2 and T cells expressing the CAR showed excellent pDC removal ability
  • CAR produced based on an antibody that specifically binds to BDCA-2 It has been confirmed that chimeric antibodies and humanized antibodies can bind to both cells expressing human BDCA-2 and cells expressing monkey BDCA-2, for the prevention or treatment of autoimmune diseases such as systemic lupus erythematosus (Lupus). It can be useful.
  • Figure 1 shows the results of confirming and comparing the BDCA-2 recognition effect of a novel antibody (201A) that specifically binds to BDCA-2 according to the present invention and AC144, a known antibody.
  • Figure 2 shows the results of treating peripheral blood mononuclear cells (PBMC) with TLR7 and TLR9 activators and confirming their secretion of IFN- ⁇ .
  • PBMC peripheral blood mononuclear cells
  • Figure 3 shows the secretion of IFN- ⁇ after treating PBMC with TLR7 and TLR9 activators, a novel antibody (201A) that specifically binds to BDCA-2 according to the present invention, and AC144, a known antibody. This is the confirmed result.
  • FIGS 4 and 5 show peripheral blood mononuclear cells (PBMC) treated with TLR7 and TLR9 activators and a novel antibody specifically binding to BDCA-2 according to the present invention; This is the result of confirming cells expressing BDCA-2 on the cell surface.
  • PBMC peripheral blood mononuclear cells
  • Figure 6 is a schematic diagram showing the structure of the CAR construct for the production of CAR-T targeting BDCA-2.
  • Figure 7 shows after transducing CAR lentivirus targeting BDCA-2 into cells, cells confirming CAR expression (A) and cells confirming BDCA-2 antigen expression (B) were mixed and co-cultured at various ratios. , This is the result of confirming the activation of CAR expressing cells (C).
  • Figure 8 shows the production of CAR-T cells targeting BDCA-2 and confirmation of their CAR expression (A). After co-culturing CAR-T cells and BDCA-2 antigen-expressing cells for 4 hours, the IFN- in the culture medium This is the result of measuring the concentration of ⁇ (Interferon-gamma) (B) and the concentration of IFN- ⁇ after 24 hours of co-culture (C).
  • Figure 9 shows the results of pDC enrichment from PBMC (A) and CAR-T cells expressing CAR according to the present invention (B) to confirm the pDC removal ability of CAR-T cells targeting BDCA-2.
  • the PBMC and CAR-T cells were derived from the same volunteer.
  • Figure 10 shows the % (A) occupied by pDC and the CAR- This is the result of checking the %(B) occupied by T. At this time, staining was performed with CD123 and BDCA-2 antibodies to confirm whether changes occurred in the pDC population due to CAR-T cells.
  • Figure 11 shows the secretion of IFN- ⁇ after co-culturing concentrated pDC and CAR-T according to the present invention for 4 hours to confirm whether CAR-T cells targeting BDCA-2 are activated by pDC. It is a result.
  • Figure 12 shows the results of confirming the sizes of the chimeric antibody against BDCA-2 and the humanized antibody against BDCA-2 according to the present invention.
  • Figure 13 shows the results of confirming the GFP fluorescence contained in the expression vector in the established cell line expressing the cynomolgus monkey BDCA-2 gene.
  • Figure 14 shows the results of confirming the expression of monkey BDCA-2 after staining with a known antibody in an established cell line expressing the monkey BDCA-2 gene.
  • Figure 15 shows the results of treating cells expressing human BDCA-2 with the chimeric and humanized antibodies against BDCA-2 according to the present invention at various concentrations and confirming whether they bind to human BDCA-2.
  • Figure 16 shows the results of treating cells expressing monkey BDCA-2 with the chimeric and humanized antibodies against BDCA-2 according to the present invention at various concentrations and confirming whether they bind to monkey BDCA-2.
  • the present invention provides a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 1; and an antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 (Blood dendritic cell antigen 2), including a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 2.
  • the BDCA-2 is a primate BDCA-2, and the primate is a human, a monkey, a chimpanzee, a gorilla or an orangutan, and more preferably a human or a monkey.
  • the antibody is characterized in that it consists of a light chain containing the amino acid sequence shown in SEQ ID NO: 3 and a heavy chain containing the amino acid sequence shown in SEQ ID NO: 4.
  • the term “antibody” refers to an anti-BDCA-2 antibody that specifically binds to BDCA-2.
  • the scope of the present invention includes not only complete antibody forms that specifically bind to BDCA-2, but also antigen-binding fragments of the antibody molecule.
  • a complete antibody has a structure of two full-length light chains and two full-length heavy chains, with each light chain connected to the heavy chain by a disulfide bond.
  • the term “heavy chain” refers to a full-length heavy chain comprising a variable region domain VH and three constant region domains CH1, CH2, and CH3, including an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen, and This means all fragments of this.
  • the term “light chain” as used herein refers to a full-length light chain and fragments thereof comprising a variable region domain VL and a constant region domain CL containing an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen. It all means.
  • the term “antigen-binding fragment” refers to a portion of the above-described intact antibody, and is a sequence that is at least one sequence shorter in length than the amino acid sequence of the intact antibody. In terms of functionality, it contains at least some activities or functions of an intact antibody or parent antibody, such as Fab, F(ab) 2 , Fab', F(ab') 2 , F(ab') 3 , Fd, Fv. and domain antibodies, but are not limited thereto.
  • Fab has a structure that includes the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region (CH1) of the heavy chain, and has one antigen binding site.
  • Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain.
  • F(ab') 2 is generated when the cysteine residue in the hinge region of Fab' forms a disulfide bond.
  • the Fv corresponds to the minimum antibody fragment containing only the heavy chain variable region and the light chain variable region.
  • Double-chain Fv two-chain Fv
  • single-chain Fv single-chain Fv
  • the regions can be covalently linked or linked directly at the C-terminus to form a dimer-like structure, such as double-chain Fv.
  • antibody fragments can be created using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of the complete antibody with papain, and F(ab') 2 can be obtained by digestion with pepsin) or genetic recombination technology. It can be produced using .
  • the “Fv” fragment is an antibody fragment that contains the complete antibody recognition and binding site. This region is a dimer of one heavy chain variable domain and one light chain variable domain.
  • the “Fab” fragment includes the variable and constant domains of the light chain and the variable and first constant domains of the heavy chain (CH1).
  • F(ab') 2 antibody fragments generally comprise a pair of Fab' fragments covalently linked by a cysteine in the hinge region present at the C-terminus of the Fab' fragments.
  • the antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 of the present invention may be a monoclonal antibody.
  • the term “monoclonal antibody” or “monoclonal antibody” refers to an antibody molecule of single molecular composition obtained from a population of substantially identical antibodies, and a monoclonal antibody exhibits a single binding specificity and affinity for a specific epitope.
  • Monoclonal antibodies are produced by fusing myeloma cells with spleen cells derived from immunized mammals, and can be produced by various methods known in the art.
  • the antibody according to the present invention may additionally be a functional substance selected from the group consisting of therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological modifiers, drugs, and PEG (polyethylene glycol). It may be provided in conjugation with. Additionally, it can be manufactured using various methods depending on the type of material being conjugated.
  • the therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological modifiers and drugs may be any commonly used in the art as long as they can achieve the desired effect.
  • binding refers to the affinity of the antibody or antigen-binding fragment for the antigen.
  • “specific binding” can typically be distinguished from non-specific background binding if the dissociation constant (Kd) is less than 1x10 -5 M or less than 1x10 -6 M or less than 1x10 -7 M.
  • Kd dissociation constant
  • Specific binding can be detected by methods known in the art, such as ELISA, SPR (Surface plasmon resonance), immunoprecipitation, coprecipitation, etc., and non-specific binding and specific binding can be distinguished. Include an appropriate control group for differentiation.
  • the antibodies of the present invention may exist as multimers such as dimers, trimers, tetramers, and pentamers that contain at least part of the antigen-binding ability of the monomer. These multimers also include homomultimers and heteromultimers. Antibody multimers have superior antigen-binding ability compared to monomers because they contain multiple antigen-binding sites. Antibody multimers also facilitate the production of multifunctional (bifunctional, trifunctional, tetrafunctional) antibodies.
  • multifunctionality refers to an antibody having two or more activities or functions (e.g., antigen-binding ability, enzyme activity, ligand or receptor-binding ability).
  • the antibody herein is a polypeptide with enzyme activity, such as For example, it can be combined with luciferase, acetyltransferase, galactosidase, etc.
  • Multifunctional antibodies also include antibodies in multivalent or multispecific (bispecific, trispecific, etc.) forms.
  • the present invention provides a nucleic acid encoding an antibody or antigen-binding fragment thereof that specifically binds to BDCA-2.
  • An antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 can be recombinantly produced by isolating the nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention.
  • Nucleic acids include, for example, DNA, cDNA, RNA, or recombinant or synthesized DNA or RNA.
  • the nucleic acid molecule is cDNA.
  • Nucleic acids may also be the corresponding genomic DNA or fragments thereof.
  • the nucleic acid sequence encoding the antibody or part or fragment thereof according to the present application may be different due to redundancy in the nucleic acid sequence encoding amino acids, and such sequences are also included herein.
  • the DNA encoding the antibody can be easily isolated or synthesized using conventional molecular biology techniques (for example, by using an oligonucleotide probe that can specifically bind to the antibody and the DNA encoding the heavy and light chains). This can be done by isolating the nucleic acid and inserting it into a replicable vector for further cloning (DNA amplification) or further expression. Based on this, the present invention also provides a recombinant expression vector containing a nucleic acid encoding an antibody that specifically binds to BDCA-2 or an antigen-binding fragment thereof.
  • the term "vector” refers to a means for expressing a gene of interest in a host cell, including viral vectors such as plasmid vectors, cosmid vectors, bacteriophage vectors, adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, etc. Includes.
  • Components of a vector generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more antibiotic resistance marker genes, an enhancer element, a promoter, and a transcription termination sequence. Nucleic acids encoding antibodies are operably linked, such as promoter and transcription termination sequences.
  • operably linked refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the control sequence is connected to the other nucleic acid. It regulates the transcription and/or translation of the sequence.
  • a nucleic acid expression control sequence e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites
  • a strong promoter capable of advancing transcription e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL ⁇ promoter, pR ⁇ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.
  • a ribosome binding site for initiation of translation e.g., amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.
  • promoters derived from the genome of mammalian cells e.g., metallothionein promoter, ⁇ -actin promoter, human heroglobin promoter, and human muscle creatine promoter
  • mammalian Promoters derived from viruses e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, Promoters of Moloney virus (promoters of Epstein-Barr virus (EBV) and promoters of Rouss sarcoma virus (RSV)) can be used and generally have a polyadenylation sequence as the transcription termination sequence.
  • viruses e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of H
  • the vector may be fused with other sequences to facilitate purification of the antibody expressed therefrom.
  • Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA).
  • the vector contains an antibiotic resistance gene commonly used in the art as a selection marker, for example, for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline. There is a resistance gene.
  • the present invention provides host cells transfected with the above recombinant expression vector.
  • Host cells used to produce the antibodies of the present invention may be prokaryotic, yeast, or higher eukaryotic cells, but are not limited thereto. Additionally, hosts with high efficiency of introducing DNA and high expression efficiency of introduced DNA are usually used.
  • Known eukaryotic and prokaryotic hosts such as Escherichia coli, Pseudomonas, Bacillus, Streptomyces, fungi and yeast, insect cells such as Spodoptera frugiperda (SF9), animal cells such as CHO and mouse cells, COS 1, COS 7, African green monkey cells such as BSC 1, BSC 40 and BMT 10, and tissue cultured human cells are examples of host cells that can be used. In one embodiment of the present invention, 293T cells were used as host cells. Of course, it should be understood that not all vectors and expression control sequences are equally functional in expressing the DNA sequence of the present invention. Likewise, not all hosts perform equally well for the same expression system.
  • those skilled in the art can make an appropriate selection among various vectors, expression control sequences, and hosts without excessive experimental burden and without departing from the scope of the present invention.
  • the host when choosing a vector, the host must be considered, because the vector must replicate within it.
  • the copy number of the vector, the ability to control copy number and the expression of other proteins encoded by the vector, such as antibiotic markers, should also be considered.
  • various factors must be considered. For example, the relative strength of the sequences, their controllability and their compatibility with the DNA sequences of the invention should be considered, especially in relation to possible secondary structures.
  • the single cell host must be prepared by the selected vector, the toxicity of the product encoded by the DNA sequence of the present invention, the secretion characteristics, the ability to correctly fold the protein, the culture and fermentation requirements, and the product encoded by the DNA sequence of the present invention from the host. It must be selected taking into account factors such as ease of purification. Within these parameters, one skilled in the art can select various vector/expression control sequence/host combinations capable of expressing the DNA sequence of the present invention in fermentation or large-scale animal culture.
  • transfection may mean “transformation.”
  • transformation means introducing DNA into a host so that the DNA can be replicated as an extrachromosomal factor or through completion of chromosomal integration.
  • the present invention includes the steps of culturing the host cells to produce antibodies; and isolating and purifying the produced antibody.
  • a method for producing an antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 is provided.
  • the host cells can be cultured in various media. Any commercially available medium can be used as a culture medium without limitation. All other necessary supplements known to those skilled in the art may be included in suitable concentrations. Culture conditions, such as temperature, pH, etc., are already used with host cells selected for expression and will be clear to those skilled in the art.
  • the antibody or antigen-binding fragment thereof can be recovered by, for example, centrifugation or ultrafiltration to remove impurities, and the resulting product can be purified using, for example, affinity chromatography. Additional other purification techniques may be used, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, etc.
  • the present invention provides a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 1; and a single-chain variable fragment (scFv) containing a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 2. It provides a BDCA-2 specific chimeric antigen receptor (CAR).
  • a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 1; and a single-chain variable fragment (scFv) containing a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 2. It provides a BDCA-2 specific chimeric antigen receptor (CAR).
  • CAR BDCA-2 specific chimeric antigen receptor
  • the light chain variable region and the heavy chain variable region of the single chain variable fragment (scFv) may be connected by a (GGGGS)m linker, where m is 1 to 10.
  • m may be 1 to 5, and more preferably, m of the linker may be 3 and may include the amino acid sequence represented by SEQ ID NO: 5.
  • the scFv may have a light chain variable region, a linker, and a heavy chain variable region sequentially linked, and the scFv may have a heavy chain variable region, a linker, and a light chain variable region sequentially linked.
  • the scFv may include the amino acid sequence represented by SEQ ID NO: 6 or SEQ ID NO: 7.
  • the scFv containing the amino acid sequence shown in SEQ ID NO: 6 is encoded in the base sequence shown in SEQ ID NO: 8
  • the scFv containing the amino acid sequence shown in SEQ ID NO: 7 is encoded in the base sequence shown in SEQ ID NO: 9. It can be.
  • the BDCA-2 specific CAR of the present invention may further include one or more selected from the group consisting of a signal peptide, stem domain, transmembrane domain, and intracellular signaling domain.
  • the BDCA-2 specific CAR of the present invention includes a signal peptide, a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 1; and a single chain variable fragment (scFv) including a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 2, a stem domain, a transmembrane domain, and an intracellular signaling domain may be sequentially linked.
  • a signal peptide a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 1
  • scFv single chain variable fragment
  • the signal peptide serves to send the nascent protein to the endoplasmic reticulum
  • any polypeptide known to have the same or similar function as the above can be used without limitation regardless of whether it is from a natural or synthetic source. It is not limited thereto, but may be one or more selected from the group consisting of CD8, CD28, GM-CSF, CD4, and CD137. In the present invention, a signal peptide derived from CD8 was used.
  • the term “stem domain” refers to any polypeptide that functions to link the transmembrane domain to the scFv of the present invention. If the polypeptide is known to have the same or similar function as the above, it may be from a natural or synthetic source. It can be used without limitation regardless of origin. It is not limited thereto, but may be one selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, and CD8 hinge. One embodiment of the present invention In the example, the CD8 hinge domain was used.
  • transmembrane domain refers to any polypeptide that allows the CAR according to the present invention to be expressed on the surface membrane of a cell.
  • Suitable transmembrane domains for CARs disclosed herein include (a) the ability to be expressed on the surface of cells, such as immune cells, such as, but not limited to, lymphoid cells or natural killer (NK) cells, and (b) has the ability to interact with the scFv and intracellular signaling domains according to the invention to direct the cellular response of immune cells against predefined target cells.
  • the transmembrane domain can also be used without limitation, regardless of whether it is from a natural or synthetic source, as long as it is a polypeptide known to have the same or similar function as the above.
  • Fc ⁇ R Fc ⁇ R
  • ICOS CD278
  • 4-1BB CD137
  • OX40 CD134
  • CD27, CD28, IL-2R ⁇ , CD40, DAP10 MHC class I molecule, TNF receptor protein, Immunoglobulin-like protein , cytokine receptor, integrin, SLAM protein, activated NK cell receptor, BTLA, Toll ligand receptor, CD2, CD7, CD30, CDS, ICAM-1, B7-H3, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 ⁇ , CD8 ⁇ , ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a , LFA-1, ITGAM, CD103,
  • intracellular signaling domain refers to the portion of a protein that transmits effector signal function signals and instructs cells to perform specialized functions. This means that after the scFv according to the present invention binds to the target, It is responsible for intracellular signaling and causes T cell activation.Intracellular signaling domain.
  • any polypeptide known to have the same or similar function as the above can be used without limitation, regardless of whether it is from a natural or synthetic source.
  • the intracellular signaling domains include CD3 ⁇ , Fc ⁇ R, ICOS (CD278), 4-1BB (CD137), OX40 (CD134), CD27, CD28, IL-2R ⁇ , IL-15R- ⁇ , MyD88, DAP10, DAP12, MHC class I molecule, TNF receptor protein, Immunoglobulin-like protein, cytokine receptor, integrin, SLAM protein, activated NK cell receptor, BTLA, Toll ligand receptor, CD2, CD7, CD30, CD40, CDS, ICAM- 1, B7-H3, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6,
  • the BDCA-2 specific CAR of the present invention may further include a tag peptide for confirming or purifying the expression of scFv binding to BDCA-2 according to the present invention.
  • a tag peptide for confirming or purifying the expression of scFv binding to BDCA-2 according to the present invention.
  • Any known tag peptide can be used without limitation, and in one embodiment of the present invention, Flag peptide (DYKDDDDK) (SEQ ID NO: 23) was used.
  • the present invention provides a nucleic acid encoding the BDCA-2 specific CAR according to the present invention.
  • the BDCA-2-specific CAR can be recombinantly produced by isolating the nucleic acid encoding the BDCA-2-specific CAR of the present invention.
  • the present invention provides a recombinant expression vector containing a nucleic acid encoding the BDCA-2 specific CAR according to the present invention.
  • the present invention provides host cells transfected with the above recombinant expression vector.
  • Redundant information regarding nucleic acids, recombinant expression vectors, and host cells is omitted to avoid excessive complexity of the specification.
  • the present invention provides transformed T cells expressing the BDCA-2 specific CAR according to the present invention.
  • the T cells may be alpha beta T cells, gamma delta T cells, or NKT cells.
  • the T cells may be allogeneic T cells, autologous T cells, engineered autologous T cells (eACT), or tumor-infiltrating lymphocytes (TIL).
  • the present invention uses a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 1 and a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 2 of the antibody that specifically binds to BDCA-2 according to the present invention. Therefore, it has a technical feature in that it can easily produce chimeric antibodies or humanized antibodies for treatment and research purposes of autoimmune diseases.
  • the present invention provides a light chain comprising the amino acid sequence represented by SEQ ID NO: 10; And a chimeric antibody against BDCA-2 comprising a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 11 is provided.
  • the light chain containing the amino acid sequence shown in SEQ ID NO: 10 is encoded by the base sequence shown in SEQ ID NO: 12, and the heavy chain containing the amino acid sequence shown in SEQ ID NO: 11 is encoded by the base sequence shown in SEQ ID NO: 13.
  • the “chimeric antibody” refers to one in which at least part of the variable region, i.e., the antigen-binding site, and the constant region of the antibody (including CL1 for light chains and CH1, CH2, and CH3 regions for heavy chains) are derived from different species. it means.
  • the variable region may be of mouse origin, and the constant region may be of human origin.
  • it also refers to a class switched antibody, for example, an antibody converted from an IgG type to an IgE type.
  • the present invention provides a light chain comprising the amino acid sequence shown in SEQ ID NO: 14; and a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 15.
  • the light chain containing the amino acid sequence shown in SEQ ID NO: 14 is encoded by the base sequence shown in SEQ ID NO: 16, and the heavy chain containing the amino acid sequence shown in SEQ ID NO: 15 is encoded by the base sequence shown in SEQ ID NO: 17.
  • the “humanized antibody” means that the antibody framework is a human antibody and part of the CDR region is modified to include only the part essential for specific binding to the antigen among the CDRs of the species from which the antibody molecule was originally derived.
  • the remaining CDR regions and light and heavy chain frameworks, excluding those essential for specific binding to antigens, are replaced with human antibodies.
  • the present invention also provides a chimeric antibody against BDCA-2 according to the present invention.
  • a nucleic acid encoding a humanized antibody against BDCA-2 according to the present invention is provided.
  • the present invention provides a chimeric antibody against BDCA-2 according to the present invention.
  • a recombinant expression vector containing a nucleic acid encoding a humanized antibody against BDCA-2 according to the present invention is provided.
  • the present invention also provides a chimeric antibody against BDCA-2 according to the present invention.
  • host cells transfected with a recombinant expression vector containing a nucleic acid encoding a humanized antibody against BDCA-2 according to the present invention are provided.
  • the present invention provides a chimeric antibody against BDCA-2 according to the present invention; or producing an antibody by culturing a host cell transfected with a recombinant expression vector containing a nucleic acid encoding a humanized antibody against BDCA-2 according to the present invention; It provides a method for producing a chimeric antibody against BDCA-2 or a humanized antibody against BDCA-2, comprising the step of isolating and purifying the produced antibody.
  • Redundant information regarding nucleic acids, recombinant expression vectors, and host cells is omitted to avoid excessive complexity of the specification.
  • SEQ ID NO: 1 SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, and SEQ ID NO: 15
  • amino acid mutations are made based on the relative similarity of amino acid side chain substitutions, such as hydrophobicity, hydrophilicity, charge, size, etc.
  • arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan, and tyrosine can be said to be biologically equivalent in function.
  • the hydrophobic index of the amino acid may be considered.
  • Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); leucine (+3.8); phenylalanine (+2.8); Cysteine/Cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); histidine (-3.2); glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); and arginine (-4.5).
  • the hydrophobic amino acid index is very important in imparting interactive biological functions to proteins or peptides. It is a known fact that similar biological activity can be maintained only when substituted with an amino acid having a similar hydrophobic index.
  • substitution is made between amino acids showing a difference in the hydrophobicity index, preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5.
  • hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Asphaltate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); threonine (-0.4); Proline (-0.5 ⁇ 1); Alanine (-0.5); histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); leucine (-1.8); isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4).
  • substitution is made between amino acids showing a difference in hydrophilicity value, preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5.
  • Amino acid exchanges in proteins or peptides that do not overall alter the activity of the molecule are known in the art.
  • the most common exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
  • any one or more amino acid sequences selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 15 are interpreted to also include sequences showing substantial identity with the sequences described in the sequence listing.
  • SEQ ID NO: 1 SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, and sequences of the present invention.
  • at least one amino acid sequence selected from the group consisting of number 15 and any other sequence are aligned to match as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art, at least 80% or more It means a sequence showing homology, more preferably 90% or more homology.
  • any method known in the art can be used without limitation.
  • the present invention may include functional equivalents of one or more base sequences selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, and SEQ ID NO: 17.
  • the “functional equivalent” refers to SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, and SEQ ID NO: 8 as a result of deletion, substitution, or insertion of a base.
  • the “% sequence homology” for a polynucleotide is determined by comparing a comparison region with two optimally aligned sequences, where a portion of the polynucleotide sequence in the comparison region is a reference sequence (additions or deletions) for the optimal alignment of the two sequences. may contain additions or deletions (i.e. gaps) compared to those that do not contain .
  • the novel antibody specifically binding to BDCA-2 according to the present invention showed an effect of recognizing BDCA-2 to a degree similar to that of known antibodies, and was effective against human plasmacytoid dendritic cells ( It showed the effect of suppressing the expression of type I interferon by mediating the internalization of BDCA-2 from the surface of plasmacytoid dendritic cell (pDC). In particular, it was confirmed that the effect was superior to that of known antibodies.
  • CAR produced based on an antibody that specifically binds to BDCA-2 and T cells expressing the CAR showed excellent pDC removal ability.
  • chimeric antibodies and humanized antibodies produced based on the antibody specifically binding to BDCA-2 are used in cells expressing human BDCA-2 and monkeys expressing BDCA-2. It was confirmed that all cells could bind.
  • the present invention provides an antibody that specifically binds to BDCA-2 according to the present invention; BDCA-2 specific CAR according to the present invention; Transformed T cells according to the present invention; Chimeric antibody against BDCA-2 according to the present invention; Or, it provides a pharmaceutical composition for preventing or treating autoimmune diseases, comprising at least one selected from the group consisting of humanized antibodies against BDCA-2 according to the present invention.
  • the present invention provides an antibody that specifically binds to BDCA-2 according to the present invention; BDCA-2 specific CAR; Transduced T cells expressing BDCA-2 specific CAR; Chimeric antibody against BDCA-2; and administering to an individual at least one selected from the group consisting of humanized antibodies against BDCA-2.
  • the autoimmune diseases include systemic lupus erythematosus (Lupus), type 1 diabetes, rheumatoid arthritis, celiac disease-sprue, IgA deficiency, Crohn's disease, multiple sclerosis, and Sjögren's syndrome.
  • scleroderma polymyositis, chronic active hepatitis, mixed connective tissue disease, primary biliary cirrhosis, pernicious anemia, autoimmune thyroiditis, idiopathic Addison's disease, vitiligo, gluten-sensitive enteropathy, Grave's disease, myasthenia gravis, autoimmune neutropenia, Characterized by one or more types selected from the group consisting of idiopathic thrombocytopenic purpura, cirrhosis, pemphigus vulgaris, autoimmune infertility, Goodpasture syndrome, bullous pemphigoid, ulcerative colitis, psoriasis, Hashimoto's disease, and dense deposit disease, More preferably, it may be systemic lupus erythematosus (Lupus).
  • Liupus systemic lupus erythematosus
  • the subject is preferably a mammal, including a human, and may include patients in need of treatment for an autoimmune disease, including patients undergoing treatment, patients who have previously received treatment, and patients in need of treatment.
  • the pharmaceutical composition may be in the form of a capsule, tablet, granule, injection, ointment, powder, or beverage, and the pharmaceutical composition may be intended for human subjects.
  • the pharmaceutical composition is not limited to these, but can be formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, and aqueous suspensions, external preparations, suppositories, and sterile injection solutions according to conventional methods. .
  • the pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, etc. for oral administration.
  • buffers, preservatives, and analgesics can be used.
  • Topics, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives, etc. can be used.
  • the dosage form of the pharmaceutical composition according to the present invention can be prepared in various ways by mixing it with a pharmaceutically acceptable carrier as described above.
  • oral administration it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be manufactured in the form of unit dosage ampoules or multiple dosage forms. there is.
  • examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil may be used.
  • fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.
  • the route of administration of the pharmaceutical composition according to the present invention is not limited to these, but is oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, and topical. , sublingual or rectal.
  • parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • the pharmaceutical composition according to the present invention can also be administered in the form of a suppository for rectal administration.
  • the effective dosage and administration period of the pharmaceutical composition according to the present invention may vary depending on the desired therapeutic effect, taking into account the specific patient, the type of antibody contained in the composition, and the administration method, etc., and should not cause toxicity to the patient. .
  • the actual dosage for each patient should be selected in consideration of various factors such as the activity of the composition used, administration route, administration time, secretion rate, other drugs used together, gender, age, weight, overall health, underlying disease, etc.
  • the antibody of the present invention may be administered in an amount of about 1 to 100 mg/kg body weight, for example, about 10, 20, 30, 40, or 50 mg/kg body weight for the treatment or prevention of disease. , in some cases, it may be administered in an amount of up to about 100 mg/kg.
  • the administration interval of the pharmaceutical composition according to the present invention may be administered at appropriate intervals of daily, weekly, or monthly basis in consideration of the half-life of the administered antibody.
  • composition herein may be formulated in a suitable pharmaceutically acceptable dosage form, such as a hydrated form, such as an aqueous solution, or a lyophilized form, regardless of the route of administration.
  • a suitable pharmaceutically acceptable dosage form such as a hydrated form, such as an aqueous solution, or a lyophilized form, regardless of the route of administration.
  • Administering the composition to a subject can be performed in any convenient manner, including nebulization, injection, swallowing, infusion, implantation, or implantation.
  • the compositions described herein can be administered to a patient by subcutaneous, intradermal, intratumoral, intranodal, intraspinal, intramuscular, intravenous (i.v.) injection or intraperitoneally.
  • the pharmaceutical composition of the present invention can be administered to a patient by intradermal or subcutaneous injection.
  • the pharmaceutical composition when comprising transformed T cells, uses a method described herein or known in the art to expand transformed T cells of the invention to therapeutic levels.
  • Cells activated and expanded using other methods may be administered to a patient in combination (e.g., previously simultaneously or subsequently) with any relevant treatment form.
  • the humanized antibody against BDCA-2 or the pharmaceutical composition of the present invention can be used in combination with known treatment methods such as chemotherapy, radiation, anticancer agents, and immunotherapy agents.
  • Example 1 Amino acid sequence analysis of a monoclonal antibody that specifically binds to BDCA-2 (Blood dendritic cell antigen 2)
  • a mouse antibody binding to human BDCA-2 was purchased from Biolegend, and amino acid sequencing was performed using REmAb®, a monoclonal sequencing solution from Rapid Novor.
  • mouse monoclonal antibody 201A which binds to human BDCA-2 including heavy chain IgG2A and light chain ⁇ (kappa), was obtained.
  • Monoclonal antibody 201A which specifically binds to human BDCA-2 obtained in Example 1, mediates internalization of BDCA-2 from the surface of human plasmacytoid dendritic cells (pDC), producing type I interferon. It was confirmed whether the expression of could be suppressed.
  • PBMC peripheral blood mononuclear cells
  • pDCs present in the PBMC were treated with anti-CD123 (6H6, Biolegend) and anti-BDCA-2 (anti-BDCA-2). It was confirmed by double staining with BDCA-2 (AC144, Miltenyi) antibody.
  • TLR7 Toll-like receptor numbers 7 (TLR7) and 9 (TLR9) using an IFN- ⁇ ELISA (IFN- ⁇ ELISA). It was confirmed using Thermo scientific). TLR7 was activated by treatment with R848 (Invivogen) at a concentration of 2.5-10 ⁇ M, and TLR9 was activated by treatment with ODN2216 (Invivogen) at a concentration of 0.5-2 ⁇ M. As a result, as shown in Figure 2, it was confirmed that the PBMCs of the two volunteers secreted high levels of IFN- ⁇ in all treatment concentration conditions in R848 and ODN2216.
  • R848 Invivogen
  • ODN2216 Invivogen
  • PBMCs were stimulated by treatment with 5 ⁇ M of R848 or 1 ⁇ M of ODN2216, and at the same time, they were treated with the 201A antibody according to the present invention and reacted for 16 hours. From this, it was confirmed whether the 201A antibody according to the present invention can inhibit the secretion of IFN- ⁇ by TLR7 or TLR9 activity.
  • the AC144 antibody a well-known clone that can mediate the internalization of BDCA-2 and thereby inhibit the secretion of IFN- ⁇ by TLR7 or TLR9 activity, was used as a positive control.
  • the AC144 antibody inhibited the secretion of IFN- ⁇ induced by R848 or ODN2216 in a dose-dependent manner.
  • the experimental group treated with the 201A antibody of the present invention also showed an effect of inhibiting the secretion of IFN- ⁇ induced by R848 or ODN2216 in a concentration-dependent manner.
  • the inhibitory ability was superior to that of AC144 at the same antibody concentration.
  • the CAR construct shown in Figure 6 was designed to produce CAR-T targeting cells expressing BDCA-2. Fusion with CD8 signal sequence (signal sequence, signal seg), hinge, and transmembrane domain (TM) to express scFv (single chain variable fragment) that can bind to BDCA-2 on the surface of T cells.
  • CD8 signal sequence signal sequence, signal seg
  • TM transmembrane domain
  • a structure was designed that fused 41BB and CD3Z as an intracellular signaling domain for T cells to proliferate after binding to BDCA-2.
  • the N-terminus of scFv was tagged with Flag peptide (DYKDDDDK) (SEQ ID NO: 23).
  • the sequences of these genes are shown in Table 1 below, and each gene was synthesized and inserted into a lenti vector (pELPS3) using a known method to produce lentivirus.
  • the scFv that can bind to BDCA-2 is GGGGSGGGGSGGGGS (SEQ ID NO: 5), in which GGGGS (SEQ ID NO: 24) is repeated three times in the variable region of the light chain and heavy chain of the antibody. They were connected with a linker consisting of and were named 201HL (SEQ ID NO: 6) and 201LH (SEQ ID NO: 7) according to the arrangement order of the light chain and heavy chain.
  • Example 4 Production of CAR lentivirus targeting BDCA-2 and confirmation of CAR activity using virus
  • the lentivector produced in Example 3 was transfected into Lenti- The lentivirus produced through this was concentrated, the titer was measured, and then stored at -80°C until use.
  • lentivirus was transduced into NFAT reporter (luc)-Jurkat cells (purchased from BPS Bioscience), and 3 days later, the cells were incubated with anti-Flag-APC (L5, After staining with Biolegend), the expression of CAR was confirmed using a FACS device. At this time, cells not transduced with lentivirus (UTD) were used as a negative control (see A in Figure 7, effector cells).
  • NFAT reporter (luc)-Jurkat cells expressing the identified CAR increases when the cells are activated by antigen, luciferase substrate was added and luminescence was quantified. . Therefore, each NFAT reporter (luc)-Jurkat cell (effector cell, 1 ⁇ 10 4 cells) in which CAR expression was confirmed in A in Figure 7 was divided into 293 cells (293/BDCA-2) expressing BDCA-2 antigen ( After culturing with target cells (see B in Figure 7) at various ratios for 4 hours, the degree of activation of the effector cells was confirmed.
  • the BDCA-2 specific CAR according to the present invention can activate T cells by reacting specifically to the BDCA-2 antigen.
  • the target cells were prepared as follows.
  • the BDCA-2 (CLEC4C) gene expression vector (Sino Biological) was transformed into HEK 293 cells using lipofectamine 2000, and then subcultured in medium supplemented with 400 ⁇ g/mL of G418. Through this, cells expressing BDCA-2 were selected and target cells were established. To confirm whether the selected target cells expressed BDCA-2, the cells were stained using anti-BDCA-2-APC (Miltenyi, AC144) and then confirmed by FACS (see B in Figure 7).
  • CAR-T was produced using the lentivirus whose CAR function was confirmed in Example 4 above.
  • blood was provided from a healthy volunteer and PBMCs were isolated according to a known procedure using the Ficoll density gradient method. Isolated PBMCs were cultured in medium containing TransAct (Miltenyi) for 2 days to activate T cells, and after adding lentivirus, transduction was performed according to a known procedure using a spinoculation method. Afterwards, culture medium containing IL-2 (Interleukin-2) was added every 2-3 days to proliferate CAR-T cells.
  • TransAct MicroAct
  • IL-2 Interleukin-2
  • Example 5 The activity of CAR-T cells produced through Example 5 was confirmed. 293 cells (293/BDCA-2, 2 ⁇ 10 4 cells/well) expressing BDCA-2 antigen were mixed with CAR-T cells at various ratios, and the culture medium was collected after 4 and 24 hours of co-culture. The concentration of Interferon-gamma (IFN- ⁇ ) secreted by T cells in the culture medium when activated was measured using an ELISA kit (Biolegend).
  • IFN- ⁇ Interferon-gamma
  • IFN- ⁇ could be measured from 4 hours, and increased significantly at 24 hours.
  • C in Figure 8 even when the number of CAR-T cells per target cell was added at a ratio of 0.0625, IFN- ⁇ was detected during co-culture for 24 hours.
  • the experimental group added with UTD cells that do not express CAR IFN- ⁇ was not detected under any conditions.
  • Can CAR-T cells targeting BDCA-2 according to the present invention (201 HL CAR-T cells and 201 LH CAR-T cells) recognize BDCA-2 expressed by pDCs and eliminate the pDC population? Confirmed.
  • PBMCs were isolated from the blood of volunteers who produced CAR-T cells, and pDC populations were concentrated therefrom and used as target cells. At this time, the pDC population was enriched using the plasmacytoid dendritic cell isolation kit II (Miltenyi).
  • the enriched pDC population was stained with anti-CD123-BV421 (6H6, Biolegend) and anti-BDCA-2 antibody (AC144, Miltenyi), and the results confirmed by FACS are shown in A in Figure 9.
  • the enriched pDC population (A in Figure 9, 2 ⁇ 10 5 cells/100 ⁇ L, target cells) was combined with CAR-T cells according to the present invention (B in Figure 9, 2 ⁇ 10 5 cells/100 ⁇ L, effector cells). After mixing, it was reacted for 4 hours. Afterwards, the percentage occupied by the remaining pDC and CAR-T was confirmed and shown in A and B of Figure 10.
  • the pDC population was stained with CD123 (6H6, Biolegend) and BDCA-2 antibody (AC144, Miltenyi) to confirm whether there were changes in the pDC population due to CAR-T cells.
  • the activation of CAR-T cells induced by pDC was confirmed by measuring the secretion of IFN- ⁇ using ELISA, and this is shown in Figure 11.
  • CD123+BDCA-2+ pDC cells by these CAR-T cells was closely related to the increase in IFN- ⁇ secreted in the culture medium.
  • CAR-T cells present in the mixed population were stained with anti-Flag-PE (L5, Biolegend) after 4 hours of co-culture, they were found to be similar at 15-17% in both cultures. , it was determined that the difference in the ability to eliminate CD123+BDCA-2+ cells between 201 LH CAR-T and 201 HL CAR-T cells identified above was not due to the number of CAR-T cells present in the mixed population. It has been done.
  • Example 8 Production and production of 201A chimeric antibody and humanized antibody
  • mouse monoclonal antibodies are known to be immunogenic in humans
  • chimeric antibodies and humanized antibodies of the 201A mouse antibody were produced.
  • a 201A chimeric antibody containing a light chain containing the amino acid sequence shown in SEQ ID NO: 10 and an amino acid sequence shown in SEQ ID NO: 11 was produced, hereinafter referred to as 201C.
  • the mouse antibody was humanized to produce a 201A humanized antibody comprising a light chain containing the amino acid sequence shown in SEQ ID NO: 14 and a heavy chain containing the amino acid sequence shown in SEQ ID NO: 15, hereinafter referred to as 201H. .
  • the light and heavy chains of the chimeric antibody, 201C, or the light and heavy chains of the humanized antibody, 201H, were cloned into pCDNA3.3 and Optivec, respectively. Expression vectors for each light chain and heavy chain were simultaneously transfected into Expi293 cells to produce 201C and 201H. The cell culture fluid was collected and the antibodies were purified using a protein G column. The size of the purified antibody was confirmed by performing SDS-PAGE using a known method (see Figure 12).
  • Cynomolgus monkey BDCA-2 (CLEC4C) gene (cyno BDCA-2, SEQ ID NO: 22) was synthesized and cloned into a virus expression vector (pCDH-CMV-MCS-EF1-GFP-puro) to produce the virus.
  • HEK 293 cells were transduced with a cyno BDCA-2 expression virus and subcultured in medium supplemented with 1 ⁇ g/mL of puromycin. Through this, cells expressing cyno BDCA-2 were selected. The efficiency of transduction into the cell line was confirmed by checking the fluorescence of GFP included as a surrogate marker in the expression vector under a microscope (see Figure 13).
  • both the chimeric antibody and the humanized antibody according to the present invention were able to bind to human BDCA-2 expressing 293 cells and cyno BDCA-2 expressing 293 cells in a dose dependent manner. Confirmed.
  • the present invention discovers an antibody that specifically binds to BDCA-2 and confirms the activity of CAR, CAR-T cells, chimeric antibodies, and humanized antibodies produced based on it, and BDCA-2 according to the present invention It was confirmed that the novel antibody that specifically binds to BDCA-2 can recognize BDCA-2 similarly to known antibodies, and internalization of BDCA-2 from the surface of human plasmacytoid dendritic cells (pDC) It showed an effect of suppressing the expression of type I interferon by mediating, and it was confirmed that the effect was superior to that of known antibodies.
  • pDC human plasmacytoid dendritic cells
  • CAR produced based on an antibody that specifically binds to BDCA-2 and T cells expressing the CAR showed excellent pDC removal ability
  • CAR produced based on an antibody that specifically binds to BDCA-2 It was confirmed that the chimeric antibody and the humanized antibody can bind to both cells expressing human BDCA-2 and cells expressing monkey BDCA-2.

Abstract

L'invention concerne un nouvel anticorps, se liant de manière spécifique à BDCA-2, qui reconnaît BDCA-2 à un niveau similaire à des anticorps connus, et médie l'internalisation de BDCA-2 à partir de la surface de cellules dendritiques plasmacytoïdes (pDC), présentant ainsi un effet d'inhibition de l'expression d'interférons de type I, et ledit effet a été confirmé comme étant supérieur à ceux des anticorps connus. En outre, il a été confirmé que le CAR produit sur la base de l'anticorps se liant de manière spécifique aux BDCA-2 et aux lymphocytes T exprimant le CAR a présenté une excellente capacité à éliminer les pDC, et l'anticorps chimérique produit sur la base de l'anticorps se liant de manière spécifique au BDCA-2 et un anticorps humanisé peut se lier aux deux cellules exprimant le BDCA-2 humain et les cellules exprimant le BDCA-2 de singe, et ainsi, l'anticorps selon la présente invention peut être utilisé de manière utile pour prévenir ou traiter des maladies auto-immunes telles que le lupus érythémateux disséminé.
PCT/KR2022/009887 2022-07-07 2022-07-07 Anticorps anti-bdca-2 et son utilisation WO2024010118A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130315820A1 (en) * 2010-12-13 2013-11-28 Lfb Biotechnologies Use of an antibody directed against a membrane protein
KR20160099083A (ko) * 2013-12-24 2016-08-19 아스테라스 세이야쿠 가부시키가이샤 신규 항인간 bdca-2 항체
US20200353003A1 (en) * 2018-01-04 2020-11-12 Miltenyi Biotec B.V. & Co. KG Chimeric Antigen Receptor Specific for BDCA2 Antigen
KR20200130468A (ko) * 2012-12-10 2020-11-18 바이오젠 엠에이 인코포레이티드 항-혈액 수지상 세포 항원 2 항체 및 이의 용도
WO2021023793A1 (fr) * 2019-08-05 2021-02-11 Capella Bioscience Ltd Anticorps anti bdca-2

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130315820A1 (en) * 2010-12-13 2013-11-28 Lfb Biotechnologies Use of an antibody directed against a membrane protein
KR20200130468A (ko) * 2012-12-10 2020-11-18 바이오젠 엠에이 인코포레이티드 항-혈액 수지상 세포 항원 2 항체 및 이의 용도
KR20160099083A (ko) * 2013-12-24 2016-08-19 아스테라스 세이야쿠 가부시키가이샤 신규 항인간 bdca-2 항체
US20200353003A1 (en) * 2018-01-04 2020-11-12 Miltenyi Biotec B.V. & Co. KG Chimeric Antigen Receptor Specific for BDCA2 Antigen
WO2021023793A1 (fr) * 2019-08-05 2021-02-11 Capella Bioscience Ltd Anticorps anti bdca-2

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