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WO2025216667A1 - Monoclonal antibody that specifically binds to bdca-2 - Google Patents

Monoclonal antibody that specifically binds to bdca-2

Info

Publication number
WO2025216667A1
WO2025216667A1 PCT/RU2025/050092 RU2025050092W WO2025216667A1 WO 2025216667 A1 WO2025216667 A1 WO 2025216667A1 RU 2025050092 W RU2025050092 W RU 2025050092W WO 2025216667 A1 WO2025216667 A1 WO 2025216667A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
acid sequence
variable domain
chain variable
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/RU2025/050092
Other languages
French (fr)
Inventor
Alexey Konstantinovich MISORIN
Aleksandra Dmitrievna AZARIAN
Andrey BUKHAREV
Tatiana Andreevna VODOPIANOVA
Elizaveta Olegovna ERMOLAEVA
Aleksandr Aleksandrovich CHEBLOKOV
Elena Vladimirovna VINOGRADOVA
Dmitry Valentinovich MOROZOV
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Joint Stock Co <<biocad>>
Original Assignee
Joint Stock Co <<biocad>>
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from RU2024110026A external-priority patent/RU2024110026A/en
Application filed by Joint Stock Co <<biocad>> filed Critical Joint Stock Co <<biocad>>
Publication of WO2025216667A1 publication Critical patent/WO2025216667A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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

Definitions

  • the present invention relates to the field of biotechnology and medicine, in particular to a monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 (blood dendritic cell antigen 2).
  • the invention further relates to nucleic acids encoding said antibody, expression vectors, host cells and methods for producing same, methods for producing the antibodies according to the invention, pharmaceutical compositions comprising the antibody according to the invention, pharmaceutical compositions comprising the antibody according to the invention and other therapeutically active compounds, methods for treating BDCA-2-mediated diseases or syndromes, uses of the antibodies or pharmaceutical compositions thereof for treating BDCA-2-mediated diseases or syndromes, and uses of the antibodies and other therapeutically active compounds for treating BDCA-2-mediated diseases or syndromes.
  • BDCA-2 blood dendritic cell antigen 2 is a C-type lectin found on the surface of plasmacytoid dendritic cells (pDCs). It is also known as the C-type lectin domain family 4 member C (CLEC4C) and CD303. BDCA-2 is an inhibitory receptor capable of modulating the function of pDCs, including influencing the production of type I interferons (Dzionek et al. The Journal of experimental medicine, 2001, 194(12), 1823-1834).
  • BDCA-2 can act as a signaling receptor that activates protein tyrosine kinases and mobilizes intracellular calcium (Dzionek et al. The Journal of experimental medicine, 2001, 194(12), 1823-1834).
  • Plasmocytoid dendritic cells are key cells in innate immunity involved in the fight against viruses and other pathogens. They are producers of type I interferons and cytokines, which have an important antiviral effect and stimulate the adaptive immune response.
  • BDCA-2 can exacerbate many pathological conditions.
  • production of type I interferons by pDCs is associated with some autoimmune diseases, including systemic lupus erythematosus.
  • BDCA-2 when bound to an antibody, sends an inhibitory signal to pDCs, reducing the production of type I interferon.
  • a monoclonal antibody against BDCA-2 is being developed and the efficacy thereof is being investigated for the treatment of systemic lupus erythematosus, including cutaneous systemic lupus erythematosus (Cho et al. Expert Opinion on Investigational Drugs, 2023, 32(5), 345-353; Cho & Furie. Immunotherapy, 2024, 16(1), 15-20).
  • pDCs and type I interferons have also been studied in connective tissue diseases that can affect the skin and mucous membranes. Increased pDCs have been demonstrated in skin lesions with dermatomyositis, as well as increased IFN-I signaling, which was closely associated with interferon-inducible protein (IP10)/CXCL10 expression and recruitment of potentially autoreactive CXCR3+ lymphocytes (Wenzel et al. Clinical and experimental dermatology, 2006, 31(4), 576-582).
  • IP10 interferon-inducible protein
  • pDCs have been shown to infiltrate lesional skin in the both diseases (McNiff & Kaplan. Journal of cutaneous pathology, 2008, 35(5), 452-456).
  • pDCs are also known to be involved in the pathogenesis of systemic sclerosis due to their ability to penetrate the skin and directly secrete interferons, interleukin-6 and other proinflammatory chemokines directly or through the IFN-I response in resident cells and, hence, the effect of a monoclonal antibody against BDCA-2 on cells from patients with systemic sclerosis was investigated in vitro (Corinaldesi et al. Arthritis and Rheumatology, 2018, Vol. 70, No. S9).
  • Neutrophil extracellular traps have been shown to activate pDCs to secrete IFN-a in the skin of patients with hidradenitis suppurativa (Byrd et al. Science translational medicine, 2019, 11(508), eaav5908). Further, the effect of IFN-I present in patients with this disease is primarily limited to the affected skin.
  • pDCs and IFN-I are known to be involved in proximal events of the innate immunity cascade that triggers psoriatic inflammation (Nestle et al. The Journal of experimental medicine, 2005, 202(1), 135-143). Although pDCs are virtually absent in long-standing psoriatic lesions, pDCs have been shown to infiltrate the skin in the early stages of psoriasis and their presence correlates with the expression of markers typical of the early phases of psoriasis (Albanesi et al. Journal of Experimental Medicine, 2009, 206(1), 249-258).
  • pDCs were reported in all individuals with lichen striatum; further, the presence of MxA protein (a surrogate marker of local type I interferon production and, therefore, an indirect assessment of pDC activity) was identical in all patients with lichen planus and lichen striatum (Badr et al. Pediatric Dermatology, 2016, 33(3), 301 - 306).
  • pDCs are a central component in the inflammatory infiltrate in lichenoid parapsoriasis (Karouni et al. Clinical and Experimental Dermatology, 2018, 43(4), 404-409). It is suggested that this disease, analogously to other similar skin diseases, has a pathological process controlled by pDCs through the production of IFN-I, which leads to a cytotoxic attack.
  • the BDCA-2 receptor like other pDC markers, is considered a potential target for cancer therapy, especially in cases where pDCs are involved in maintaining the tumor microenvironment.
  • malignant proliferation of pDCs is involved in the pathogenesis of some oncohematological diseases, in particular, blastic plasmacytoid dendritic cell neoplasm (BPDCN) and acute myeloid dendritic cell leukemia (Wilson et al. Leukemia & lymphoma, 2022, 63(1), 19-30).
  • BPDCN blastic plasmacytoid dendritic cell neoplasm
  • acute myeloid dendritic cell leukemia Wilson et al. Leukemia & lymphoma, 2022, 63(1), 19-30.
  • pDCs were also reported to be consistently and abundantly present in keratoacanthomas (Abbas et al. Journal of the American Academy of Dermatology, 2014, 70(6), 1142-1145).
  • pDCs were found in squamous cell and basal cell carcinomas (Pinto et al. Oncoimmunology, 2012, 1(5), 726-734). pDCs significantly influence the immune cascade and can promote both tumor progression and regression. For example, the number of pDCs recruited to imiquimod-treated actinic keratosis significantly correlated with the therapeutic effect, indicating that skin-penetrating pDCs are critically involved in the regression of the disease (Ogawa et al. Journal of Dermatological Science, 2014, 76(1), 67-69). pDC recruitment was also reported to be a recurrent factor in the early stages of melanoma development (Monti et al.
  • pDCs were also reported to influence the development and course of Kaposi's sarcoma (West et al. Journal of virology, 2011, 85(2), 895-904). pDCs are also critical in infectious diseases. Accordingly, pDCs are important in the polarization of antifungal Thl7 cells and can shift the immune balance towards tolerance or inflammation for various mycoses, including chromoblastomycosis, lobomycosis, and paracoccidioidomycosis (Verma et al. Cold Spring Harbor perspectives in medicine, 2015, 5(3), a019612). In addition, BDCA-2+ cells have also been found in patients with leprosy (Hansen disease) (Pinheiro et al. Frontiers in immunology, 2018, 9, 518).
  • BDCA-2 is highly significant as a molecular target for the correction of pDC function in various diseases, which fact may lead to a significant increase in the efficacy of the therapy.
  • Monoclonal antibodies in the form of chimeric, humanized or fully human molecules have proven the value thereof as effective drugs for the treatment of a number of syndromes and diseases.
  • the authors of the present group of inventions have developed antibodies that specifically bind to BDCA-2 and have high parameters of binding affinity to BDCA-2, including on the surface of the BDCA-2+ cell line.
  • the antibodies against BDCA-2 of the invention do not activate the NF AT signaling pathway through interaction with the FcyRIIIa receptor, which reduces the likelihood of adverse events, but specifically activate the NF AT signaling pathway through interaction with FcyRIIa, which may indicate increased functional activity thereof in patients carrying the FcyRIIa R/R131 allotype.
  • the antibodies against BDCA-2 of the invention are capable of effectively blocking IFN-a production and induce BDCA-2 internalization following binding to this target on the surface of pDCs.
  • the antibodies against BDCA-2 of the invention inhibit the release of interleukin-6 by pDCs and do not induce antibody-dependent death of BDCA-2 + and BDCA-2" cell lines. Also, the antibodies against BDCA-2 of the invention demonstrated anti-inflammatory activity in in vivo experiments on the imiquimod-induced psoriasis-like skin inflammation model in mice.
  • KD in this description refers to the affinity constant (or equilibrium dissociation constant), which is calculated from the ratio of Kd to Ka (i.e. Kd/Ka), and it is expressed as a molar concentration (M).
  • Binding affinity generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g. an antibody) and its binding partner (e.g. an antigen). Unless indicated otherwise, "binding affinity” refers to intrinsic (characteristic, true) binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g. antibody and antigen).
  • the affinity of a molecule X for its binding partner Y can generally be represented by the equilibrium dissociation constant (KD).
  • the preferred Kd value is about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM, or less.
  • Affinity can be measured by common methods known in the art, including those described in the present description. Low- affinity antibodies typically bind an antigen slowly and tend to dissociate readily, whereas high- affinity antibodies typically bind an antigen faster and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, any one of these methods may be used for the purposes of the present invention.
  • Kd refers to the off rate constant of a particular interaction between a binding molecule and antigen.
  • the koff dissociation rate constant can be measured using bio-layer interferometry, for example, using the OctetTM system; as well as by surface plasmon resonance: for example, using the BiacoreTM system.
  • Ka "kon” or "on-rate” refers to the association rate constant.
  • in vitro refers to a biological entity, a biological process, or a biological reaction outside the body under artificial conditions.
  • a cell grown in vitro is to be understood as a cell grown in an environment outside the body, e.g. in a test tube, a culture vial, or a microtiter plate.
  • ED50 EC50 (50% effective dose/concentration) refers to concentrations of a formulation producing 50% biological effect (which may include cytoxicity).
  • Antigen a protein which the antibody (antibodies) in question specifically binds to.
  • Genetic construct an artificially created circular DNA molecule comprising various elements necessary for the expression of target genes and replication within organisms.
  • Reporter cell line a cell line carrying a reporter gene for evaluation of intracellular signaling.
  • Transient production the production of protein from a temporary producer.
  • Transfection a method for DNA delivery where foreign DNA molecules are delivered to a cell using chemical or physical methods: as part of liposomal complexes, as a result of electroporation, and the like.
  • Chromatography a method of separation and analysis of mixtures of substances, as well as a study of physico-chemical properties of substances. It is based on distribution of substances between two phases - the stationary phase (solid phase or liquid coated on an inert carrier) and the mobile phase (gas or liquid phase, eluent).
  • ADCC antibody-dependent cellular cytotoxicity
  • CD16 cluster of differentiation 16, also FcyRIIIa, has 2 isoforms: V158 (or V176) — high affinity receptor, F158 (or F176) — low affinity receptor.
  • CD32 cluster of differentiation 32, also FcyRIIA, has 2 isoforms: H131 — high affinity receptor, R131 — low affinity receptor
  • CHO Chinese hamster ovary cell line.
  • Fc fragment crystallizable
  • FcyRs receptors for the immunoglobulin G Fc fragment (receptors for the IgG Fc fragment).
  • Tris-HCl — buffer solution comprising tri s(hydroxymethyl)aminom ethane and hydrochloric acid (HC1).
  • the present invention relates to a monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2.
  • mAb refers to an antibody that is synthesized and isolated as an individual clonal population of cells.
  • the antibody of the invention is a recombinant antibody.
  • recombinant antibody refers to an antibody that is expressed in a cell or cell line comprising nucleotide sequence(s) encoding an antibody, wherein said nucleotide sequence(s) is (are) not associated with the cell in nature.
  • the present invention relates to an isolated monoclonal antibody or antigenbinding fragment thereof that specifically binds to BDCA-2 and comprises:
  • a heavy chain variable domain comprising:
  • CDR1 with the amino acid sequence of SEQ ID NO: 1
  • CDR2 with the amino acid sequence WIX1PX2SGGSSSPQKFQG, where
  • X 2 N or W; iii) CDR3 with the amino acid sequence DRDX3LX4GAAFDV, where
  • a light chain variable domain comprising: i) CDR1 with the amino acid sequence GGNNIGXsKXeVH, where
  • X 5 S or Y
  • X 6 S or W or Y; ii) CDR2 with the amino acid sequence of SEQ ID NO: 12, iii) CDR3 with the amino acid sequence QVWDX7SSDHSWV, where
  • X7 S or Q or L or W or Y.
  • the antibody according to the invention is an isolated antibody.
  • isolated used to describe various antibodies according to the present description refers to an antibody which has been identified and isolated and/or regenerated from a cell or cell culture, in which the antibody is expressed.
  • Impurities contaminant components
  • the isolated polypeptide is typically prepared by at least one purification step.
  • antibody or “immunoglobulin” (Ig) as used in the present description includes whole antibodies.
  • antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain comprises a heavy chain variable region (abbreviated referred to in the present description as VH) and a heavy chain constant region.
  • Each light chain consists of a light chain variable region (abbreviated referred to in the present description as VL) and light chain constant region.
  • the light chain constant domain can be CK (kappa light chain constant domain) or CL (lambda light chain constant domain).
  • the light chain is a kappa (K) light chain
  • the light chain constant domain is preferably CK.
  • Antibodies according to the invention may be of any class (e.g., IgA, IgD, IgE, IgG, and IgM, preferably IgG), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2, preferably IgGl).
  • class e.g., IgA, IgD, IgE, IgG, and IgM, preferably IgG
  • subclass e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2, preferably IgGl.
  • VL and VH regions may be further subdivided into hyper-variability regions called complementarity determining regions (CDRs), interspersed between regions that are more conserved, termed framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of heavy and light chains form a binding domain that interacts with an antigen.
  • the constant regions of antibodies may mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (e.g. effector cells) and the first component (Clq) of the classical complement system.
  • various cells of the immune system e.g. effector cells
  • the first component (Clq) of the classical complement system e.g. Clq
  • antigen-binding portion of antibody or antigen-binding fragment refers to one or more antibody fragments that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of antibody can be performed by fragments of a full-length antibody.
  • binding fragments which are included within the term "antigen-binding portion" of an antibody include (i) Fab-fragment, monovalent fragment, consisting of VL, VH, CL and CHI domains; (ii) F(ab')2 fragment, a bivalent fragment comprising two Fab-fragments linked by a disulfide bridge at the hinge region; (iii) Fd-fragment consisting of VH and CHI domains; (iv) Fv-fragment consisting of VL and VH domains of a single arm of an antibody; (v) dAb-fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH/VHH domain.
  • VL and VH two regions of the Fv-fragment, VL and VH, are encoded by different genes, they can be joined using recombinant methods using a synthetic linker that enables to receive them as a single protein chain in which the VL and VH regions are paired to form monovalent molecules (known as a single-chain Fv (scFv); see e.g. Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). It is assumed that such single-stranded molecules are also included within the term "antigen -binding portion" of antibody. Such antibody fragments are produced using conventional techniques known to those skilled in the art, and these fragments are screened in the same manner as intact antibodies are.
  • Kabat numbering scheme or “numbering according to Kabat” as used in the present application refers to the system for numbering of amino acid residues that are more variable (i.e. hypervariable) than other amino acid residues in variable regions of heavy and light chains of antibody (Kabat et al. Ann. N.Y. Acad. Sci., 190:382-93 (1971); Kabat et al. Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991)).
  • the antibody of the present invention "which specifically binds" to a target antigen refers to an antibody that binds an antigen with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent targeting a protein or cell or tissue expressing the antigen.
  • the term "specifically binds to" a particular polypeptide or an epitope on a particular target polypeptide may be described by example of a molecule having a Kd for the target of at least about 200 nM, or at least about 150 nM, or at least about 100 nM, or at least about 60 nM, or at least about 50 nM, or at least about 40 nM, or at least about 30 nM, or at least about 20 nM, or at least about 10 nM, or at least about 8 nM, or at least about 6 nM, or at least about 4 nM, or at least about 2 nM, or at least about 1 nM, or at least about 100 pM or less.
  • the term "specific binding” refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or epitope on a polypeptide.
  • the isolated monoclonal antibody or antigen-binding fragment thereof includes a heavy chain variable domain that comprises CDR2 with an amino acid sequence that is selected from the group: SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
  • the isolated monoclonal antibody or antigen-binding fragment thereof includes a heavy chain variable domain that comprises CDR3 with an amino acid sequence that is selected from the group: SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.
  • the isolated monoclonal antibody or antigen-binding fragment thereof includes a heavy chain variable domain comprising:
  • CDR2 with an amino acid sequence that is selected from the group: SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4;
  • the isolated monoclonal antibody or antigen-binding fragment thereof includes:
  • the isolated monoclonal antibody or antigen-binding fragment thereof includes a light chain variable domain that comprises CDR1 with an amino acid sequence that is selected from the group: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.
  • the isolated monoclonal antibody or antigen-binding fragment thereof includes a light chain variable domain that comprises CDR3 with an amino acid sequence that is selected from the group: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.
  • the isolated monoclonal antibody or antigen-binding fragment thereof includes a light chain variable domain comprising:
  • CDR1 with an amino acid sequence that is selected from the group: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11;
  • CDR3 with an amino acid sequence that is selected from the group: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.
  • the isolated monoclonal antibody or antigen-binding fragment thereof includes:
  • the isolated monoclonal antibody or antigen-binding fragment thereof includes:
  • CDR2 with an amino acid sequence that is selected from the group: SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4;
  • CDR3 with an amino acid sequence that is selected from the group: SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.
  • CDR1 with an amino acid sequence that is selected from the group: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11;
  • CDR3 with an amino acid sequence that is selected from the group: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.
  • the isolated monoclonal antibody or antigen-binding fragment thereof includes:
  • CDR1 with the amino acid sequence of SEQ ID NO: 8
  • CDR2 with the amino acid sequence of SEQ ID NO: 12
  • CDR3 with the amino acid sequence of SEQ ID NO: 13;
  • a heavy chain variable domain comprising: CDR1 with the amino acid sequence of SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 2 and CDR3 with the amino acid sequence of SEQ ID NO: 7; and (b) a light chain variable domain comprising:
  • CDR1 with the amino acid sequence of SEQ ID NO: 8
  • CDR2 with the amino acid sequence of SEQ ID NO: 12
  • CDR3 with the amino acid sequence of SEQ ID NO: 13;
  • CDR1 with the amino acid sequence of SEQ ID NO: 1
  • CDR2 with the amino acid sequence of SEQ ID NO: 4
  • CDR3 with the amino acid sequence of SEQ ID NO: 5
  • a light chain variable domain comprising:
  • CDR1 with the amino acid sequence of SEQ ID NO: 8
  • CDR2 with the amino acid sequence of SEQ ID NO: 12
  • CDR3 with the amino acid sequence of SEQ ID NO: 13;
  • a heavy chain variable domain comprising: CDR1 with the amino acid sequence of SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 2 and CDR3 with the amino acid sequence of SEQ ID NO: 5; and (b) a light chain variable domain comprising:
  • CDR1 with the amino acid sequence of SEQ ID NO: 8
  • CDR2 with the amino acid sequence of SEQ ID NO: 12
  • CDR3 with the amino acid sequence of SEQ ID NO: 14 or
  • CDR1 with the amino acid sequence of SEQ ID NO: 8
  • CDR2 with the amino acid sequence of SEQ ID NO: 12
  • CDR3 with the amino acid sequence of SEQ ID NO: 15;
  • CDR1 with the amino acid sequence of SEQ ID NO: 8
  • CDR2 with the amino acid sequence of SEQ ID NO: 12
  • CDR3 with the amino acid sequence of SEQ ID NO: 16;
  • (ix) (a) a heavy chain variable domain comprising: CDR1 with the amino acid sequence of SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 2 and CDR3 with the amino acid sequence of SEQ ID NO: 5; and (b) a light chain variable domain comprising:
  • CDR1 with the amino acid sequence of SEQ ID NO: 8
  • CDR2 with the amino acid sequence of SEQ ID NO: 12
  • CDR3 with the amino acid sequence of SEQ ID NO: 17;
  • (x) (a) a heavy chain variable domain comprising: CDR1 with the amino acid sequence of SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 2 and CDR3 with the amino acid sequence of SEQ ID NO: 5; and (b) a light chain variable domain comprising:
  • the isolated monoclonal antibody or antigen-binding fragment thereof includes the heavy chain variable domain comprises an amino acid sequence that is selected from the group: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23.
  • the isolated monoclonal antibody or antigen-binding fragment thereof includes a light chain variable domain comprises an amino acid sequence that is selected from the group: SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31.
  • the isolated monoclonal antibody or antigen-binding fragment thereof includes:
  • the heavy chain variable domain comprises an amino acid sequence that is selected from the group: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23; and
  • a light chain variable domain comprises an amino acid sequence that is selected from the group: SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31.
  • the isolated monoclonal antibody or antigen-binding fragment thereof includes:
  • the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:
  • the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 24;
  • the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:
  • the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 24;
  • the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:
  • the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 24;
  • the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:
  • the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 24;
  • the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:
  • the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 24;
  • the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:
  • the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 24;
  • the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 19 and
  • the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 25;
  • the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 19 and
  • the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 26;
  • the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 19 and
  • the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 27;
  • the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:
  • the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 28; or
  • the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:
  • the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 29;
  • the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 19 and
  • the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 30;
  • the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 19 and
  • the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 31.
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is a full-length IgG antibody.
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is a full-length IgG antibody that is of human IgGl, IgG2, IgG3 or IgG4 isotype.
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is a full-length IgG antibody that is of human IgGl isotype.
  • the isolated monoclonal antibody comprises mutations that are selected from the group: i) Q1E; or ii) M252Y, S254T and T256E; or iii) G236A; or iv) I332E and G236A; or v) S239D, I332E and G236A; or vi) Y300L; or vii) D270E; or viii) N325S and L328F; or ix) L235M; or x) S298M; or xi) N325W; or xii) S239F; or xiii) deletion 446G and 447K; or xiv) a combination of i) and any mutation selected from the group comprising ii)-xiii); or xv) a combination of i), any mutation selected from the group comprising ii)-xii) and xiii), according to the EU numbering scheme
  • the isolated monoclonal antibody includes a heavy chain comprising an amino acid sequence that is selected from the group: SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 49.
  • the isolated monoclonal antibody includes a light chain comprising an amino acid sequence that is selected from the group: SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 or SEQ ID NO: 57.
  • the isolated monoclonal antibody includes:
  • a heavy chain comprising an amino acid sequence that is selected from the group: SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 49, and
  • a light chain comprising the amino acid sequence that is selected from the group: SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 or SEQ ID NO: 57.
  • the isolated monoclonal antibody includes:
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is an antibody that is selected from the group: 01-008, 05-001, 05-002, 05-003, 05- 004, 05-005, 05-006, 05-007, 05-008, 05-009, 05-010, 05-011, 05-012, 05-016, 05-017, 05-018, 05- 019, 05-020, 05-021, 05-022, 05-023, 05-024, 05-025 or 05-026.
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 01-008.
  • Antibody 01-008 includes:
  • Antibody 01-008 includes:
  • Antibody 01-008 includes:
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-001.
  • Antibody 05-001 includes:
  • Antibody 05-001 includes:
  • Antibody 05-001 includes:
  • a heavy chain variable domain comprising: (i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-002.
  • Antibody 05-002 includes:
  • Antibody 05-002 includes:
  • Antibody 05-002 includes:
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-003.
  • Antibody 05-003 includes:
  • Antibody 05-003 includes:
  • Antibody 05-003 includes:
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-004.
  • Antibody 05-004 includes:
  • Antibody 05-004 includes:
  • Antibody 05-004 includes:
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-005.
  • Antibody 05-005 includes:
  • Antibody 05-005 includes: (a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 18; and
  • Antibody 05-005 includes:
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-006.
  • Antibody 05-006 includes:
  • Antibody 05-006 includes:
  • Antibody 05-006 includes:
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-007.
  • Antibody 05-007 includes:
  • Antibody 05-007 includes:
  • Antibody 05-007 includes:
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-008.
  • Antibody 05-008 includes:
  • Antibody 05-008 includes:
  • Antibody 05-008 includes:
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-009.
  • Antibody 05-009 includes:
  • Antibody 05-009 includes:
  • Antibody 05-009 includes:
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-010.
  • Antibody 05-010 includes:
  • Antibody 05-010 includes:
  • Antibody 05-010 includes:
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-011.
  • Antibody 05-011 includes:
  • Antibody 05-011 includes:
  • Antibody 05-011 includes:
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-012.
  • Antibody 05-012 includes:
  • Antibody 05-012 includes:
  • Antibody 05-012 includes:
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-016.
  • Antibody 05-016 includes:
  • Antibody 05-016 includes:
  • Antibody 05-016 includes:
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-017.
  • Antibody 05-017 includes:
  • Antibody 05-017 includes:
  • Antibody 05-017 includes:
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-018.
  • Antibody 05-018 includes:
  • Antibody 05-018 includes:
  • Antibody 05-018 includes:
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-019.
  • Antibody 05-019 includes:
  • Antibody 05-019 includes:
  • Antibody 05-019 includes:
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-020.
  • Antibody 05-020 includes:
  • Antibody 05-020 includes:
  • Antibody 05-020 includes:
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-021.
  • Antibody 05-021 includes:
  • Antibody 05-021 includes:
  • Antibody 05-021 includes: (a) a heavy chain variable domain comprising:
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-022.
  • Antibody 05-022 includes:
  • Antibody 05-022 includes:
  • Antibody 05-022 includes:
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-023.
  • Antibody 05-023 includes:
  • Antibody 05-023 includes:
  • Antibody 05-023 includes:
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-024.
  • Antibody 05-024 includes:
  • Antibody 05-024 includes:
  • Antibody 05-024 includes:
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-025.
  • Antibody 05-025 includes:
  • Antibody 05-025 includes: (a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 19; and
  • Antibody 05-025 includes:
  • the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-026.
  • Antibody 05-026 includes:
  • Antibody 05-026 includes:
  • Antibody 05-026 includes:
  • hypervariable regions of variable domains of light and heavy chains (LCDR1, 2, 3 and HCDR1, 2, 3) of all the above antibodies are provided in accordance with the Kabat nomenclature. Those skilled will appreciate that the hypervariable regions of variable domains of light and heavy chains (LCDR1, 2, 3 and HCDR1, 2, 3) may also be represented in accordance with other commonly known numbering scheme, for example, IMGT, Chothia or AbM. Thus, all of the above antibodies which are characterized by means of hypervariable regions of variable domains of light and heavy chains (LCDR1, 2, 3 and HCDR1, 2, 3) using the IMGT, Chothia or AbM numbering scheme are also encompassed by the present invention.
  • the antibodies of the invention which specifically bind to BDCA-2 have high binding affinity parameters with respect to BDCA-2, including on the surface of BDCA-2+ cell line.
  • the antibodies against BDCA-2 of the invention do not activate the NFAT signaling pathway through interaction with the FcyRIIIa receptor, which reduces the likelihood of adverse events, but specifically activate the NFAT signaling pathway through interaction with FcyRIIa, which may indicate increased functional activity thereof in patients carrying the FcyRIIa R/R131 allotype.
  • the antibodies against BDCA-2 of the invention are capable of effectively blocking IFN-a production and induce BDCA-2 internalization following binding to this target on the surface of pDCs.
  • the antibodies against BDCA-2 of the invention inhibit the release of interleukin-6 by pDCs and do not induce antibody-dependent death of BDCA-2 + and BDCA-2" cell lines. Also, the antibodies against BDCA- 2 of the invention demonstrated anti-inflammatory activity in in vivo experiments on the imiquimod- induced psoriasis-like skin inflammation model in mice.
  • the present invention relates to a nucleic acid that encodes any one of the above antibody or antigen-binding fragment thereof that specifically binds to BDCA-2.
  • the nucleic acid molecules may be isolated.
  • nucleic acid means a precise sequence of nucleotides, modified or not, determining a fragment or a region of a nucleic acid, containing unnatural nucleotides or not, and being either a double-strand DNA or RNA, a single-strand DNA or RNA, or transcription products of said DNAs.
  • nucleotide sequence encompasses its complement.
  • a nucleic acid having a particular sequence should be understood as one which encompasses the complementary strand thereof with the complementary sequence thereof.
  • nucleic acid molecule is one which is identified and separated from at least one nucleic acid molecule-impurity.
  • An isolated nucleic acid molecule is different from the form or set in which it is found under natural conditions.
  • an isolated nucleic acid molecule is different from a nucleic acid molecule that exists in cells under natural conditions.
  • the present invention relates to a nucleic acid molecule comprising a nucleotide sequence that encodes an amino acid sequence selected from SEQ ID NO: 1-57.
  • a nucleic acid molecule may also comprise any combination of said nucleotide sequences.
  • DNA sequences can encode the amino acid sequence of the light chain or heavy chain of the antibody according to the invention or fragments thereof (VH, VL, CDR, etc.). It is well within the skill of those trained in the art to create these alternative DNA sequences encoding one and the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention.
  • the isolated nucleic acid is DNA.
  • the nucleic acid molecule of the invention may be isolated from any source that produces the monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2.
  • the nucleic acid molecule of the invention may be synthesized by way of chemical synthesis, rather than isolated.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain variable domain of antibodies 01-008, 05-001, 05-002, 05- 003, 05-004, 05-005, 05-006, 05-007, 05-008, 05-009, 05-010, 05-011, 05-012, and includes a nucleotide sequence with SEQ ID NO: 58.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain variable domain of antibodies 05-020, 05-021, 05-022, 05- 023, 05-024, 05-025, 05-026, and includes a nucleotide sequence with SEQ ID NO: 59.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain variable domain of antibody 05-016, and includes a nucleotide sequence with SEQ ID NO: 60.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain variable domain of antibody 05-017, and includes a nucleotide sequence with SEQ ID NO: 61.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain variable domain of antibody 05-018, and includes a nucleotide sequence with SEQ ID NO: 62.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain variable domain of antibody 05-019, and includes a nucleotide sequence with SEQ ID NO: 63.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain variable domain of antibodies 01-008, 05-001, 05-002, 05- 003, 05-004, 05-005, 05-006, 05-007, 05-008, 05-009, 05-010, 05-011, 05-012, and includes a nucleotide sequence with SEQ ID NO: 64.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain variable domain of antibody 05-020, and includes a nucleotide sequence with SEQ ID NO: 65.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain variable domain of antibody 05-021, and includes a nucleotide sequence with SEQ ID NO: 66.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain variable domain of antibody 05-022, and includes a nucleotide sequence with SEQ ID NO: 67.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain variable domain of antibody 05-023, and includes a nucleotide sequence with SEQ ID NO: 68.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain variable domain of antibody 05-024, and includes a nucleotide sequence with SEQ ID NO: 69.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain variable domain of antibody 05-025, and includes a nucleotide sequence with SEQ ID NO: 70.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain variable domain of antibody 05-026, and includes a nucleotide sequence with SEQ ID NO: 71.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 01-008, and includes a nucleotide sequence with SEQ ID NO: 72.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-001, and includes a nucleotide sequence with SEQ ID NO: 73.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-002, and includes a nucleotide sequence with SEQ ID NO: 74. In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-003, and includes a nucleotide sequence with SEQ ID NO: 75.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-004, and includes a nucleotide sequence with SEQ ID NO: 76.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-005, and includes a nucleotide sequence with SEQ ID NO: 77.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-006, and includes a nucleotide sequence with SEQ ID NO: 78.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-007, and includes a nucleotide sequence with SEQ ID NO: 79.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-008, and includes a nucleotide sequence with SEQ ID NO: 80.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-009, and includes a nucleotide sequence with SEQ ID NO: 81.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-010, and includes a nucleotide sequence with SEQ ID NO: 82.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-011, and includes a nucleotide sequence with SEQ ID NO: 83.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-012, and includes a nucleotide sequence with SEQ ID NO: 84.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-016, and includes a nucleotide sequence with SEQ ID NO: 85. In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-017, and includes a nucleotide sequence with SEQ ID NO: 86.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-018, and includes a nucleotide sequence with SEQ ID NO: 87.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-019, and includes a nucleotide sequence with SEQ ID NO: 88.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibodies 05-020, 05-021, 05-022, 05-023, 05-024, 05- 025, 05-026, and includes a nucleotide sequence with SEQ ID NO: 89.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain of antibodies 01-008, 05-001, 05-002, 05-003, 05-004, 05- 005, 05-006, 05-007, 05-008, 05-009, 05-010, 05-011, 05-012, 05-016, 05-017, 05-018, 05-019, and includes a nucleotide sequence with SEQ ID NO: 90.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain of antibody 05-020, and includes a nucleotide sequence with SEQ ID NO: 91.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain of antibody 05-021, and includes a nucleotide sequence with SEQ ID NO: 92.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain of antibody 05-022, and includes a nucleotide sequence with SEQ ID NO: 93.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain of antibody 05-023, and includes a nucleotide sequence with SEQ ID NO: 94.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain of antibody 05-024, and includes a nucleotide sequence with SEQ ID NO: 95.
  • the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain of antibody 05-025, and includes a nucleotide sequence with SEQ ID NO: 96. In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain of antibody 05-026, and includes a nucleotide sequence with SEQ ID NO: 97.
  • the nucleic acid molecules may be used to express the monoclonal antibody or antigenbinding fragment thereof that specifically binds to BDCA-2.
  • the present invention relates to an expression vector comprising any one of the above nucleic acid molecules that encode the corresponding amino acid sequences of the antibody that specifically binds to BDCA-2, or portions thereof (for example, heavy chain and/or light chain binding domain sequences).
  • the present invention relates to a vector suitable for the expression of any one of nucleotide sequences described herein.
  • vector means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • expression is defined as the transcription and/or translation of a particular nucleotide sequence.
  • the vector is a plasmid, i.e. a circular double stranded piece of DNA into which additional DNA segments may be inserted.
  • the vector is a viral (expression) vector, wherein additional DNA segments may be inserted into the viral genome.
  • the vectors are capable of autonomous replication in a host cell into which they have been introduced (e.g. bacterial vectors having a bacterial site of replication origin and episomal vectors).
  • vectors e.g. non- episomal vectors
  • certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors").
  • expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses, such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, and the like.
  • DNA molecules may be inserted into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of DNA.
  • An expression vector and expression control sequences may be chosen to be compatible with the expression host cell used.
  • DNA molecules encoding partially or fully heavy and light chain sequences can be inserted into distinct vectors. In one embodiment, any combination of the above DNA molecules is introduced into the same expression vector.
  • DNA molecules may be introduced into an expression vector by standard methods (e.g. ligation of complementary restriction sites on a gene fragment of antibody and vector, or blunt end ligation if no restriction sites are present).
  • a suitable vector is one that includes restriction sites such that any VH or VL sequence can easily be inserted and expressed, as described above.
  • a recombinant expression vector can also encode a signal peptide that facilitates secretion of an antibody chain from a host cell.
  • An antibody chain gene may be cloned into a vector such that the signal peptide is linked in-frame to the N-terminus of an immunoglobulin chain.
  • a signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e. a signal peptide from a non-immunoglobulin protein).
  • the vector may include an expression control sequence.
  • expression control sequence refers to polynucleotide sequences that are necessary to effect the expression and processing of coding sequences to which they are inserted. It will be understood by those skilled in the art that the design of an expression vector, including the selection of expression control sequences, may depend on such factors as the choice of the type of a host cell to be transformed, the required level of expression of antibody, and so forth.
  • Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion.
  • the nature of such expression control sequences differs depending upon the host organism; in prokaryotes, such expression control sequences typically include a promoter, a ribosome binding site, as well as transcription termination sequences; in eukaryotes, such expression control sequences typically include promoters and transcription termination sequences.
  • Preferred expression control sequences for an expression host cell in a mammal include viral elements that ensure high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from a retroviral LTR (long terminal repeat), cytomegalovirus (CMV) (such as a CMV promoter/enhancer), simian virus 40 (SV40) (such as a SV40 promoter/enhancer), adenovirus, (e.g. the major late promoter adenovirus (AdMLP)), polyomavirus and strong mammalian promoters such as TTR promoter, native immunoglobulin promoter or actin promoter.
  • CMV cytomegalovirus
  • SV40 simian virus 40
  • adenovirus e.g. the major late promoter adenovirus (AdMLP)
  • AdMLP major late promoter adenovirus
  • Expression control sequences encompass at least all components whose presence is important for expression and processing
  • the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of a vector in host cells (e.g. origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates the selection of host cells into which a vector has been introduced.
  • the present invention relates to a method for producing a host cell to produce any above antibody or antigen-binding fragment thereof that specifically binds to BDCA-2, and includes transformation of the cell with the above vector.
  • the present invention relates to a host cell for producing any above antibody or antigen-binding fragment thereof that specifically binds to BDCA-2, comprising any one of the above nucleic acids.
  • host cell refers to a cell into which a recombinant expression vector has been introduced.
  • the present invention further relates to host cells that comprise, for example, a nucleotide sequence encoding a heavy chain or antigen-binding portions thereof, a nucleotide sequence encoding a light chain or antigen-binding portions thereof, or both.
  • host cell refers not only to a particular subject cell but to the progeny of such cell as well. Since modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to a parental cell; however, such cells are still included within the scope of the term "host cell” as used herein.
  • Nucleic acid molecules encoding the monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 according to the invention and vectors comprising these nucleic acid molecules may be used for transfection of a mammalian cell, plant cell, bacterial cell, or yeast cell. Transfection may be carried out by any known method for introducing polynucleotides into a host cell.
  • Methods for introducing heterologous polynucleotides into mammalian cells include dextran-mediated transfection, cationic polymer-nucleic acid complex transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, encapsulation of the polynucleotides in liposomes, and direct microinjection of DNA into nuclei.
  • the nucleic acid molecules may be introduced into mammalian cells by viral (expression) vectors.
  • Mammalian cell lines used as hosts for transformation include a plurality of immortalized cell lines. These include, e.g., Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HEK-293T cells, FreeStyle 293 cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549, SK-HEP1, HUH7, Hep-RG cells and a number of other cell lines. Cell lines are selected by way of determining which cell lines have high expression levels and provide for necessary characteristics of the protein being produced.
  • Other cell lines that may be used are insect cell lines, such as Sf9 or Sf21 cells.
  • insect cell lines such as Sf9 or Sf21 cells.
  • the antibodies or fragments thereof are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibodies or fragments thereof in host cells or, more preferably, secretion of the antibodies or fragments thereof into the culture medium in which the host cells are grown.
  • the monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 may be isolated from culture medium using standard protein purification techniques.
  • Plant host cells include e.g. Nicotiana, Arabidopsis.
  • Bacterial host cells include Escherichia and Streptomyces species.
  • Yeast host cells include Schizosaccharomyces pombe. Saccharomyces cerevisiae and Pichia pastor is.
  • level of production of the monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 from a production cell line may be enhanced using a number of known techniques.
  • the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions.
  • the monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 from various cell lines will have a different glycosylation profile as compared to one another.
  • the monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 encoded by nucleic acid molecules described herein, or comprising amino acid sequences provided herein are part of the present invention, regardless of the glycosylation of the binding molecules, and, in general, regardless of the presence or absence of post-translational modifications.
  • the above host cell does not relate to a host cell produced using human embryos.
  • the above host cell does not relate to a host cell produced by modifying the genetic integrity of human germline cells.
  • the present invention relates to a method for producing the antibody or antigenbinding fragment thereof that specifically binds to BDCA-2, comprising culturing the above host cell in a growth medium under conditions sufficient to produce said antibody or fragment thereof, followed by isolation and purification of the resulting antibody or fragment thereof.
  • compositions Another aspect of the invention is a pharmaceutical composition comprising, as an active ingredient (or as the only active ingredient), the monoclonal antibody according to the present invention or antigen-binding fragment thereof that specifically binds to BDCA-2.
  • the present invention relates to a pharmaceutical composition that comprises any above-mentioned antibody or antigen-binding fragment thereof in combination with one or more pharmaceutically acceptable excipients.
  • the present invention relates to a pharmaceutical composition used for treating a BDCA-2-mediated disease or syndrome, which comprises any above antibody or antigen-binding fragment thereof in combination with one or more pharmaceutically acceptable excipients.
  • the present invention relates to a pharmaceutical composition used for treating a BDCA-2-mediated disease or syndrome, which comprises any above antibody or antigen-binding fragment thereof in a therapeutically effective amount in combination with one or more pharmaceutically acceptable excipients.
  • “Pharmaceutical composition” means a composition comprising the antibody according to the invention and at least one of components selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible fillers, solvents, diluents, carriers, auxiliary, distributing and sensing agents, delivery agents.
  • pharmaceutically acceptable refers to one or more compatible liquid or solid components that are suitable for administration in a mammal, preferably in a human.
  • excipient is used herein to describe any ingredient other than the antibody according to the present invention. These are substances of inorganic or organic nature which are used in the pharmaceutical production/manufacturing in order to give drug products the necessary physicochemical properties.
  • compositions are intended to improve the condition or treat diseases or syndromes that may be mediated by BDCA-2.
  • disease or syndrome mediated by BDCA-2 refers to any disease or syndrome that is either directly, or indirectly associated with BDCA-2, including pathogenesis, progression, recurrence or chronification of a disease or syndrome.
  • Treatment refers to a method of alleviating or abrogating a biological disorder and/or at least one of attendant symptoms thereof.
  • the term “syndrome” means any condition that would benefit from treatment according to the present invention.
  • the definition of the term includes chronic and acute symptoms or diseases including those pathological conditions that predispose a mammal to the impairment in question.
  • “Therapeutically effective amount” refers to that amount of the therapeutic agent being administered during treatment which will relieve to some extent one or more of the symptoms of the disease being treated.
  • a therapeutically effective amount may vary according to factors such as the particular condition being treated, the age, sex and weight of the patient, and whether the monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 is being administered as a stand-alone treatment or in combination with one or more additional drug or treatment.
  • the subject of treatment, or patient is a mammal, preferably a human subject.
  • Said subject may be either male or female, of any age.
  • compositions of the present invention and methods of preparation thereof will be undoubtedly apparent to those skilled in the art.
  • the pharmaceutical compositions should preferably be manufactured in compliance with the GMP (Good Manufacturing Practice) requirements.
  • the pharmaceutical composition may include a buffer composition, tonicity agents (osmolyte or osmotic agent), stabilizers and/or solubilizers.
  • tonicity agents osmolyte or osmotic agent
  • stabilizers osmolyte or osmotic agent
  • solubilizers osmolyte or solubilizers
  • the pharmaceutical composition according to the invention is a stable composition.
  • a pharmaceutical composition is "stable" if the active agent retains physical stability and/or chemical stability and/or biological activity thereof during the specified shelf life at a storage temperature, for example, of 2-8 °C.
  • the active agent retains both physical and chemical stability, as well as biological activity. Storage period is adjusted based on the results of stability test in accelerated or natural aging conditions.
  • the pharmaceutical composition is an injectable dosage form.
  • the injectable dosage form is an infusion solution.
  • the injectable dosage form is a solution for subcutaneous administration.
  • Injectable formulations may be manufactured without limitation, in unit dosage form, such as in ampoules, vials, plastic containers, pre-filled syringes, autoinjection devices.
  • the pharmaceutical composition is a pharmaceutical composition provided in dry, i.e. powder or granular, form for reconstitution with a suitable solvent (e.g., sterile pyrogen-free water) prior to administration.
  • a suitable solvent e.g., sterile pyrogen-free water
  • Such medicinal formulation may be prepared by, for example, lyophilization, i.e. a process, which is known in the art as freeze drying, and which involves freezing a product followed by removal of solvent from frozen material.
  • the pharmaceutical composition is a lyophilizate for preparing a solution for infusion. In some embodiments, the pharmaceutical composition is a lyophilizate for preparing a solution for subcutaneous administration.
  • the pharmaceutical composition is a concentrate for preparing a solution for infusion.
  • the pharmaceutical composition is a concentrate for preparing a solution for subcutaneous administration.
  • the present invention relates to a pharmaceutical composition that comprises a monoclonal antibody according to the present invention or antigen-binding fragment thereof that specifically binds to BDCA-2 and at least one other therapeutically active compound.
  • the present invention relates to a pharmaceutical composition for treating a BDCA-2-mediated disease or syndrome, comprising any above antibody or antigen-binding fragment thereof and at least one other therapeutically active compound.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising any above antibody or antigen-binding fragment thereof and further at least one other therapeutically active compound.
  • the present invention relates to a pharmaceutical composition for treating a BDCA-2-mediated disease or syndrome, comprising any above antibody or antigen-binding fragment thereof and further at least one other therapeutically active compound.
  • the present invention relates to a pharmaceutical composition for treating a BDCA-2-mediated disease or syndrome, comprising any above antibody or antigen-binding fragment thereof and at least one other therapeutically active compound, which is an antibody, a small molecule, a hormone therapy agent or a combination thereof.
  • the other therapeutically active compound is an antibody that specifically binds to CD20.
  • the antibody that specifically binds to CD20 is divosilimab, rutiximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab, tositumomab, ublituximab, ocaratuzumab, ripertamab, veltuzumab, or zuberitamab.
  • the BDCA-2-mediated disease or syndrome is selected from the group: systemic lupus erythematosus, cutaneous systemic lupus erythematosus, discoid lupus erythematosus, chronic cutaneous lupus erythematosus, blast tumor of plasmacytoid dendritic cells, acute myeloid dendritic cell leukemia, blastic plasmacytoid dendritic cell neoplasm, progressive systemic sclerosis (systemic scleroderma), dermatomyositis (Wagner disease), psoriasis, hidradenitis suppurativa, lichen planus, lichen linearis (lichen striatum), parapsoriasis lichenoides, tinea versicolor, atopic dermatitis, polymorphic light eruption, polymorphic photodermatitis, actinic keratosis (senile keratosis
  • the antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 is used in the treatment of diseases or syndromes mediated by BDCA-2.
  • the subject of treatment, or patient is a mammal, preferably a human subject.
  • Said subject may be either male or female, of any age.
  • the present invention relates to a method for treating a BDCA-2-mediated disease or syndrome, comprising administering in a subject in need of such treatment any above antibody or antigen-binding fragment thereof or said pharmaceutical composition, in a therapeutically effective amount.
  • the present invention relates to a method for treating a BDCA-2-mediated disease or syndrome, comprising administering in a subject in need of such treatment any above antibody or antigen-binding fragment thereof and at least one other therapeutically active compound in a therapeutically effective amount.
  • the BDCA-2-mediated disease or syndrome is selected from the group: systemic lupus erythematosus, cutaneous systemic lupus erythematosus, discoid lupus erythematosus, chronic cutaneous lupus erythematosus, blast tumor of plasmacytoid dendritic cells, acute myeloid dendritic cell leukemia, blastic plasmacytoid dendritic cell neoplasm, progressive systemic sclerosis (systemic scleroderma), dermatomyositis (Wagner disease), psoriasis, hidradenitis suppurativa, lichen planus, lichen linearis (lichen striatum), parapsoriasis lichenoides, tinea versicolor, atopic dermatitis, polymorphic light eruption, polymorphic photodermatitis, actinic keratosis (senile keratos)
  • the other therapeutically active compound is an antibody, small molecule, hormone therapy agent, or any combination thereof.
  • the other therapeutically active compound is an antibody that specifically binds to CD20.
  • the antibody that specifically binds to CD20 is divosilimab, rutiximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab, tositumomab, ublituximab, ocaratuzumab, ripertamab, veltuzumab, or zuberitamab.
  • the present invention relates to the use of the above antibody or antigen-binding fragment thereof or the above pharmaceutical composition for treating in a subject in need of such treatment a BDCA-2-mediated disease or syndrome.
  • the present invention relates to the use of the above antibody or antigen-binding fragment thereof and at least one other therapeutically active compound for treating in a subject in need of such treatment a BDCA-2-mediated disease or syndrome.
  • the BDCA-2-mediated disease or syndrome is selected from the group: systemic lupus erythematosus, cutaneous systemic lupus erythematosus, discoid lupus erythematosus, chronic cutaneous lupus erythematosus, blast tumor of plasmacytoid dendritic cells, acute myeloid dendritic cell leukemia, blastic plasmacytoid dendritic cell neoplasm, progressive systemic sclerosis (systemic scleroderma), dermatomyositis (Wagner disease), psoriasis, hidradenitis suppurativa, lichen planus, lichen linearis (lichen striatum), parapsoriasis lichenoides, tinea versicolor, atopic dermatitis, polymorphic light eruption, polymorphic photodermatitis, actinic keratosis (senile keratosis),
  • the other therapeutically active compound is an antibody, small molecule, hormone therapy agent, or a combination thereof.
  • the other therapeutically active compound is an antibody that specifically binds to CD20.
  • the antibody that specifically binds to CD20 is divosilimab, rutiximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab, tositumomab, ublituximab, ocaratuzumab, ripertamab, veltuzumab, or zuberitamab.
  • the antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 may be administered without further therapeutic treatment, i.e. as an independent therapy.
  • parenteral administration in the form of sterile medicinal products intended for administration into the body of a subject by breaching the integrity of the skin or mucous membranes, bypassing the gastrointestinal tract by means of injection or infusion.
  • parenteral administration includes, inter alia, subcutaneous, intraperitoneal, intramuscular, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intrasynovial, transdermal injection or infusion; and kidney dialytic infusion techniques.
  • the antibody or antigen-binding fragment that specifically binds to BDCA-2 or the pharmaceutical composition is administered intravenously.
  • intravenous administration is carried out by using infusion, prolonged infusion, or long-lasting continuous infusion.
  • the antibody or antigen-binding fragment that specifically binds to BDCA-2 or the pharmaceutical composition is administered subcutaneously.
  • subcutaneous administration is carried out by using subcutaneous injection.
  • a suitable dose of the monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 according to the present invention will range from 0.1 to 200 mg/kg.
  • Figure l is a map of the expression plasmid pEE-HC for the generation of the heavy chain of antibodies.
  • Figure 2 is a map of the expression plasmid pEE-CK for the generation of the light chain of antibodies.
  • Figure 3 is a histogram showing the results of assessment of the ability of the antibody 01- 008 to BDCA-2 to inhibit IFN-a release.
  • IFN-a concentration pg/ml
  • the values of IFN-a concentration are plotted along the ordinate axis.
  • the following groups are plotted along the abscissa axis:
  • Figure 4 is the results of assessment of ability of antibody 05-008 to induce internalization of the BDCA-2 receptor from the surface of human pDCs.
  • Antibody concentrations pg/ml are plotted along the abscissa axis.
  • the values of the median fluorescence intensity in a.u. (arbitrary units) are plotted along the ordinate axis.
  • the point of the graph marked with an arrow reflects the effect where there are no antibodies.
  • ⁇ - a four-parameter logistic regression model describing the induction of BDCA-2 receptor internalization from the surface of human pDCs caused by antibody 05-008.
  • Figure 5 is the results of assessment of ability of antibody 05-008 to inhibit the release of IL- 6 by human pDCs following activation via Fc-independent pathway.
  • Figure 6 is the results of assessment of ability of antibody 05-008 to induce antibodydependent cell death against BDCA-2 + cell line and BDCA-2" cell line, PBMC donor with the FcyRIIIa receptor (allelic variant F/F176).
  • Antibody concentrations (pg/ml) are plotted along the abscissa axis. Cytotoxicity percentage is plotted along the ordinate axis. The points of the graph marked with an arrow reflect the effect where there are no antibodies.
  • Figure 7 is a histological assessment of inflammatory infiltration of the skin.
  • Desired gene segments were prepared from oligonucleotides made by chemical synthesis.
  • the gene segments of 300-1400 bp long, flanked by singular restriction sites, were assembled by annealing and ligation of oligonucleotides including PCR amplification and subsequently cloned via the restriction sites.
  • the DNA sequences of the cloned gene fragments were confirmed by DNA sequencing.
  • DNA sequences were determined by Sanger sequencing.
  • the Unipro's UGENE suite version 1.29 and SnapGene version 6.1 were used for sequence creation, mapping, analysis, annotation and illustration.
  • the vectors contained all the elements necessary for the expression of protein in eukaryotic cells, as well as all the elements necessary for maintaining plasmid copy number in prokaryotic cells.
  • the fusion genes comprising the subject antibody chains as described below were generated by PCR and/or gene synthesis and assembled using known recombinant methods and techniques by connection of the according nucleic acid segments, e.g. using unique restriction sites in the corresponding vectors. The resulting nucleic acid sequences were verified by DNA sequencing. For eukaryotic cell transfections, larger quantities of the plasmids were prepared by preparation thereof from transformed E. coli cultures.
  • the sequence encoding the extracellular domain of human, cynomolgus monkey and rhesus macaque BDCA-2 was cloned into the pEE plasmid with various peptide tags for protein generation using the Sall/Notl restriction sites.
  • the required quantities of the plasmid were cultured in E.coli cells and purified using conventional techniques. Protein products of antigens were produced by transient transfection of the CHO-T suspension cell line, according to published protocols [Cytotechnology (2012) 64:613-622], Suspension culture was conducted in flasks on orbital shaker using serum-free media supplemented with 8 mmol L- glutamine and 1 g/1 pluronic 68.
  • Polyethylenimine (PEI) was used as a transfection reagent. DNA/PEI ratio was 1 :3/1 :10. 5-7 days following transfection, the culture liquid was clarified using a filtration system. Target proteins were isolated from culture liquid by affine HPLC.
  • the recombinant protein comprising a peptide tag at the protein C-terminus was isolated by affinity chromatography.
  • the purified product was dialized into a storage buffer (membrane pore diameter (kDa) was selected individually taking into account the size of the protein product). If and when necessary, the purified product was concentrated using centrifugal concentrators (membrane pore diameter (kDa) was selected individually taking into account the size of the protein product).
  • Example 2 Obtaining of sequences of variable domains of antibodies against BDCA-2 using human antibody selection in a naive Fab library.
  • Human BDCA-2 was immobilized on the surface of specifically treated tubes at a concentration of 20 pg/ml for the first round of selection by phage display (see below), 5 pg/ml for the second round, 2 pg/ml for the third round, 1 pg/ml for the fourth round, 0.5 pg/ml for the fifth round, 0.25 pg/ml for the sixth round, 0.125 pg/ml for the seventh round, and 0.0625 pg/ml for the eighth round.
  • the plastic was then washed with a phosphate-buffer saline supplemented with 0.1% Tween 20 (PBST) and blocked with a solution of non-fat milk or solution of bovine serum albumin (BSA) in the same phosphate-buffer saline.
  • BSA bovine serum albumin
  • a naive library of human antibodies was used as a library for selection. To this end, it was diluted in a phosphate-buffer saline supplemented with skimmed milk and an additional neutral protein with peptide tags used in the above BDCA-2 antigens (for screening out non-specifically binding phage particles). Immunoassay test tubes with the immobilized antigen were then incubated with libraries.
  • PBST PBST
  • Phage particles bound to the antigen were eluted with a buffer glycine HC1 solution (pH 2.2) for 15 minutes while stirring, then the solution was neutralized with 1 M Tris-HCl solution (pH 7.6).
  • E. coli cells were infected with phages, grown in culture medium and used in the next selection round.
  • Polyclonal phage enzyme-linked immunosorbent assay (ELISA) was used for enrichment of libraries against target antigens and assessment of presence of non-specifically binding phage particles.
  • target human BDCA-2 antigens with tags from Example 1 were immobilized on high-absorption plates (Greiner-Bio). The analysis was performed according to the standard ELISA protocol.
  • Phagemid DNA was isolated from E. coli culture according to the manufacturer's protocol (Qiagen). Phagemid DNA was then used to transform electrocompetent E. coli cells, followed by sorting to monoclones and determination of an unambiguous sequence of genetic constructs by Sanger sequencing. As a result of sequencing, a plurality clones was analyzed, which contained 8 unique sequences of ScFv fragments of heavy and light chains of antibodies. Of these, one sequence 01-008 was selected for further study based on its non-obvious structural features, degree of identity to the human sequence and other criteria.
  • the heavy chain variable domain was cloned into the vector pEE-HC using Sall/Nhel restriction sites. The assembly quality was assessed by Sanger sequencing. The resulting genetic constructs comprised all the elements necessary for protein expression in eukaryotes.
  • Two primers were constructed for each fragment of the DNA nanomatrix: an ordinary one on one side, and, on the other side, one carrying a sequence (18-25 nucleotides) at the 5' end, complementary to the end of the other fragment. DNA fragments in an equimolar amount and 2 pairs of primers were mixed in the reaction. Amplification was performed in 20-25 cycles. DNA molecules consisting of two fragments were detected by electrophoretic analysis in agarose gel. Then, DNA fragments were isolated from the gel. The resulting fragment was then used in a molecular cloning reaction. The fragments were cloned into the expression vector pEE-HC using Sall/BamHI sites. Genetic constructs were verified by Sanger sequencing.
  • oligonucleotides are hybridized onto each other and combined in a polymerase chain reaction into an entire fragment.
  • the fragment produced in the first round is amplified using flanking primers (22 cycles).
  • amplified are also sequences comprising mutations, i.e. substitutions, insertions and deletions of one or more nucleotides.
  • NEB T7 Endonuclease I
  • a third round is carried out, in which the target sequence is once again amplified using flanking oligonucleotides (20 cycles); then, the fragment can be used in a molecular cloning reaction.
  • the third round is necessary to increase the number of correct sequences.
  • the heavy chain variable domain was cloned into the vector pEE-HC comprising the sequence of the IgGl constant domain using Sall/Nhel restriction sites.
  • the light chain variable domain was cloned into the vector pEE-CK using Sall/BsiWI restriction sites. The assembly quality was verified by Sanger sequencing.
  • the resulting genetic constructs comprised all the elements necessary for protein expression in eukaryotes.
  • Example 5 Generation and purification of antibodies against BDCA-2.
  • variable domains of antibodies were cloned into expression vectors comprising sequences of constant domains of human IgGl according to the standard procedure.
  • the desired overlaps (sequence regions identical for insertion and vector) of 20-30 nucleotide long were added to the 5’ and 3’ ends of the molecule for further molecular cloning by ligase-free methods.
  • the heavy chain variable domain was cloned into the vector pEE-HC comprising the sequence of the IgGl constant domain using Sall/Nhel restriction sites.
  • the resulting vector comprised all the elements necessary for generation of protein in eukaryotic cells (for example, in Chinese hamster ovary (CHO) cells).
  • Figure 1 is a schematic map of such vector.
  • the light chain variable domain was cloned by SLIC into the vector pEE-CK using Sall/BsiWI restriction sites.
  • the resulting vector comprised all the elements necessary for generation of protein in eukaryotic cells (for example, in Chinese hamster ovary (CHO) cells).
  • Figure 2 is a schematic map of such vector.
  • the assembly quality was verified by Sanger sequencing.
  • the resulting genetic constructs comprised all the elements necessary for protein expression in eukaryotes.
  • Antibodies were generated in the CHO-T suspension cell line by transient transfection using the above genetic constructs. Preparation of a cell culture prior to transient transfection and the culturing of transfected cell culture has been described in Example 1.
  • Antibody purification for screening analytical purposes was carried out on a Freedom EVO 150 robotic station (Tecan) using dedicated Protein A columns.
  • the sorbent in the columns was equilibrated with a buffer solution of 50 mmol tris-HCl, 150 mmol NaCl, pH 7.5; clarified culture liquid was applied; the sorbent was washed with a buffer solution of 20 mmol tris-HCl, pH 7.5 and the target fraction was eluted using a buffer solution of 100 mmol glycine, pH 3.5.
  • Antibodies 01-008, 05-001, 05-002, 05-003, 05-004, 05-005, 05-006, 05-007, 05-008, 05-009, 05-010, 05-011, 05-012, 05-016, 05-017, 05-018, 05-019, 05-020, 05-021, 05-022, 05-023, 05-024, 05-025, 05-026 were checked on the Octet (ForteBio) using ProA sensors (ForteBio), AHC (ForteBio) and Anti-hlgG Fc Capture (AHC) (Sartorius).
  • Resutling antibodies were checked using the following antigens: human BDCA-2, cynomolgus monkey BDCA-2, and rhesus macaque BDCA- 2.
  • baseline recording protein immobilization on the sensor surface
  • baseline recording protein immobilization on the sensor surface
  • analyte association recording dissociation recording.
  • the baseline was recorded in a kinetic buffer solution (4.3 mmol Na2HPO4, 136.9 mmol NaCl, 1.5 mmol KH2PO4, 2.7 mmol KC1, 0.1% Tween 20, 0.1% BSA, pH 7.4).
  • Concentration of immobilization protein was 10 pg/ml.
  • Concentration of antigens and antibodies was 10 pg/ml.
  • Antibody 01-008 specifically interacts with human BDCA-2 wit i kD of 4.37 nM.
  • Antibody 01-008 specifically interacts with cynomolgus BDCA-2 with kD of 28 nM.
  • Antibodies 05-001, 05-002, 05-003, 05-004, 05-005, 05-006, 05-007, 05-008, 05-009, 05-010, 05-011, 05-012 specifically interact with human BDCA-2 with nanomolar affinity.
  • Antibody 05-008 specifically interacts with human BDCA-2 with affinity of 74 nM.
  • Table 8 - Kinetic constants for the interaction of antibodies 05-016, 05-017, 05-018, 05-019, 05-020, 05-021, 05-022, 05-023, 05-024, 05-025, 05-026 with human BDCA-2 obtained using Anti- hlgG Fc Capture (AHC) sensors (Sartorius).
  • AHC Anti- hlgG Fc Capture
  • Antibodies 05-016, 05-017, 05-018, 05-019, 05-020, 05-021, 05-022, 05-023, 05-024, 05-025, 05-026 specifically interact with human BDCA-2 with nanomolar affinity.
  • Test antibody specifically interacts with rhesus macaque BDCA-2.
  • Example 7 Evaluation of biological activity of antibodies against BDCA-2 through BDCA-2 + cell line/human pDC binding.
  • BDCA-2 + BDCA-2 +
  • pDCs human plasmocytoid dendritic cells
  • BDCA-2 + or BDCA-2-negative cell line (BDCA-2") or pDC cells isolated by negative magnetic separation from human peripheral blood mononuclear cells (PBMCs) at a concentration of 0.5* 10 6 cells/ml was incubated in a staining buffer (PBS+0.5% BSA) for 20 minutes at +4°C with serial dilutions of test antibodies. After the specified period of time, the cells were washed with a cold (+4°C) staining buffer; bound antibodies were detected by staining with antihuman Fc secondary antibodies labelled with Phycoerythrin for 20 minutes at +4°C. Antibody -free wells and wells with only secondary antibodies were used as controls.
  • a staining buffer PBS+0.5% BSA
  • the cells were washed with a cold (+4°C) staining buffer and resuspended in 100 pl of a cold (+4°C) staining buffer.
  • the samples were then analyzed on a flow cytometer.
  • Forward and side scatter (FSC and SSC) gating was performed to distinguish the target cell population; thereafter, forward scatter height and forward scatter area (FSC-H and FSC-A) gating was performed to identify single (singlet) cells in the population.
  • the median fluorescence intensity was determined for singlet cells gate events.
  • EC50 half-maximal effective concentration
  • Example 8 Study of ability of antibodies to BDCA-2 to induce cell death mediated by activation of FcyRIIIa receptor (CD16 allele variants V/V176 and F/F176) with respect to BDCA-2 + cell line and study of ability of antibodies to BDCA-2 to induce antibody-dependent cellular phagocytosis mediated by activation of FcyRIIA receptor (CD32 allele variant R/R131).
  • Ability of the product to inhibit IFN-I production is due to the internalization of the BDCA- 2 and FcyRIIA receptors, which is achieved through the interaction of a crystallizable fragment of IgGl immunoglobulin with FcyRIIA.
  • Ability of the product to induce antibody-dependent cell death is due to the interaction of the crystallizable fragment of IgGl immunoglobulin with FcyRIIIa.
  • BDCA-2-positive plasmocytoid dendritic cells by human immune cells is undesirable, since it can theoretically increase the susceptibility of patients receiving anti-BDCA-2 therapy to viral infections due to elimination of pDCs, being one of the main antiviral units of the immune system.
  • creation of an effectorless antibody to BDCA-2 which has a low affinity for Fey receptors, in order to reduce adverse events, will lead to a decrease in efficacy of the product also due to decreased affinity for FcyRIIA (CD32) [Pellerin A. et al. Anti-BDCA 2 monoclonal antibody inhibits plasmacytoid dendritic cell activation through Fc-dependent and Fc-independent mechanisms //EMBO molecular medicine, 2015, Vol.7, No.4, P.464-476.].
  • allelic isoforms of FCGR3 A gene differing in amino acid substitution at position 176.
  • allelic variant VI 76 characterized by the presence of valine at position 176 in the second immunoglobulin-like domain, determines the high sensitivity of the receptor to IgG, compared to the variant Fl 76, characterized by the presence of phenylalanine at position 176 [Castro- Dopico T., Clatworthy M.R. IgG and Fey receptors in intestinal immunity and inflammation//Frontiers in Immunology, 2019, Vol. 10],
  • Ability of the antibody to interact with FcyRIIIa and induce antibody-dependent cell death of pDCs can be investigated in a reporter assay by assessing the activation of the FcyRIIIa receptor signaling pathway mediated by the interaction of the antibody with BDCA-2 on the surface of a BDCA-2 + cell line and with FcyRIIIa on the surface of a modified cell line with surface presentation of FcyRIIIa and the luciferase gene under the promoter of the nuclear factor of activated T-cells (NFAT).
  • NFAT nuclear factor of activated T-cells
  • the modified Jurkat FcyRIIIa line was produced based on the Jurkat line (TIB-152, American Type Culture Collection) by transfection with target plasmids using the standard protocol from the Neon system (Neon Electroporation System, Thermo Fisher Scientific).
  • the Jurkat FcyRIIIa cell line carries a luciferase-encoding gene under the control of NFAT-sensitive elements, and has on its surface the CD16 receptor allele variant V176 (Jurkat FcyRIIIa V176), or the CD16 receptor allele variant F176 (Jurkat FcyRIIIa F176).
  • Activation of the FcyRIIIa receptor leads to increased concentration of intracellular calcium and translocation of the NFAT nuclear transcription factor into the nucleus, where NFAT interacts with promoter regions of genes related to the induction of antibody-dependent death of target cells [Oh-hora M., Rao A. The calcium/NFAT pathway: role in development and function of regulatory T cells//Microbes and Infection, 2009, Vol. 11, No. 5, P. 612- 619.].
  • activation of the NFAT-dependent signaling pathway leads to the production of luciferase in the cell.
  • BDCA-2 luciferase-specific substrate
  • the activation of the FcyRIIIa receptor signaling pathway was measured using luminescence detection using the BDCA-2 + cell line or BDCA-2" cell line and the Jurkat FcyRIIIa line (VI 76 or Fl 76).
  • a suspension of BDCA-2 + or BDCA-2" cell lines at a concentration of 0.6* 10 6 cells/ml was introduced into a 96-well white-sided clear plate with flat bottom using a growth medium supplemented with bovine serum with a low content of IgG immunoglobulins (DMEM/F12, PanEco; 2 mM L-glutamine, PanEco; Fetal Bovine Serum ultra-low IgG, Gibco).
  • the plate was incubated at 37°C, 5% CO2 for 12 hours. After incubation, serial dilutions of antibodies and a suspension of Jurkat FcyRIIIa (V176 or F176) cells at a concentration of 6*10 6 cells/ml were added to the wells of the plate. The plate was incubated at 37°C, 5% CO2 for 22 hours.
  • Activation of the FcyRIIIa signaling pathway was measured using a commercial luciferase detection kit in accordance with the manufacturer's protocol (BioGio, Promega). Changes in signal intensity were detected using a spectrophotometer. The half-maximal effective concentration (EC50) was calculated using a four-parameter logistic regression model, the data are shown in Tables 13, 14, 15, 16.
  • test antibody does not activate the NF AT signaling pathway through interaction with the FcyRIIIa receptor on the surface of lurkat FcyRIIIa V176/F176 cells. Accordingly, test antibody does not interact with FcyRIIIa (CD16 allele variant V/V176), which means it reduces the likelihood of adverse events associated with possible pDC depletion.
  • allelic isoforms of FCGR2A gene differing in amino acid substitution at position 131.
  • the allelic variant H131 characterized by the presence of histidine at position 131 in the second immunoglobulin-like domain, determines the high sensitivity of the receptor to IgGl and IgG2, compared to the variant R131 , characterized by the presence of arginine at position 131 [Castro- Dopico T., Clatworthy M.R. IgG and Fey receptors in intestinal immunity and inflammation//Frontiers in Immunology, 2019, Vol. 10.].
  • Ability of the antibody to interact with FcyRIIa and induce antibody-dependent phagocytosis of pDCs can be investigated in a reporter assay by assessing the activation of the FcyRIIa receptor signaling pathway mediated by the interaction of the antibody with BDCA-2 on the surface of a BDCA-2 + cell line and with FcyRIIa on the surface of a modified cell line with surface presentation of FcyRIIa and the luciferase gene under the promoter of the nuclear factor of activated T-cells (NFAT).
  • NFAT nuclear factor of activated T-cells
  • the modified urkat FcyRIIa line was produced based on the urkat line (TIB-152, American Type Culture Collection) by transfection with target plasmids using the standard protocol from the Neon system (Neon Electroporation System, Thermo Fisher Scientific).
  • the urkat FcyRIIa cell line carries a luciferase-encoding gene under the control of NFAT response elements and has on the surface thereof the allelic variant R131 of the FcyRIIa receptor ( urkat FcyRIIIa R131).
  • Activation of the FcyRIIa receptor leads to increased concentration of intracellular calcium and translocation of the NF AT nuclear transcription factor into the nucleus, where NF AT interacts with promoter regions of genes related to the induction of antibody-dependent phagocytosis of target cells [Oh-hora M., Rao A. The calcium/NFAT pathway: role in development and function of regulatory T cells//Microbes and Infection, 2009, Vol. 11, No. 5, P. 612-619.].
  • activation of the NFAT-dependent signaling pathway leads to the production of luciferase in the cell.
  • BDCA-2 luciferase-specific substrate
  • the activation of the FcyRIIa receptor signaling pathway was measured using luminescence detection using the BDCA-2 + cell line or BDCA-2" cell line and the Jurkat FcyRIIa line (R131).
  • a cell suspension of BDCA-2 + cell line at a concentration of 0.6* 10 6 cells/ml was introduced into a 96-well white-sided clear plate with flat bottom using a growth medium supplemented with bovine serum with a low content of IgG immunoglobulins (DMEM/F12, PanEco; 2 mM L-glutamine, PanEco; Fetal Bovine Serum ultra-low IgG, Gibco).
  • the plate was incubated at 37°C, 5% CO2 for 12 hours. After incubation, serial dilutions of antibodies and a suspension of Jurkat FcyRIIa (R131) cells at a concentration of 6*10 6 cells/ml were added to the wells of the plate. The plate was incubated at 37°C, 5% CO2 for 22 hours.
  • EC50 from Table 18 can be extrapolated to other anti-BDCA-2 antibodies of the invention, in particular 01-008, 05-001, 05-002, 05-003, 05-004, 05-005, 05-006, 05-007, 05-009, 05-010, 05-011, 05-012, since the antigen-binding fragment sequences of antibodies 01-008, 05-001, 05-002, 05-003, 05-004, 05-005, 05-006, 05-007, 05-009, 05-010, 05-011, 05-012 are identical to that of 05-008.
  • Example 9 Measurement of biological activity of antibodies against BDCA-2 due to inhibition of IFN-a by plasmocytoid dendritic cells.
  • Oligonucleotide CpG-A (ODN 2216, Invivogen) is an agonist of Toll -like receptor 9 (TLR9 agonist) and activates plasmocytoid dendritic cells (pDCs) isolated by negative magnetic separation or pDCs being part of peripheral blood mononuclear cells (PBMCs), leading to type I IFN production.
  • TLR9 agonist Toll -like receptor 9
  • pDCs plasmocytoid dendritic cells isolated by negative magnetic separation or pDCs being part of peripheral blood mononuclear cells (PBMCs), leading to type I IFN production.
  • PBMCs peripheral blood mononuclear cells
  • Activation using CpG-A reflects the activation mechanism of the TLR9 signaling pathway, independent of the antibody Fc fragment, since the TLR9 agonist is able to enter the cell without internalizing the FcyRIIA Fc receptor (CD32a).
  • pDCs can be activated by immune complexes (IC), which are a complex of anti-Sm/RNP antibodies with Sm/RNP antigen, a ligand associated with the disease, which reflects the mechanism of Fc-dependent activation of Toll-like receptor 7 (TLR7) through the interaction of the Fc fragment of IC with FcyRIIa followed by internalization of the IC complex with FcyRIIa.
  • IC immune complexes
  • TLR7 Toll-like receptor 7
  • the biological activity of antibody 01-008 was measured in a system with isolated pDCs from PBMCs.
  • Peripheral blood mononuclear cells PBMCs
  • PBMCs Peripheral blood mononuclear cells
  • pDCs plasmacytoid dendritic cells
  • a suspension of pDCs with a concentration of 8* 10 5 cells/ml was incubated in serum-free AIM-V medium (ThermoFisher) with the CpG-A activator at a concentration of 1 pM for 10 minutes at 20°C.
  • Test antibodies were diluted in AIM-V medium to a concentration of 10 pg/ml and added to the cell suspension.
  • the resulting solutions were incubated in a plate for 18-20 hours at 37°C in a CO2 incubator, during which time IFN-a was released from the pDCs.
  • the supernatant was collected and enzyme-linked immunosorbent assay (ELISA) was performed.
  • ELISA enzyme-linked immunosorbent assay
  • the antibody against BDCA-2 according to the invention is able to block IFN-a production.
  • IFN-a production was reduced 17-fold ( Figure 3).
  • the biological activity of antibody 05-008 was measured in a system with pDCs being part of PBMCs.
  • PBMCs Peripheral blood mononuclear cells
  • a suspension of PBMCs at a concentration of 20* 10 6 cells/ml was incubated in AIM-V medium with the CpG-A activator at a concentration of 1 pM for 10 minutes at 20°C, or a suspension of PBMCs at a concentration of 48* 10 6 cells/ml was incubated in AIM-V medium with the immune complexes of Table 16 for 90 minutes at 37°C.
  • Test antibodies were serially diluted in AIM-V medium and added to the cell suspension.
  • the resulting solutions were incubated in a plate for 18-20 hours at 37°C in a CO2 incubator, during which time IFN-a was released from pDCs being part of PBMCs. The supernatant was collected and ELISA was performed. The half-maximal inhibitory concentration (IC50) was calculated using a four- parameter logistic regression model. The data are shown in Table 19.
  • Test antibody against BDCA-2 is able to effectively block IFN-a production of different allotypes of the FcyRIIa receptor (CD32a) on the surface of pDCs.
  • Example 10 Ability of antibody to induce BDCA-2 internalization from the surface of human pDCs.
  • PDCs plasmacytoid dendritic cells
  • the cell precipitation procedure was repeated and then the cells were resuspended in a solution with secondary antibodies against human Fc fragment with a phycoerythrin tag for 15 minutes at +4°C. Antibody -free wells and wells with only secondary antibodies were used as controls. After the time, the cells were washed 2 times with cold (+4°C) staining buffer. The samples were then analyzed on a flow cytometer. Forward and side scatter (FSC and SSC) gating was performed to distinguish the target cell population; thereafter, forward scatter height and forward scatter area (FSC-H and FSC-A) gating was performed to identify single (singlet) cells in the population. The median fluorescence intensity was determined for singlet cells gate events.
  • FSC and SSC Forward and side scatter
  • FSC-H and FSC-A forward scatter height and forward scatter area
  • IC50 half-maximal inhibitory concentration
  • antibody 05-008 induces the internalization of BDCA-2 following binding to the target on the surface of human pDCs ( Figure 4).
  • the median fluorescence intensity value is inversely related to the concentration of antibody 05-008.
  • Example 11 Ability of antibodies to BDCA-2 to inhibit IL-6 release by human pDCs.
  • Interleukin-6 (IL-6) is central to the survival and differentiation of plasmablasts, the precursors of plasma cells [Jourdan M. et al. IL-6 supports the generation of human long-lived plasma cells in combination with either APRIL or stromal cell-soluble factors //Leukemia, 2014, Vol.28., No.8., P.1647-1656], In vivo studies in animal models have shown that anti-IL-6 antibodies can lead to a decrease in autoantibody levels.
  • Immune complexes in the human body formed by the interaction of autoantibodies with the human cognate antigens, can activate IL-6 production by pDCs, which, in turn, maintains the level of autoreactive B cells and the expansion of plasmablasts, which then leads to the differentiation and accumulation of autoantibody-producing plasma cells [Davison L. M., Jorgensen T. N. New treatments for systemic lupus erythematosus on the horizon: targeting plasmacytoid dendritic cells to inhibit cytokine production //Journal of clinical & cellular immunology, 2017, Vol.8, No. 6] .
  • PBMCs Peripheral blood mononuclear cells
  • a suspension of pDCs at a concentration of 0.36* 10 6 cells/ml was incubated in AIM-V medium (ThermoFisher) with a CpG-A activator (ODN 2216, Invivogen) at a concentration of 1 pM for 10 minutes at 20°C.
  • Test antibodies were serially diluted in AIM-V medium and added to the cell suspension. The resulting solutions were incubated in a plate for 18-20 hours at 37°C in a CO2 incubator, during which time IL-6 was released from the pDCs. The supernatant was collected and ELISA was performed.
  • antibody 05-008 inhibits the release of interleukin-6 (IL-6) by human pDCs following activation via Fc-independent pathway (Figure 5).
  • Concentration of IL-6 released by human pDCs is inversely related to the concentration of the product.
  • Figure 5 is the results of one-way ANOVA followed by pairwise comparisons of means using Tukey's test (*p-value ⁇ 0.05).
  • Example 12 Results of assessing the ability of antibodies to BDCA-2 to induce cell death mediated by human mononuclear cells carrying the FcyRIIIa receptor (allelic variant F/F176) on surface against BDCA-2 + or BDCA-2" cell lines.
  • Cell death assessment was performed using human PBMCs and BDCA-2 + cell line or BDCA- 2" cell line as negative control.
  • PBMCs were isolated from whole blood of healthy donors by density gradient centrifugation of a polysucrose solution.
  • a suspension of BDCA-2 + or BDCA-2" cell lines at a concentration of 0.6* 10 6 cells/ml using AIM-V medium was added to a 96-well flat-bottom culture plate.
  • the plate was incubated at 37°C in a CO2 incubator for 12 hours.
  • serial dilutions of antibodies and a suspension of human PBMCs at a concentration of 20* 10 6 cells/ml were added to the wells of the plate.
  • the plate was incubated at 37°C in a CO2 incubator for 22 hours.
  • Lactate dehydrogenase (LDH) release was measured. Change in optical density was detected using a spectrophotometer at 492 nm and 690 nm, the main and reference wavelengths, respectively. Cytotoxicity was measured using the formula given in the manufacturer's protocol. The half-maximal effective concentration (EC50) was calculated using a four-parameter logistic regression model.
  • the antibody 05-008 does not cause antibodydependent cell death due to interaction with FcyRIIIa (CD16 allele variant F/F176) on the surface of PBMCs against the BDCA-2 + cell line, hence reduced likelihood of adverse events due to possible depletion of pDCs.
  • Antibody 05-008 does not induce antibody-dependent cell death of the BDCA-2" cell line ( Figure 6).
  • Example 13 Specific activity of antibodies to BDCA-2 in a model of imiquimod-induced psoriasis-like skin inflammation in mice.
  • Anti-inflammatory activity was measured on immunodeficient NSIG mice humanized with human plasmacytoid dendritic cells.
  • a cream containing 5% imiquimod was applied three times to a shaved area of the back skin of mice.
  • the antibody 05-008 product was administered intraperitoneally at a dose of 20 mg/kg 12 hours following the first application of the cream.
  • Animals in the control group received a placebo (buffer solution of 05-008 product).
  • Human plasmacytoid dendritic cells isolated from leukapheresis material were injected into the tail vein of mice at a concentration of 3 x io 5 cells/mouse the day after the first application of the cream.

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Abstract

The present invention relates to the field of biotechnology and medicine, in particular to a monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2. The invention further relates to nucleic acids encoding said antibody, expression vectors, host cells and methods for producing same, methods for producing the antibodies according to the invention, pharmaceutical compositions comprising the antibody according to the invention, pharmaceutical compositions comprising the antibody according to the invention and other therapeutically active compounds, methods for treating BDCA-2-mediated diseases or syndromes, uses of the antibodies or pharmaceutical compositions thereof for treating BDCA-2-mediated diseases or syndromes, and uses of the antibodies and other therapeutically active compounds for treating BDCA-2-mediated diseases or syndromes.

Description

MONOCLONAL ANTIBODY THAT SPECIFICALLY BINDS TO BDCA-2
Field of the invention
The present invention relates to the field of biotechnology and medicine, in particular to a monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 (blood dendritic cell antigen 2). The invention further relates to nucleic acids encoding said antibody, expression vectors, host cells and methods for producing same, methods for producing the antibodies according to the invention, pharmaceutical compositions comprising the antibody according to the invention, pharmaceutical compositions comprising the antibody according to the invention and other therapeutically active compounds, methods for treating BDCA-2-mediated diseases or syndromes, uses of the antibodies or pharmaceutical compositions thereof for treating BDCA-2-mediated diseases or syndromes, and uses of the antibodies and other therapeutically active compounds for treating BDCA-2-mediated diseases or syndromes.
Background of the invention
BDCA-2 (blood dendritic cell antigen 2) is a C-type lectin found on the surface of plasmacytoid dendritic cells (pDCs). It is also known as the C-type lectin domain family 4 member C (CLEC4C) and CD303. BDCA-2 is an inhibitory receptor capable of modulating the function of pDCs, including influencing the production of type I interferons (Dzionek et al. The Journal of experimental medicine, 2001, 194(12), 1823-1834). It also specifically recognizes non-sialylated galactose-terminated biantennary glycans containing the trisaccharide epitope Gal(betal- 3/4)GlcNAc(betal-2)Man and binds to serum IgG (Riboldi et al. Journal of Biological Chemistry, 2011, 286(41), 35329-35333; Jegouzo et al. Journal of Biological Chemistry, 2015, 290(27), 16759- 16771). Furthermore, BDCA-2 can act as a signaling receptor that activates protein tyrosine kinases and mobilizes intracellular calcium (Dzionek et al. The Journal of experimental medicine, 2001, 194(12), 1823-1834).
Plasmocytoid dendritic cells are key cells in innate immunity involved in the fight against viruses and other pathogens. They are producers of type I interferons and cytokines, which have an important antiviral effect and stimulate the adaptive immune response.
At the disease level, abnormal activation or dysfunction of pDCs and associated therewith BDCA-2 can exacerbate many pathological conditions. For example, production of type I interferons by pDCs is associated with some autoimmune diseases, including systemic lupus erythematosus. It is believed that BDCA-2, when bound to an antibody, sends an inhibitory signal to pDCs, reducing the production of type I interferon. With that said, a monoclonal antibody against BDCA-2 is being developed and the efficacy thereof is being investigated for the treatment of systemic lupus erythematosus, including cutaneous systemic lupus erythematosus (Cho et al. Expert Opinion on Investigational Drugs, 2023, 32(5), 345-353; Cho & Furie. Immunotherapy, 2024, 16(1), 15-20).
The role of pDCs and type I interferons has also been studied in connective tissue diseases that can affect the skin and mucous membranes. Increased pDCs have been demonstrated in skin lesions with dermatomyositis, as well as increased IFN-I signaling, which was closely associated with interferon-inducible protein (IP10)/CXCL10 expression and recruitment of potentially autoreactive CXCR3+ lymphocytes (Wenzel et al. Clinical and experimental dermatology, 2006, 31(4), 576-582). In addition to the general cutaneous microscopic findings that are common to systemic lupus erythematosus and dermatomyositis, pDCs have been shown to infiltrate lesional skin in the both diseases (McNiff & Kaplan. Journal of cutaneous pathology, 2008, 35(5), 452-456). pDCs are also known to be involved in the pathogenesis of systemic sclerosis due to their ability to penetrate the skin and directly secrete interferons, interleukin-6 and other proinflammatory chemokines directly or through the IFN-I response in resident cells and, hence, the effect of a monoclonal antibody against BDCA-2 on cells from patients with systemic sclerosis was investigated in vitro (Corinaldesi et al. Arthritis and Rheumatology, 2018, Vol. 70, No. S9).
Neutrophil extracellular traps have been shown to activate pDCs to secrete IFN-a in the skin of patients with hidradenitis suppurativa (Byrd et al. Science translational medicine, 2019, 11(508), eaav5908). Further, the effect of IFN-I present in patients with this disease is primarily limited to the affected skin.
Activated pDCs and IFN-I are known to be involved in proximal events of the innate immunity cascade that triggers psoriatic inflammation (Nestle et al. The Journal of experimental medicine, 2005, 202(1), 135-143). Although pDCs are virtually absent in long-standing psoriatic lesions, pDCs have been shown to infiltrate the skin in the early stages of psoriasis and their presence correlates with the expression of markers typical of the early phases of psoriasis (Albanesi et al. Journal of Experimental Medicine, 2009, 206(1), 249-258).
In the last few years, several studies have shown that IFN-I and pDCs are critical in the development of lichen planus (Wenzel et al. Journal of cutaneous pathology, 2006, 33(10), 672-678; Santoro et al. The Journal of Pathology: A Journal of the Pathological Society of Great Britain and Ireland, 2005, 205(4), 426-434). The demonstration of an increase in pDCs in lichen planus in close association with the recruitment of CXCR3+ granzyme B+ lymphocytes and strong expression of the interferon-inducible protein IP10/CXCL10 suggests that pDCs play a significant role in the development of this disease by way of the recruitment of cytotoxic effector lymphocytes via IP10/CXCR3 interactions (Wenzel et al. Journal of cutaneous pathology, 2006, 33(10), 672-678). pDCs are central component in the inflammatory infiltrate in lichen striatum (linearis), indicating a significant role in the pathogenesis of the disease. pDCs were reported in all individuals with lichen striatum; further, the presence of MxA protein (a surrogate marker of local type I interferon production and, therefore, an indirect assessment of pDC activity) was identical in all patients with lichen planus and lichen striatum (Badr et al. Pediatric Dermatology, 2016, 33(3), 301 - 306).
Patients with atopic dermatitis have been found to have higher levels of circulating pDCs in the blood compared to controls, whereas pDC recruitment to atopic dermatitis lesions is significantly lower compared to other inflammatory dermatoses, such as psoriasis (Tomasini et al. Journal of cutaneous pathology, 2010, 37(11), 1132-1139). Affected skin in this disease had pDCs in close proximity to vessels expressing peripheral nerve addressins, which are physiological ligands of L- selectin, highly expressed by pDCs (Hashizume et al. The Journal of Immunology, 2005, 174(4), 2396-2403).
Study of the presence of pDCs in photoexposed skin in polymorphic light eruption revealed that this cell type may be critical in the development of the disease (Rossi et al. Journal of the European Academy of Dermatology and Venereology, 2018, 32(6), 985-991). Further, the dermal distribution of pDCs in skin biopsies of patients appears to be similar to that of cutaneous systemic lupus erythematosus.
Furthermore, pDCs are a central component in the inflammatory infiltrate in lichenoid parapsoriasis (Karouni et al. Clinical and Experimental Dermatology, 2018, 43(4), 404-409). It is suggested that this disease, analogously to other similar skin diseases, has a pathological process controlled by pDCs through the production of IFN-I, which leads to a cytotoxic attack.
In a study of skin biopsy materials from patients diagnosed with graft-versus-host disease, increased BDCA-2+ cell levels were reported in the skin of patients (Malard et al. Journal of leukocyte biology, 2013, 94(6), 1337-1343). Furthermore, a strong expression of the IFN-inducible protein Mxl was observed in the skin of patients with this disease compared with that in patients without the disease, allowing to suggest that it was the pDCs that produced IFN-I.
On the other hand, the BDCA-2 receptor, like other pDC markers, is considered a potential target for cancer therapy, especially in cases where pDCs are involved in maintaining the tumor microenvironment. For example, malignant proliferation of pDCs is involved in the pathogenesis of some oncohematological diseases, in particular, blastic plasmacytoid dendritic cell neoplasm (BPDCN) and acute myeloid dendritic cell leukemia (Wilson et al. Leukemia & lymphoma, 2022, 63(1), 19-30). pDCs were also reported to be consistently and abundantly present in keratoacanthomas (Abbas et al. Journal of the American Academy of Dermatology, 2014, 70(6), 1142-1145).
Furthermore, pDCs were found in squamous cell and basal cell carcinomas (Pinto et al. Oncoimmunology, 2012, 1(5), 726-734). pDCs significantly influence the immune cascade and can promote both tumor progression and regression. For example, the number of pDCs recruited to imiquimod-treated actinic keratosis significantly correlated with the therapeutic effect, indicating that skin-penetrating pDCs are critically involved in the regression of the disease (Ogawa et al. Journal of Dermatological Science, 2014, 76(1), 67-69). pDC recruitment was also reported to be a recurrent factor in the early stages of melanoma development (Monti et al. Cells, 2020, 9(2), 417). pDC were also reported to influence the development and course of Kaposi's sarcoma (West et al. Journal of virology, 2011, 85(2), 895-904). pDCs are also critical in infectious diseases. Accordingly, pDCs are important in the polarization of antifungal Thl7 cells and can shift the immune balance towards tolerance or inflammation for various mycoses, including chromoblastomycosis, lobomycosis, and paracoccidioidomycosis (Verma et al. Cold Spring Harbor perspectives in medicine, 2015, 5(3), a019612). In addition, BDCA-2+ cells have also been found in patients with leprosy (Hansen disease) (Pinheiro et al. Frontiers in immunology, 2018, 9, 518).
Thus, BDCA-2 is highly significant as a molecular target for the correction of pDC function in various diseases, which fact may lead to a significant increase in the efficacy of the therapy.
Monoclonal antibodies in the form of chimeric, humanized or fully human molecules have proven the value thereof as effective drugs for the treatment of a number of syndromes and diseases.
International application WO2014093396 provides an antibody that specifically binds to BDCA-2.
In connection with the above, there is a need for novel antibodies that specifically bind to BDCA-2.
Description
The authors of the present group of inventions have developed antibodies that specifically bind to BDCA-2 and have high parameters of binding affinity to BDCA-2, including on the surface of the BDCA-2+ cell line. In addition, the antibodies against BDCA-2 of the invention do not activate the NF AT signaling pathway through interaction with the FcyRIIIa receptor, which reduces the likelihood of adverse events, but specifically activate the NF AT signaling pathway through interaction with FcyRIIa, which may indicate increased functional activity thereof in patients carrying the FcyRIIa R/R131 allotype. The antibodies against BDCA-2 of the invention are capable of effectively blocking IFN-a production and induce BDCA-2 internalization following binding to this target on the surface of pDCs. In addition, the antibodies against BDCA-2 of the invention inhibit the release of interleukin-6 by pDCs and do not induce antibody-dependent death of BDCA-2+ and BDCA-2" cell lines. Also, the antibodies against BDCA-2 of the invention demonstrated anti-inflammatory activity in in vivo experiments on the imiquimod-induced psoriasis-like skin inflammation model in mice.
Definitions and general methods
Unless defined otherwise herein, all technical and scientific terms used in connection with the present invention will have the same meaning as is commonly understood by those skilled in the art.
Furthermore, unless otherwise required by context, singular terms shall include plural terms, and the plural terms shall include the singular terms. Typically, the present classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthesis chemistry, medical and pharmaceutical chemistry, as well as hybridization and chemistry of protein and nucleic acids described herein are well known by those skilled and widely used in the art. Enzyme reactions and purification methods are performed according to the manufacturer's guidelines, as is common in the art, or as described herein.
The term "KD" in this description refers to the affinity constant (or equilibrium dissociation constant), which is calculated from the ratio of Kd to Ka (i.e. Kd/Ka), and it is expressed as a molar concentration (M).
"Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g. an antibody) and its binding partner (e.g. an antigen). Unless indicated otherwise, "binding affinity" refers to intrinsic (characteristic, true) binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g. antibody and antigen). The affinity of a molecule X for its binding partner Y can generally be represented by the equilibrium dissociation constant (KD). The preferred Kd value is about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM, or less. Affinity can be measured by common methods known in the art, including those described in the present description. Low- affinity antibodies typically bind an antigen slowly and tend to dissociate readily, whereas high- affinity antibodies typically bind an antigen faster and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, any one of these methods may be used for the purposes of the present invention.
The term "Kd", "koff" or "kdis" refers to the off rate constant of a particular interaction between a binding molecule and antigen. The koff dissociation rate constant can be measured using bio-layer interferometry, for example, using the Octet™ system; as well as by surface plasmon resonance: for example, using the Biacore™ system.
The term "Ka", "kon" or "on-rate" refers to the association rate constant.
The term "in vitro" refers to a biological entity, a biological process, or a biological reaction outside the body under artificial conditions. For example, a cell grown in vitro is to be understood as a cell grown in an environment outside the body, e.g. in a test tube, a culture vial, or a microtiter plate.
The term "ED50" (EC50) (50% effective dose/concentration) refers to concentrations of a formulation producing 50% biological effect (which may include cytoxicity).
Antigen — a protein which the antibody (antibodies) in question specifically binds to.
Genetic construct (plasmid) — an artificially created circular DNA molecule comprising various elements necessary for the expression of target genes and replication within organisms.
Reporter cell line — a cell line carrying a reporter gene for evaluation of intracellular signaling.
Transient production — the production of protein from a temporary producer.
Transfection — a method for DNA delivery where foreign DNA molecules are delivered to a cell using chemical or physical methods: as part of liposomal complexes, as a result of electroporation, and the like.
Chromatography — a method of separation and analysis of mixtures of substances, as well as a study of physico-chemical properties of substances. It is based on distribution of substances between two phases - the stationary phase (solid phase or liquid coated on an inert carrier) and the mobile phase (gas or liquid phase, eluent).
ADCC — antibody-dependent cellular cytotoxicity.
CD16 — cluster of differentiation 16, also FcyRIIIa, has 2 isoforms: V158 (or V176) — high affinity receptor, F158 (or F176) — low affinity receptor.
CD32 — cluster of differentiation 32, also FcyRIIA, has 2 isoforms: H131 — high affinity receptor, R131 — low affinity receptor
CHO — Chinese hamster ovary cell line.
Fc (fragment crystallizable) — constant fragment region of an antibody.
FcyRs — receptors for the immunoglobulin G Fc fragment (receptors for the IgG Fc fragment).
Tris-HCl — buffer solution comprising tri s(hydroxymethyl)aminom ethane and hydrochloric acid (HC1).
As used in the present description and claims that follow, unless otherwise dictated by the context, the words "include" and "comprise", or variations thereof such as "includes", "including", "comprises", or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
Antibody
The present invention relates to a monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2.
The term "monoclonal antibody" or "mAb" refers to an antibody that is synthesized and isolated as an individual clonal population of cells.
The antibody of the invention is a recombinant antibody.
The term "recombinant antibody" refers to an antibody that is expressed in a cell or cell line comprising nucleotide sequence(s) encoding an antibody, wherein said nucleotide sequence(s) is (are) not associated with the cell in nature.
In one aspect, the present invention relates to an isolated monoclonal antibody or antigenbinding fragment thereof that specifically binds to BDCA-2 and comprises:
1) a heavy chain variable domain comprising:
1) CDR1 with the amino acid sequence of SEQ ID NO: 1, ii) CDR2 with the amino acid sequence WIX1PX2SGGSSSPQKFQG, where
Xi = N or I;
X2 = N or W; iii) CDR3 with the amino acid sequence DRDX3LX4GAAFDV, where
X3 = L or W;
X4 = F or I;
2) a light chain variable domain comprising: i) CDR1 with the amino acid sequence GGNNIGXsKXeVH, where
X5 = S or Y;
X6 = S or W or Y; ii) CDR2 with the amino acid sequence of SEQ ID NO: 12, iii) CDR3 with the amino acid sequence QVWDX7SSDHSWV, where
X7 = S or Q or L or W or Y.
In one embodiment of the invention, the antibody according to the invention is an isolated antibody.
The term "isolated" used to describe various antibodies according to the present description refers to an antibody which has been identified and isolated and/or regenerated from a cell or cell culture, in which the antibody is expressed. Impurities (contaminant components) from natural environment are materials which typically interfere with diagnostic or therapeutic uses of the polypeptide, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. The isolated polypeptide is typically prepared by at least one purification step.
The term "antibody" or "immunoglobulin" (Ig) as used in the present description includes whole antibodies. The term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated referred to in the present description as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated referred to in the present description as VL) and light chain constant region. The light chain constant domain can be CK (kappa light chain constant domain) or CL (lambda light chain constant domain). Preferably the light chain is a kappa (K) light chain, and the light chain constant domain is preferably CK.
Antibodies according to the invention may be of any class (e.g., IgA, IgD, IgE, IgG, and IgM, preferably IgG), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2, preferably IgGl).
VL and VH regions may be further subdivided into hyper-variability regions called complementarity determining regions (CDRs), interspersed between regions that are more conserved, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of heavy and light chains form a binding domain that interacts with an antigen.
The constant regions of antibodies may mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (e.g. effector cells) and the first component (Clq) of the classical complement system.
The term "antigen-binding portion" of antibody or "antigen-binding fragment", as used in the present description, refers to one or more antibody fragments that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of antibody can be performed by fragments of a full-length antibody. Examples of binding fragments which are included within the term "antigen-binding portion" of an antibody include (i) Fab-fragment, monovalent fragment, consisting of VL, VH, CL and CHI domains; (ii) F(ab')2 fragment, a bivalent fragment comprising two Fab-fragments linked by a disulfide bridge at the hinge region; (iii) Fd-fragment consisting of VH and CHI domains; (iv) Fv-fragment consisting of VL and VH domains of a single arm of an antibody; (v) dAb-fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH/VHH domain. In addition, two regions of the Fv-fragment, VL and VH, are encoded by different genes, they can be joined using recombinant methods using a synthetic linker that enables to receive them as a single protein chain in which the VL and VH regions are paired to form monovalent molecules (known as a single-chain Fv (scFv); see e.g. Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). It is assumed that such single-stranded molecules are also included within the term "antigen -binding portion" of antibody. Such antibody fragments are produced using conventional techniques known to those skilled in the art, and these fragments are screened in the same manner as intact antibodies are.
"Kabat numbering scheme" or "numbering according to Kabat" as used in the present application refers to the system for numbering of amino acid residues that are more variable (i.e. hypervariable) than other amino acid residues in variable regions of heavy and light chains of antibody (Kabat et al. Ann. N.Y. Acad. Sci., 190:382-93 (1971); Kabat et al. Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991)).
The antibody of the present invention "which specifically binds" to a target antigen refers to an antibody that binds an antigen with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent targeting a protein or cell or tissue expressing the antigen.
The term "specifically binds to" a particular polypeptide or an epitope on a particular target polypeptide may be described by example of a molecule having a Kd for the target of at least about 200 nM, or at least about 150 nM, or at least about 100 nM, or at least about 60 nM, or at least about 50 nM, or at least about 40 nM, or at least about 30 nM, or at least about 20 nM, or at least about 10 nM, or at least about 8 nM, or at least about 6 nM, or at least about 4 nM, or at least about 2 nM, or at least about 1 nM, or at least about 100 pM or less.
In one embodiment, the term "specific binding" refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or epitope on a polypeptide.
In some embodiments of the invention, the isolated monoclonal antibody or antigen-binding fragment thereof includes a heavy chain variable domain that comprises CDR2 with an amino acid sequence that is selected from the group: SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
In some embodiments of the invention, the isolated monoclonal antibody or antigen-binding fragment thereof includes a heavy chain variable domain that comprises CDR3 with an amino acid sequence that is selected from the group: SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.
In some embodiments of the invention, the isolated monoclonal antibody or antigen-binding fragment thereof includes a heavy chain variable domain comprising:
(i) CDR1 with the amino acid sequence of SEQ ID NO: 1;
(ii) CDR2 with an amino acid sequence that is selected from the group: SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; and
(iii) CDR3 with an amino acid sequence that is selected from the group: SEQ ID NO: 5, SEQ
ID NO: 6 or SEQ ID NO: 7.
In some embodiments of the invention, the isolated monoclonal antibody or antigen-binding fragment thereof includes:
(i) a heavy chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 1,
CDR2 with the amino acid sequence of SEQ ID NO: 2 and
CDR3 with the amino acid sequence of SEQ ID NO: 5; or
(ii) a heavy chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 1,
CDR2 with the amino acid sequence of SEQ ID NO: 2 and
CDR3 with the amino acid sequence of SEQ ID NO: 6; or
(iii) a heavy chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 1,
CDR2 with the amino acid sequence of SEQ ID NO: 2 and
CDR3 with the amino acid sequence of SEQ ID NO: 7; or
(iv) a heavy chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 1,
CDR2 with the amino acid sequence of SEQ ID NO: 3 and
CDR3 with the amino acid sequence of SEQ ID NO: 5; or
(v) a heavy chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 1,
CDR2 with the amino acid sequence of SEQ ID NO: 4 and
CDR3 with the amino acid sequence of SEQ ID NO: 5.
In some embodiments of the invention, the isolated monoclonal antibody or antigen-binding fragment thereof includes a light chain variable domain that comprises CDR1 with an amino acid sequence that is selected from the group: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.
In some embodiments of the invention, the isolated monoclonal antibody or antigen-binding fragment thereof includes a light chain variable domain that comprises CDR3 with an amino acid sequence that is selected from the group: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17. In some embodiments of the invention, the isolated monoclonal antibody or antigen-binding fragment thereof includes a light chain variable domain comprising:
(i) CDR1 with an amino acid sequence that is selected from the group: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11;
(ii) CDR2 with the amino acid sequence of SEQ ID NO: 12; and
(iii) CDR3 with an amino acid sequence that is selected from the group: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.
In some embodiments of the invention, the isolated monoclonal antibody or antigen-binding fragment thereof includes:
(i) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 8,
CDR2 with the amino acid sequence of SEQ ID NO: 12 and
CDR3 with the amino acid sequence of SEQ ID NO: 13; or
(ii) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 8,
CDR2 with the amino acid sequence of SEQ ID NO: 12 and
CDR3 with the amino acid sequence of SEQ ID NO: 14 or
(iii) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 8,
CDR2 with the amino acid sequence of SEQ ID NO: 12 and
CDR3 with the amino acid sequence of SEQ ID NO: 15; or
(iv) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 8,
CDR2 with the amino acid sequence of SEQ ID NO: 12 and
CDR3 with the amino acid sequence of SEQ ID NO: 16; or
(v) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 8,
CDR2 with the amino acid sequence of SEQ ID NO: 12 and
CDR3 with the amino acid sequence of SEQ ID NO: 17; or
(vi) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 9,
CDR2 with the amino acid sequence of SEQ ID NO: 12 and
CDR3 with the amino acid sequence of SEQ ID NO: 13; or (vii) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 10,
CDR2 with the amino acid sequence of SEQ ID NO: 12 and
CDR3 with the amino acid sequence of SEQ ID NO: 13; or
(viii) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 11,
CDR2 with the amino acid sequence of SEQ ID NO: 12 and
CDR3 with the amino acid sequence of SEQ ID NO: 13.
In some embodiments of the invention, the isolated monoclonal antibody or antigen-binding fragment thereof includes:
(a) a heavy chain variable domain comprising:
(i) CDR1 with the amino acid sequence of SEQ ID NO: 1;
(ii) CDR2 with an amino acid sequence that is selected from the group: SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; and
(iii) CDR3 with an amino acid sequence that is selected from the group: SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.
(b) a light chain variable domain comprising:
(i) CDR1 with an amino acid sequence that is selected from the group: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11;
(ii) CDR2 with the amino acid sequence of SEQ ID NO: 12; and
(iii) CDR3 with an amino acid sequence that is selected from the group: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.
In some embodiments of the invention, the isolated monoclonal antibody or antigen-binding fragment thereof includes:
(i) (a) a heavy chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 1,
CDR2 with the amino acid sequence of SEQ ID NO: 2 and
CDR3 with the amino acid sequence of SEQ ID NO: 5; and
(b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 8,
CDR2 with the amino acid sequence of SEQ ID NO: 12 and
CDR3 with the amino acid sequence of SEQ ID NO: 13; or (ii) (a) a heavy chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 1,
CDR2 with the amino acid sequence of SEQ ID NO: 2 and
CDR3 with the amino acid sequence of SEQ ID NO: 6; and
(b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 8, CDR2 with the amino acid sequence of SEQ ID NO: 12 and CDR3 with the amino acid sequence of SEQ ID NO: 13; or
(iii) (a) a heavy chain variable domain comprising: CDR1 with the amino acid sequence of SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 2 and CDR3 with the amino acid sequence of SEQ ID NO: 7; and (b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 8, CDR2 with the amino acid sequence of SEQ ID NO: 12 and CDR3 with the amino acid sequence of SEQ ID NO: 13; or
(iv) (a) a heavy chain variable domain comprising: CDR1 with the amino acid sequence of SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 3 and CDR3 with the amino acid sequence of SEQ ID NO: 5; and (b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 8,
CDR2 with the amino acid sequence of SEQ ID NO: 12 and
CDR3 with the amino acid sequence of SEQ ID NO: 13; or
(v) (a) a heavy chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 4 and CDR3 with the amino acid sequence of SEQ ID NO: 5; and (b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 8, CDR2 with the amino acid sequence of SEQ ID NO: 12 and CDR3 with the amino acid sequence of SEQ ID NO: 13; or
(vi) (a) a heavy chain variable domain comprising: CDR1 with the amino acid sequence of SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 2 and CDR3 with the amino acid sequence of SEQ ID NO: 5; and (b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 8, CDR2 with the amino acid sequence of SEQ ID NO: 12 and CDR3 with the amino acid sequence of SEQ ID NO: 14 or
(vii) (a) a heavy chain variable domain comprising: CDR1 with the amino acid sequence of SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 2 and CDR3 with the amino acid sequence of SEQ ID NO: 5; and (b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 8, CDR2 with the amino acid sequence of SEQ ID NO: 12 and CDR3 with the amino acid sequence of SEQ ID NO: 15; or
(viii) (a) a heavy chain variable domain comprising: CDR1 with the amino acid sequence of SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 2 and CDR3 with the amino acid sequence of SEQ ID NO: 5; and (b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 8, CDR2 with the amino acid sequence of SEQ ID NO: 12 and CDR3 with the amino acid sequence of SEQ ID NO: 16; or
(ix) (a) a heavy chain variable domain comprising: CDR1 with the amino acid sequence of SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 2 and CDR3 with the amino acid sequence of SEQ ID NO: 5; and (b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 8, CDR2 with the amino acid sequence of SEQ ID NO: 12 and CDR3 with the amino acid sequence of SEQ ID NO: 17; or
(x) (a) a heavy chain variable domain comprising: CDR1 with the amino acid sequence of SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 2 and CDR3 with the amino acid sequence of SEQ ID NO: 5; and (b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 9,
CDR2 with the amino acid sequence of SEQ ID NO: 12 and
CDR3 with the amino acid sequence of SEQ ID NO: 13; or
(xi) (a) a heavy chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 1,
CDR2 with the amino acid sequence of SEQ ID NO: 2 and
CDR3 with the amino acid sequence of SEQ ID NO: 5; and
(b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 10,
CDR2 with the amino acid sequence of SEQ ID NO: 12 and
CDR3 with the amino acid sequence of SEQ ID NO: 13; or
(xii) (a) a heavy chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 1,
CDR2 with the amino acid sequence of SEQ ID NO: 2 and
CDR3 with the amino acid sequence of SEQ ID NO: 5; and
(b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 11,
CDR2 with the amino acid sequence of SEQ ID NO: 12 and
CDR3 with the amino acid sequence of SEQ ID NO: 13.
In some embodiments of the invention, the isolated monoclonal antibody or antigen-binding fragment thereof includes the heavy chain variable domain comprises an amino acid sequence that is selected from the group: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23.
In some embodiments of the invention, the isolated monoclonal antibody or antigen-binding fragment thereof includes a light chain variable domain comprises an amino acid sequence that is selected from the group: SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31.
In some embodiments of the invention, the isolated monoclonal antibody or antigen-binding fragment thereof includes:
(a) the heavy chain variable domain comprises an amino acid sequence that is selected from the group: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23; and (b) a light chain variable domain comprises an amino acid sequence that is selected from the group: SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31.
In some embodiments of the invention, the isolated monoclonal antibody or antigen-binding fragment thereof includes:
(i) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:
18 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 24; or
(ii) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:
19 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 24; or
(iii) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:
20 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 24; or
(iv) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:
21 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 24; or
(v) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:
22 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 24; or
(vi) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:
23 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 24; or
(vii) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 19 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 25; or
(viii) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 19 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 26; or
(ix) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 19 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 27; or
(x) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:
19 and (b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 28; or
(xi) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:
19 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 29; or
(xii) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 19 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 30; or
(xiii) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 19 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 31.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is a full-length IgG antibody.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is a full-length IgG antibody that is of human IgGl, IgG2, IgG3 or IgG4 isotype.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is a full-length IgG antibody that is of human IgGl isotype.
In some embodiments of the invention, the isolated monoclonal antibody comprises mutations that are selected from the group: i) Q1E; or ii) M252Y, S254T and T256E; or iii) G236A; or iv) I332E and G236A; or v) S239D, I332E and G236A; or vi) Y300L; or vii) D270E; or viii) N325S and L328F; or ix) L235M; or x) S298M; or xi) N325W; or xii) S239F; or xiii) deletion 446G and 447K; or xiv) a combination of i) and any mutation selected from the group comprising ii)-xiii); or xv) a combination of i), any mutation selected from the group comprising ii)-xii) and xiii), according to the EU numbering scheme for amino acids of antibodies (Edelman G.M. et al., Proc. Natl. Acad. Sci. USA 63, 1969, pp. 78- 85).
In some embodiments of the invention, the isolated monoclonal antibody includes a heavy chain comprising an amino acid sequence that is selected from the group: SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 49.
In some embodiments of the invention, the isolated monoclonal antibody includes a light chain comprising an amino acid sequence that is selected from the group: SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 or SEQ ID NO: 57.
In some embodiments of the invention, the isolated monoclonal antibody includes:
(a) a heavy chain comprising an amino acid sequence that is selected from the group: SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 49, and
(b) a light chain comprising the amino acid sequence that is selected from the group: SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 or SEQ ID NO: 57.
In some embodiments of the invention, the isolated monoclonal antibody includes:
(i) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 32 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(ii) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(iii) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(iv) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 35 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(v) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(vi) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 37 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(vii) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(viii) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 39 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(ix) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 40 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(x) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 41 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(xi) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 42 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(xii) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 43 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(xiii) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 44 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(xiv) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 45 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(xv) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 46 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(xvi) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 47 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(xvii) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 48 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or (xviii) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 51; or
(xix) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 52; or
(xx) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 53; or
(xxi) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 54; or
(xxii) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 55; or (xxiii) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 56; or (xxiv) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 57.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is an antibody that is selected from the group: 01-008, 05-001, 05-002, 05-003, 05- 004, 05-005, 05-006, 05-007, 05-008, 05-009, 05-010, 05-011, 05-012, 05-016, 05-017, 05-018, 05- 019, 05-020, 05-021, 05-022, 05-023, 05-024, 05-025 or 05-026.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 01-008.
Antibody 01-008 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 32; and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50.
Antibody 01-008 includes:
(a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 18; and
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 24.
Antibody 01-008 includes:
(a) a heavy chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 5, and
(b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 13.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-001.
Antibody 05-001 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 33; and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50.
Antibody 05-001 includes:
(a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 18; and
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 24.
Antibody 05-001 includes:
(a) a heavy chain variable domain comprising: (i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 5, and
(b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 13.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-002.
Antibody 05-002 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 34; and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50.
Antibody 05-002 includes:
(a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 18; and
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 24.
Antibody 05-002 includes:
(a) a heavy chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 5, and
(b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 13.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-003.
Antibody 05-003 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 35; and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50.
Antibody 05-003 includes:
(a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 18; and
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 24. Antibody 05-003 includes:
(a) a heavy chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 5, and
(b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 13.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-004.
Antibody 05-004 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36; and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50.
Antibody 05-004 includes:
(a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 18; and
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 24.
Antibody 05-004 includes:
(a) a heavy chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 5, and
(b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 13.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-005.
Antibody 05-005 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 37; and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50.
Antibody 05-005 includes: (a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 18; and
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 24.
Antibody 05-005 includes:
(a) a heavy chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 5, and
(b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 13.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-006.
Antibody 05-006 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 38; and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50.
Antibody 05-006 includes:
(a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 18; and
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 24.
Antibody 05-006 includes:
(a) a heavy chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 5, and
(b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 13.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-007.
Antibody 05-007 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 39; and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 50.
Antibody 05-007 includes:
(a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 18; and
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 24.
Antibody 05-007 includes:
(a) a heavy chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 5, and
(b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 13.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-008.
Antibody 05-008 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 40; and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50.
Antibody 05-008 includes:
(a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 18; and
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 24.
Antibody 05-008 includes:
(a) a heavy chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 5, and
(b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 13.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-009. Antibody 05-009 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 41; and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50.
Antibody 05-009 includes:
(a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 18; and
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 24.
Antibody 05-009 includes:
(a) a heavy chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 5, and
(b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 13.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-010.
Antibody 05-010 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 42; and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50.
Antibody 05-010 includes:
(a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 18; and
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 24.
Antibody 05-010 includes:
(a) a heavy chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 5, and
(b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 13. In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-011.
Antibody 05-011 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 43; and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50.
Antibody 05-011 includes:
(a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 18; and
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 24.
Antibody 05-011 includes:
(a) a heavy chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 5, and
(b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 13.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-012.
Antibody 05-012 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 44; and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50.
Antibody 05-012 includes:
(a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 18; and
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 24.
Antibody 05-012 includes:
(a) a heavy chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 5, and
(b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8, (ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 13.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-016.
Antibody 05-016 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 45; and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50.
Antibody 05-016 includes:
(a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 20; and
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 24.
Antibody 05-016 includes:
(a) a heavy chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 6, and
(b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 13.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-017.
Antibody 05-017 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 46; and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50.
Antibody 05-017 includes:
(a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 21; and
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 24.
Antibody 05-017 includes:
(a) a heavy chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 7, and 1 (b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 13.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-018.
Antibody 05-018 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 47; and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50.
Antibody 05-018 includes:
(a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 22; and
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 24.
Antibody 05-018 includes:
(a) a heavy chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 3,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 5, and
(b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 13.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-019.
Antibody 05-019 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 48; and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50.
Antibody 05-019 includes:
(a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 23; and
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 24.
Antibody 05-019 includes:
(a) a heavy chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1, (ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 4,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 5, and
(b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 13.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-020.
Antibody 05-020 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49; and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 51.
Antibody 05-020 includes:
(a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 19; and
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 25.
Antibody 05-020 includes:
(a) a heavy chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 5, and
(b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 9,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 13.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-021.
Antibody 05-021 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49; and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 52.
Antibody 05-021 includes:
(a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 19; and
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 26.
Antibody 05-021 includes: (a) a heavy chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 5, and
(b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 10,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 13.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-022.
Antibody 05-022 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49; and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 53.
Antibody 05-022 includes:
(a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 19; and
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 26.
Antibody 05-022 includes:
(a) a heavy chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 5, and
(b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 11,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 13.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-023.
Antibody 05-023 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49; and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 54.
Antibody 05-023 includes:
(a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 19;
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 26. Antibody 05-023 includes:
(a) a heavy chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 5, and
(b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 14.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-024.
Antibody 05-024 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49; and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 55.
Antibody 05-024 includes:
(a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 19; and
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 26.
Antibody 05-024 includes:
(a) a heavy chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 5, and
(b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 15.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-025.
Antibody 05-025 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49; and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 56.
Antibody 05-025 includes: (a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 19; and
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 26.
Antibody 05-025 includes:
(a) a heavy chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 5, and
(b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 16.
In some embodiments of the invention, the isolated monoclonal antibody that specifically binds to BDCA-2 is antibody 05-026.
Antibody 05-026 includes:
(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49; and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 57.
Antibody 05-026 includes:
(a) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 19; and
(b) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 26.
Antibody 05-026 includes:
(a) a heavy chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 5, and
(b) a light chain variable domain comprising:
(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,
(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 12,
(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 17.
The hypervariable regions of variable domains of light and heavy chains (LCDR1, 2, 3 and HCDR1, 2, 3) of all the above antibodies are provided in accordance with the Kabat nomenclature. Those skilled will appreciate that the hypervariable regions of variable domains of light and heavy chains (LCDR1, 2, 3 and HCDR1, 2, 3) may also be represented in accordance with other commonly known numbering scheme, for example, IMGT, Chothia or AbM. Thus, all of the above antibodies which are characterized by means of hypervariable regions of variable domains of light and heavy chains (LCDR1, 2, 3 and HCDR1, 2, 3) using the IMGT, Chothia or AbM numbering scheme are also encompassed by the present invention.
The antibodies of the invention which specifically bind to BDCA-2 have high binding affinity parameters with respect to BDCA-2, including on the surface of BDCA-2+ cell line. In addition, the antibodies against BDCA-2 of the invention do not activate the NFAT signaling pathway through interaction with the FcyRIIIa receptor, which reduces the likelihood of adverse events, but specifically activate the NFAT signaling pathway through interaction with FcyRIIa, which may indicate increased functional activity thereof in patients carrying the FcyRIIa R/R131 allotype. The antibodies against BDCA-2 of the invention are capable of effectively blocking IFN-a production and induce BDCA-2 internalization following binding to this target on the surface of pDCs. In addition, the antibodies against BDCA-2 of the invention inhibit the release of interleukin-6 by pDCs and do not induce antibody-dependent death of BDCA-2+ and BDCA-2" cell lines. Also, the antibodies against BDCA- 2 of the invention demonstrated anti-inflammatory activity in in vivo experiments on the imiquimod- induced psoriasis-like skin inflammation model in mice.
Nucleic acid molecule
In one aspect, the present invention relates to a nucleic acid that encodes any one of the above antibody or antigen-binding fragment thereof that specifically binds to BDCA-2.
In any one of said embodiments, the nucleic acid molecules may be isolated.
The terms "nucleic acid", "nucleic sequence" or "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence", used interchangeably in the present description, mean a precise sequence of nucleotides, modified or not, determining a fragment or a region of a nucleic acid, containing unnatural nucleotides or not, and being either a double-strand DNA or RNA, a single-strand DNA or RNA, or transcription products of said DNAs.
Unless otherwise indicated, the term nucleotide sequence encompasses its complement. Thus, a nucleic acid having a particular sequence should be understood as one which encompasses the complementary strand thereof with the complementary sequence thereof.
An "isolated" nucleic acid molecule is one which is identified and separated from at least one nucleic acid molecule-impurity. An isolated nucleic acid molecule is different from the form or set in which it is found under natural conditions. Thus, an isolated nucleic acid molecule is different from a nucleic acid molecule that exists in cells under natural conditions. In one aspect, the present invention relates to a nucleic acid molecule comprising a nucleotide sequence that encodes an amino acid sequence selected from SEQ ID NO: 1-57. A nucleic acid molecule may also comprise any combination of said nucleotide sequences.
As would be appreciated by those skilled in the art, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the amino acid sequence of the light chain or heavy chain of the antibody according to the invention or fragments thereof (VH, VL, CDR, etc.). It is well within the skill of those trained in the art to create these alternative DNA sequences encoding one and the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention.
In some embodiments of the invention, the isolated nucleic acid is DNA.
The nucleic acid molecule of the invention may be isolated from any source that produces the monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2. In certain embodiments of the invention, the nucleic acid molecule of the invention may be synthesized by way of chemical synthesis, rather than isolated.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain variable domain of antibodies 01-008, 05-001, 05-002, 05- 003, 05-004, 05-005, 05-006, 05-007, 05-008, 05-009, 05-010, 05-011, 05-012, and includes a nucleotide sequence with SEQ ID NO: 58.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain variable domain of antibodies 05-020, 05-021, 05-022, 05- 023, 05-024, 05-025, 05-026, and includes a nucleotide sequence with SEQ ID NO: 59.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain variable domain of antibody 05-016, and includes a nucleotide sequence with SEQ ID NO: 60.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain variable domain of antibody 05-017, and includes a nucleotide sequence with SEQ ID NO: 61.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain variable domain of antibody 05-018, and includes a nucleotide sequence with SEQ ID NO: 62.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain variable domain of antibody 05-019, and includes a nucleotide sequence with SEQ ID NO: 63. In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain variable domain of antibodies 01-008, 05-001, 05-002, 05- 003, 05-004, 05-005, 05-006, 05-007, 05-008, 05-009, 05-010, 05-011, 05-012, and includes a nucleotide sequence with SEQ ID NO: 64.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain variable domain of antibody 05-020, and includes a nucleotide sequence with SEQ ID NO: 65.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain variable domain of antibody 05-021, and includes a nucleotide sequence with SEQ ID NO: 66.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain variable domain of antibody 05-022, and includes a nucleotide sequence with SEQ ID NO: 67.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain variable domain of antibody 05-023, and includes a nucleotide sequence with SEQ ID NO: 68.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain variable domain of antibody 05-024, and includes a nucleotide sequence with SEQ ID NO: 69.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain variable domain of antibody 05-025, and includes a nucleotide sequence with SEQ ID NO: 70.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain variable domain of antibody 05-026, and includes a nucleotide sequence with SEQ ID NO: 71.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 01-008, and includes a nucleotide sequence with SEQ ID NO: 72.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-001, and includes a nucleotide sequence with SEQ ID NO: 73.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-002, and includes a nucleotide sequence with SEQ ID NO: 74. In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-003, and includes a nucleotide sequence with SEQ ID NO: 75.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-004, and includes a nucleotide sequence with SEQ ID NO: 76.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-005, and includes a nucleotide sequence with SEQ ID NO: 77.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-006, and includes a nucleotide sequence with SEQ ID NO: 78.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-007, and includes a nucleotide sequence with SEQ ID NO: 79.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-008, and includes a nucleotide sequence with SEQ ID NO: 80.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-009, and includes a nucleotide sequence with SEQ ID NO: 81.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-010, and includes a nucleotide sequence with SEQ ID NO: 82.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-011, and includes a nucleotide sequence with SEQ ID NO: 83.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-012, and includes a nucleotide sequence with SEQ ID NO: 84.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-016, and includes a nucleotide sequence with SEQ ID NO: 85. In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-017, and includes a nucleotide sequence with SEQ ID NO: 86.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-018, and includes a nucleotide sequence with SEQ ID NO: 87.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibody 05-019, and includes a nucleotide sequence with SEQ ID NO: 88.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of antibodies 05-020, 05-021, 05-022, 05-023, 05-024, 05- 025, 05-026, and includes a nucleotide sequence with SEQ ID NO: 89.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain of antibodies 01-008, 05-001, 05-002, 05-003, 05-004, 05- 005, 05-006, 05-007, 05-008, 05-009, 05-010, 05-011, 05-012, 05-016, 05-017, 05-018, 05-019, and includes a nucleotide sequence with SEQ ID NO: 90.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain of antibody 05-020, and includes a nucleotide sequence with SEQ ID NO: 91.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain of antibody 05-021, and includes a nucleotide sequence with SEQ ID NO: 92.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain of antibody 05-022, and includes a nucleotide sequence with SEQ ID NO: 93.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain of antibody 05-023, and includes a nucleotide sequence with SEQ ID NO: 94.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain of antibody 05-024, and includes a nucleotide sequence with SEQ ID NO: 95.
In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain of antibody 05-025, and includes a nucleotide sequence with SEQ ID NO: 96. In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain of antibody 05-026, and includes a nucleotide sequence with SEQ ID NO: 97.
The nucleic acid molecules may be used to express the monoclonal antibody or antigenbinding fragment thereof that specifically binds to BDCA-2.
Vector
In one aspect, the present invention relates to an expression vector comprising any one of the above nucleic acid molecules that encode the corresponding amino acid sequences of the antibody that specifically binds to BDCA-2, or portions thereof (for example, heavy chain and/or light chain binding domain sequences). The present invention relates to a vector suitable for the expression of any one of nucleotide sequences described herein.
The term "vector" as used herein means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
As used in the present description, the term "expression" is defined as the transcription and/or translation of a particular nucleotide sequence.
In some embodiments of the invention, the vector is a plasmid, i.e. a circular double stranded piece of DNA into which additional DNA segments may be inserted.
In some embodiments of the invention, the vector is a viral (expression) vector, wherein additional DNA segments may be inserted into the viral genome.
In some embodiments of the invention, the vectors are capable of autonomous replication in a host cell into which they have been introduced (e.g. bacterial vectors having a bacterial site of replication origin and episomal vectors). In further embodiments of the invention, vectors (e.g. non- episomal vectors) may be integrated into the genome of a host cell upon introduction into a host cell, and thereby are replicated along with the host gene. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors").
In some embodiments of the invention, expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses, such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, and the like. DNA molecules may be inserted into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of DNA. An expression vector and expression control sequences may be chosen to be compatible with the expression host cell used.
In one embodiment of the invention, DNA molecules encoding partially or fully heavy and light chain sequences can be inserted into distinct vectors. In one embodiment, any combination of the above DNA molecules is introduced into the same expression vector.
In one embodiment of the invention, DNA molecules may be introduced into an expression vector by standard methods (e.g. ligation of complementary restriction sites on a gene fragment of antibody and vector, or blunt end ligation if no restriction sites are present).
In some embodiments of the invention, a suitable vector is one that includes restriction sites such that any VH or VL sequence can easily be inserted and expressed, as described above. A recombinant expression vector can also encode a signal peptide that facilitates secretion of an antibody chain from a host cell. An antibody chain gene may be cloned into a vector such that the signal peptide is linked in-frame to the N-terminus of an immunoglobulin chain. A signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e. a signal peptide from a non-immunoglobulin protein).
In some embodiments of the invention, the vector may include an expression control sequence. The term "expression control sequence" as used in the present description refers to polynucleotide sequences that are necessary to effect the expression and processing of coding sequences to which they are inserted. It will be understood by those skilled in the art that the design of an expression vector, including the selection of expression control sequences, may depend on such factors as the choice of the type of a host cell to be transformed, the required level of expression of antibody, and so forth. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such expression control sequences differs depending upon the host organism; in prokaryotes, such expression control sequences typically include a promoter, a ribosome binding site, as well as transcription termination sequences; in eukaryotes, such expression control sequences typically include promoters and transcription termination sequences. Preferred expression control sequences for an expression host cell in a mammal include viral elements that ensure high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from a retroviral LTR (long terminal repeat), cytomegalovirus (CMV) (such as a CMV promoter/enhancer), simian virus 40 (SV40) (such as a SV40 promoter/enhancer), adenovirus, (e.g. the major late promoter adenovirus (AdMLP)), polyomavirus and strong mammalian promoters such as TTR promoter, native immunoglobulin promoter or actin promoter. Expression control sequences encompass at least all components whose presence is important for expression and processing. In some embodiments of the invention, in addition to antibody chain genes and expression control sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of a vector in host cells (e.g. origins of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which a vector has been introduced.
Host cell
In one aspect, the present invention relates to a method for producing a host cell to produce any above antibody or antigen-binding fragment thereof that specifically binds to BDCA-2, and includes transformation of the cell with the above vector.
In one aspect, the present invention relates to a host cell for producing any above antibody or antigen-binding fragment thereof that specifically binds to BDCA-2, comprising any one of the above nucleic acids.
The term "host cell" as used herein refers to a cell into which a recombinant expression vector has been introduced. The present invention further relates to host cells that comprise, for example, a nucleotide sequence encoding a heavy chain or antigen-binding portions thereof, a nucleotide sequence encoding a light chain or antigen-binding portions thereof, or both. It should be understood that "host cell" refers not only to a particular subject cell but to the progeny of such cell as well. Since modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to a parental cell; however, such cells are still included within the scope of the term "host cell" as used herein.
Nucleic acid molecules encoding the monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 according to the invention and vectors comprising these nucleic acid molecules may be used for transfection of a mammalian cell, plant cell, bacterial cell, or yeast cell. Transfection may be carried out by any known method for introducing polynucleotides into a host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, cationic polymer-nucleic acid complex transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, encapsulation of the polynucleotides in liposomes, and direct microinjection of DNA into nuclei. In addition, the nucleic acid molecules may be introduced into mammalian cells by viral (expression) vectors.
Mammalian cell lines used as hosts for transformation are well known in the art and include a plurality of immortalized cell lines. These include, e.g., Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HEK-293T cells, FreeStyle 293 cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549, SK-HEP1, HUH7, Hep-RG cells and a number of other cell lines. Cell lines are selected by way of determining which cell lines have high expression levels and provide for necessary characteristics of the protein being produced. Other cell lines that may be used are insect cell lines, such as Sf9 or Sf21 cells. When recombinant expression vectors encoding the monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 are introduced into mammalian host cells, the antibodies or fragments thereof are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibodies or fragments thereof in host cells or, more preferably, secretion of the antibodies or fragments thereof into the culture medium in which the host cells are grown. The monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 may be isolated from culture medium using standard protein purification techniques. Plant host cells include e.g. Nicotiana, Arabidopsis. duckweed, com, wheat, potato, and the like. Bacterial host cells include Escherichia and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe. Saccharomyces cerevisiae and Pichia pastor is.
Furthermore, level of production of the monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 from a production cell line may be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions.
It is likely that the monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 from various cell lines will have a different glycosylation profile as compared to one another. However, the monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 encoded by nucleic acid molecules described herein, or comprising amino acid sequences provided herein are part of the present invention, regardless of the glycosylation of the binding molecules, and, in general, regardless of the presence or absence of post-translational modifications.
The above host cell does not relate to a host cell produced using human embryos.
The above host cell does not relate to a host cell produced by modifying the genetic integrity of human germline cells.
Method for producing antibody
In one aspect, the present invention relates to a method for producing the antibody or antigenbinding fragment thereof that specifically binds to BDCA-2, comprising culturing the above host cell in a growth medium under conditions sufficient to produce said antibody or fragment thereof, followed by isolation and purification of the resulting antibody or fragment thereof.
Pharmaceutical compositions Another aspect of the invention is a pharmaceutical composition comprising, as an active ingredient (or as the only active ingredient), the monoclonal antibody according to the present invention or antigen-binding fragment thereof that specifically binds to BDCA-2.
In one aspect, the present invention relates to a pharmaceutical composition that comprises any above-mentioned antibody or antigen-binding fragment thereof in combination with one or more pharmaceutically acceptable excipients.
In one aspect, the present invention relates to a pharmaceutical composition used for treating a BDCA-2-mediated disease or syndrome, which comprises any above antibody or antigen-binding fragment thereof in combination with one or more pharmaceutically acceptable excipients.
In one aspect, the present invention relates to a pharmaceutical composition used for treating a BDCA-2-mediated disease or syndrome, which comprises any above antibody or antigen-binding fragment thereof in a therapeutically effective amount in combination with one or more pharmaceutically acceptable excipients.
"Pharmaceutical composition" means a composition comprising the antibody according to the invention and at least one of components selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible fillers, solvents, diluents, carriers, auxiliary, distributing and sensing agents, delivery agents.
The term "pharmaceutically acceptable" refers to one or more compatible liquid or solid components that are suitable for administration in a mammal, preferably in a human.
The term "excipient" is used herein to describe any ingredient other than the antibody according to the present invention. These are substances of inorganic or organic nature which are used in the pharmaceutical production/manufacturing in order to give drug products the necessary physicochemical properties.
In some embodiments, the compositions are intended to improve the condition or treat diseases or syndromes that may be mediated by BDCA-2.
The term "disease or syndrome mediated by BDCA-2" refers to any disease or syndrome that is either directly, or indirectly associated with BDCA-2, including pathogenesis, progression, recurrence or chronification of a disease or syndrome.
"Treat", "treatment" and "therapy" refer to a method of alleviating or abrogating a biological disorder and/or at least one of attendant symptoms thereof.
The term "syndrome" means any condition that would benefit from treatment according to the present invention. The definition of the term includes chronic and acute symptoms or diseases including those pathological conditions that predispose a mammal to the impairment in question. "Therapeutically effective amount" refers to that amount of the therapeutic agent being administered during treatment which will relieve to some extent one or more of the symptoms of the disease being treated. A therapeutically effective amount may vary according to factors such as the particular condition being treated, the age, sex and weight of the patient, and whether the monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 is being administered as a stand-alone treatment or in combination with one or more additional drug or treatment.
In one aspect, the subject of treatment, or patient, is a mammal, preferably a human subject. Said subject may be either male or female, of any age.
The pharmaceutical compositions of the present invention and methods of preparation thereof will be undoubtedly apparent to those skilled in the art. The pharmaceutical compositions should preferably be manufactured in compliance with the GMP (Good Manufacturing Practice) requirements.
In some embodiments of the pharmaceutical composition, it may include a buffer composition, tonicity agents (osmolyte or osmotic agent), stabilizers and/or solubilizers.
The pharmaceutical composition according to the invention is a stable composition.
A pharmaceutical composition is "stable" if the active agent retains physical stability and/or chemical stability and/or biological activity thereof during the specified shelf life at a storage temperature, for example, of 2-8 °C. Preferably, the active agent retains both physical and chemical stability, as well as biological activity. Storage period is adjusted based on the results of stability test in accelerated or natural aging conditions.
In some embodiments, the pharmaceutical composition is an injectable dosage form.
In some embodiments, the injectable dosage form is an infusion solution.
In some embodiments, the injectable dosage form is a solution for subcutaneous administration.
Injectable formulations may be manufactured without limitation, in unit dosage form, such as in ampoules, vials, plastic containers, pre-filled syringes, autoinjection devices.
In some embodiments, the pharmaceutical composition is a pharmaceutical composition provided in dry, i.e. powder or granular, form for reconstitution with a suitable solvent (e.g., sterile pyrogen-free water) prior to administration. Such medicinal formulation may be prepared by, for example, lyophilization, i.e. a process, which is known in the art as freeze drying, and which involves freezing a product followed by removal of solvent from frozen material.
In some embodiments, the pharmaceutical composition is a lyophilizate for preparing a solution for infusion. In some embodiments, the pharmaceutical composition is a lyophilizate for preparing a solution for subcutaneous administration.
In some embodiments, the pharmaceutical composition is a concentrate for preparing a solution for infusion.
In some embodiments, the pharmaceutical composition is a concentrate for preparing a solution for subcutaneous administration.
In one aspect, the present invention relates to a pharmaceutical composition that comprises a monoclonal antibody according to the present invention or antigen-binding fragment thereof that specifically binds to BDCA-2 and at least one other therapeutically active compound.
In one aspect, the present invention relates to a pharmaceutical composition for treating a BDCA-2-mediated disease or syndrome, comprising any above antibody or antigen-binding fragment thereof and at least one other therapeutically active compound.
In one aspect, the present invention relates to a pharmaceutical composition comprising any above antibody or antigen-binding fragment thereof and further at least one other therapeutically active compound.
In one aspect, the present invention relates to a pharmaceutical composition for treating a BDCA-2-mediated disease or syndrome, comprising any above antibody or antigen-binding fragment thereof and further at least one other therapeutically active compound.
In one aspect, the present invention relates to a pharmaceutical composition for treating a BDCA-2-mediated disease or syndrome, comprising any above antibody or antigen-binding fragment thereof and at least one other therapeutically active compound, which is an antibody, a small molecule, a hormone therapy agent or a combination thereof.
In some embodiments of the pharmaceutical composition, the other therapeutically active compound is an antibody that specifically binds to CD20.
In some embodiments of the pharmaceutical composition, the antibody that specifically binds to CD20 is divosilimab, rutiximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab, tositumomab, ublituximab, ocaratuzumab, ripertamab, veltuzumab, or zuberitamab.
In some embodiments of the pharmaceutical composition, the BDCA-2-mediated disease or syndrome is selected from the group: systemic lupus erythematosus, cutaneous systemic lupus erythematosus, discoid lupus erythematosus, chronic cutaneous lupus erythematosus, blast tumor of plasmacytoid dendritic cells, acute myeloid dendritic cell leukemia, blastic plasmacytoid dendritic cell neoplasm, progressive systemic sclerosis (systemic scleroderma), dermatomyositis (Wagner disease), psoriasis, hidradenitis suppurativa, lichen planus, lichen linearis (lichen striatum), parapsoriasis lichenoides, tinea versicolor, atopic dermatitis, polymorphic light eruption, polymorphic photodermatitis, actinic keratosis (senile keratosis), graft-versus-host disease, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, melanoma, Kaposi's sarcoma, mycosis, chromoblastomycosis, lobomycosis, paracoccidioidomycosis or leprosy (Hansen disease).
Therapeutic use of monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2
In one aspect, the antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 is used in the treatment of diseases or syndromes mediated by BDCA-2.
In one aspect, the subject of treatment, or patient, is a mammal, preferably a human subject. Said subject may be either male or female, of any age.
In one aspect, the present invention relates to a method for treating a BDCA-2-mediated disease or syndrome, comprising administering in a subject in need of such treatment any above antibody or antigen-binding fragment thereof or said pharmaceutical composition, in a therapeutically effective amount.
In one aspect, the present invention relates to a method for treating a BDCA-2-mediated disease or syndrome, comprising administering in a subject in need of such treatment any above antibody or antigen-binding fragment thereof and at least one other therapeutically active compound in a therapeutically effective amount.
In some embodiments of the method of treatment, the BDCA-2-mediated disease or syndrome is selected from the group: systemic lupus erythematosus, cutaneous systemic lupus erythematosus, discoid lupus erythematosus, chronic cutaneous lupus erythematosus, blast tumor of plasmacytoid dendritic cells, acute myeloid dendritic cell leukemia, blastic plasmacytoid dendritic cell neoplasm, progressive systemic sclerosis (systemic scleroderma), dermatomyositis (Wagner disease), psoriasis, hidradenitis suppurativa, lichen planus, lichen linearis (lichen striatum), parapsoriasis lichenoides, tinea versicolor, atopic dermatitis, polymorphic light eruption, polymorphic photodermatitis, actinic keratosis (senile keratosis), graft-versus-host disease, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, melanoma, Kaposi's sarcoma, mycosis, chromoblastomycosis, lobomycosis, paracoccidioidomycosis or leprosy (Hansen disease).
In some embodiments of the method for treatment, the other therapeutically active compound is an antibody, small molecule, hormone therapy agent, or any combination thereof.
In some embodiments of the method of treatment, the other therapeutically active compound is an antibody that specifically binds to CD20.
In some embodiments of the method of treatment, the antibody that specifically binds to CD20 is divosilimab, rutiximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab, tositumomab, ublituximab, ocaratuzumab, ripertamab, veltuzumab, or zuberitamab. In one aspect, the present invention relates to the use of the above antibody or antigen-binding fragment thereof or the above pharmaceutical composition for treating in a subject in need of such treatment a BDCA-2-mediated disease or syndrome.
In one aspect, the present invention relates to the use of the above antibody or antigen-binding fragment thereof and at least one other therapeutically active compound for treating in a subject in need of such treatment a BDCA-2-mediated disease or syndrome.
In some embodiments of the use, the BDCA-2-mediated disease or syndrome is selected from the group: systemic lupus erythematosus, cutaneous systemic lupus erythematosus, discoid lupus erythematosus, chronic cutaneous lupus erythematosus, blast tumor of plasmacytoid dendritic cells, acute myeloid dendritic cell leukemia, blastic plasmacytoid dendritic cell neoplasm, progressive systemic sclerosis (systemic scleroderma), dermatomyositis (Wagner disease), psoriasis, hidradenitis suppurativa, lichen planus, lichen linearis (lichen striatum), parapsoriasis lichenoides, tinea versicolor, atopic dermatitis, polymorphic light eruption, polymorphic photodermatitis, actinic keratosis (senile keratosis), graft-versus-host disease, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, melanoma, Kaposi's sarcoma, mycosis, chromoblastomycosis, lobomycosis, paracoccidioidomycosis or leprosy (Hansen disease).
In some embodiments of the use, the other therapeutically active compound is an antibody, small molecule, hormone therapy agent, or a combination thereof.
In some embodiments of the use, the other therapeutically active compound is an antibody that specifically binds to CD20.
In some embodiments of the use, the antibody that specifically binds to CD20 is divosilimab, rutiximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab, tositumomab, ublituximab, ocaratuzumab, ripertamab, veltuzumab, or zuberitamab.
The uses or methods used herein relating to the antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 with one or more other therapeutic agents are contemplated to mean, refer to and include the following:
1) simultaneous administration of such combination of the antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 and therapeutic agent to a patient in need of treatment, when such components are formulated together into a single dosage form which releases said components at substantially the same time to said patient,
2) simultaneous administration of such combination of the antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 and therapeutic agent to a patient in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at substantially the same time by said patient, whereupon said components are released at substantially the same time to said patient,
3) sequential administration of such combination of the antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 and therapeutic agent to a patient in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at consecutive times by said patient with a significant time interval between each administration, whereupon said components are released at substantially different times to said patient; and
4) sequential administration of such combination of the antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 and therapeutic agent to a patient in need of treatment, when such components are formulated together into a single dosage form which releases said components in a controlled manner, whereupon they are concurrently, consecutively, or jointly released at the same and/or different times to said patient, where each portion may be administered by either the same or different routes.
The antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 may be administered without further therapeutic treatment, i.e. as an independent therapy.
The antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 and the pharmaceutical composition according to the present invention are suitable for parenteral administration in the form of sterile medicinal products intended for administration into the body of a subject by breaching the integrity of the skin or mucous membranes, bypassing the gastrointestinal tract by means of injection or infusion. In particular, it is contemplated that parenteral administration includes, inter alia, subcutaneous, intraperitoneal, intramuscular, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intrasynovial, transdermal injection or infusion; and kidney dialytic infusion techniques.
In some embodiments of the method of treatment or the use, the antibody or antigen-binding fragment that specifically binds to BDCA-2 or the pharmaceutical composition is administered intravenously.
In some embodiments, intravenous administration is carried out by using infusion, prolonged infusion, or long-lasting continuous infusion.
In some embodiments of the method of treatment or the use, the antibody or antigen-binding fragment that specifically binds to BDCA-2 or the pharmaceutical composition is administered subcutaneously.
In some embodiments, subcutaneous administration is carried out by using subcutaneous injection. In some embodiments of the method for treating or the use, a suitable dose of the monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2 according to the present invention will range from 0.1 to 200 mg/kg.
Brief description of drawings
Figure l is a map of the expression plasmid pEE-HC for the generation of the heavy chain of antibodies.
Figure 2 is a map of the expression plasmid pEE-CK for the generation of the light chain of antibodies.
Figure 3 is a histogram showing the results of assessment of the ability of the antibody 01- 008 to BDCA-2 to inhibit IFN-a release.
The values of IFN-a concentration (pg/ml) are plotted along the ordinate axis. The following groups are plotted along the abscissa axis:
1. pDCs activated by CpG-A
2. pDCs activated by CpG-A supplemented with a solution of antibody 01-008.
Figure 4 is the results of assessment of ability of antibody 05-008 to induce internalization of the BDCA-2 receptor from the surface of human pDCs. Antibody concentrations (pg/ml) are plotted along the abscissa axis. The values of the median fluorescence intensity in a.u. (arbitrary units) are plotted along the ordinate axis. The point of the graph marked with an arrow reflects the effect where there are no antibodies.
■ - a four-parameter logistic regression model describing the induction of BDCA-2 receptor internalization from the surface of human pDCs caused by antibody 05-008.
Figure 5 is the results of assessment of ability of antibody 05-008 to inhibit the release of IL- 6 by human pDCs following activation via Fc-independent pathway.
The values of IL-6 concentration (pg/ml) are plotted along the ordinate axis. The following groups are plotted along the abscissa axis:
1. pDCs activated by CpG-A
2. pDCs
3. pDCs activated by CpG-A supplemented with a solution of antibody 05-008.
Figure 6 is the results of assessment of ability of antibody 05-008 to induce antibodydependent cell death against BDCA-2+ cell line and BDCA-2" cell line, PBMC donor with the FcyRIIIa receptor (allelic variant F/F176).
Antibody concentrations (pg/ml) are plotted along the abscissa axis. Cytotoxicity percentage is plotted along the ordinate axis. The points of the graph marked with an arrow reflect the effect where there are no antibodies.
▼ - four-parameter logistic regression model describing the induction of antibody-dependent cell death against BDCA-2+ cell line caused by antibody 05-008
■ - four-parameter logistic regression model describing the induction of antibody-dependent cell death against BDCA-2" cell line caused by antibody 05-008.
Figure 7 is a histological assessment of inflammatory infiltration of the skin.
Examples
The following examples are provided for better understanding of the invention. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.
Materials and general methods
General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is given in: Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). Amino acids of antibody chains are numbered according to EU numbering (Edelman, G.M., et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85; Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, (1991). Recombinant DNA techniques
Standard methods were used to manipulate DNA as described in Sambrook, J. et al, Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The molecular biological reagents were used according to the manufacturer protocols.
Gene synthesis
Desired gene segments were prepared from oligonucleotides made by chemical synthesis. The gene segments of 300-1400 bp long, flanked by singular restriction sites, were assembled by annealing and ligation of oligonucleotides including PCR amplification and subsequently cloned via the restriction sites. The DNA sequences of the cloned gene fragments were confirmed by DNA sequencing.
DNA sequence determination
DNA sequences were determined by Sanger sequencing.
DNA and protein sequence analysis and sequence data management
The Unipro's UGENE suite version 1.29 and SnapGene version 6.1 were used for sequence creation, mapping, analysis, annotation and illustration.
Expression vectors
To produce the antibodies described herein, we used variants of genetic constructs intended for the expression of a transgene in eukaryotic cells (for example, in Chinese hamster ovary (CHO) cells), as well as for maintaining plasmid copy number in prokaryotic cells (for example, E. coif). In addition to the transgene encoding the sequence of a given antibody, the vectors contained all the elements necessary for the expression of protein in eukaryotic cells, as well as all the elements necessary for maintaining plasmid copy number in prokaryotic cells.
The fusion genes comprising the subject antibody chains as described below were generated by PCR and/or gene synthesis and assembled using known recombinant methods and techniques by connection of the according nucleic acid segments, e.g. using unique restriction sites in the corresponding vectors. The resulting nucleic acid sequences were verified by DNA sequencing. For eukaryotic cell transfections, larger quantities of the plasmids were prepared by preparation thereof from transformed E. coli cultures.
Example 1. Preparation of recombinant BDCA-2.
The sequence encoding the extracellular domain of human, cynomolgus monkey and rhesus macaque BDCA-2 was cloned into the pEE plasmid with various peptide tags for protein generation using the Sall/Notl restriction sites. The required quantities of the plasmid were cultured in E.coli cells and purified using conventional techniques. Protein products of antigens were produced by transient transfection of the CHO-T suspension cell line, according to published protocols [Cytotechnology (2012) 64:613-622], Suspension culture was conducted in flasks on orbital shaker using serum-free media supplemented with 8 mmol L- glutamine and 1 g/1 pluronic 68. Polyethylenimine (PEI) was used as a transfection reagent. DNA/PEI ratio was 1 :3/1 :10. 5-7 days following transfection, the culture liquid was clarified using a filtration system. Target proteins were isolated from culture liquid by affine HPLC.
The recombinant protein comprising a peptide tag at the protein C-terminus was isolated by affinity chromatography.
The purified product was dialized into a storage buffer (membrane pore diameter (kDa) was selected individually taking into account the size of the protein product). If and when necessary, the purified product was concentrated using centrifugal concentrators (membrane pore diameter (kDa) was selected individually taking into account the size of the protein product).
Example 2. Obtaining of sequences of variable domains of antibodies against BDCA-2 using human antibody selection in a naive Fab library.
Human BDCA-2 was immobilized on the surface of specifically treated tubes at a concentration of 20 pg/ml for the first round of selection by phage display (see below), 5 pg/ml for the second round, 2 pg/ml for the third round, 1 pg/ml for the fourth round, 0.5 pg/ml for the fifth round, 0.25 pg/ml for the sixth round, 0.125 pg/ml for the seventh round, and 0.0625 pg/ml for the eighth round.
The plastic was then washed with a phosphate-buffer saline supplemented with 0.1% Tween 20 (PBST) and blocked with a solution of non-fat milk or solution of bovine serum albumin (BSA) in the same phosphate-buffer saline. A naive library of human antibodies was used as a library for selection. To this end, it was diluted in a phosphate-buffer saline supplemented with skimmed milk and an additional neutral protein with peptide tags used in the above BDCA-2 antigens (for screening out non-specifically binding phage particles). Immunoassay test tubes with the immobilized antigen were then incubated with libraries.
The unbound phage particles were removed following multiple washings with a solution of PBST (pH 7.4). Phage particles bound to the antigen were eluted with a buffer glycine HC1 solution (pH 2.2) for 15 minutes while stirring, then the solution was neutralized with 1 M Tris-HCl solution (pH 7.6). E. coli cells were infected with phages, grown in culture medium and used in the next selection round. Polyclonal phage enzyme-linked immunosorbent assay (ELISA) was used for enrichment of libraries against target antigens and assessment of presence of non-specifically binding phage particles. To carry out ELISA, target human BDCA-2 antigens with tags from Example 1 were immobilized on high-absorption plates (Greiner-Bio). The analysis was performed according to the standard ELISA protocol.
By the seventh round of selection on the target BDCA-2 antigen, the polyclonal phage product ELISA showed sufficient enrichment of the library. Phagemid DNA was isolated from E. coli culture according to the manufacturer's protocol (Qiagen). Phagemid DNA was then used to transform electrocompetent E. coli cells, followed by sorting to monoclones and determination of an unambiguous sequence of genetic constructs by Sanger sequencing. As a result of sequencing, a plurality clones was analyzed, which contained 8 unique sequences of ScFv fragments of heavy and light chains of antibodies. Of these, one sequence 01-008 was selected for further study based on its non-obvious structural features, degree of identity to the human sequence and other criteria.
Example 3. Production of antibodies against BDCA-2 with substitutions in antibody Fc fragment.
Mutations were introduced into the Fc fragment of antibody 01-008 using conventional techniques.
Positions of substitutions, name of antibodies comprising these substitutions are shown in OimidKa! HCTOHHHK CCMJIKH He Haimem.
Table 1 - Substitutions introduced into antibody 01-008 to increase biological activity The nucleotide sequence of the heavy chain variable domain of antibody 01-008 was produced by polymerase chain reaction. The phagemid isolated from a monoclone produced following seven rounds of selection was used as a matrix. In the course of sequence synthesis, the desired overlaps (sequence regions identical for insertion and vector) of 20-30 nucleotide long were added to the 5’ and 3’ ends of the molecule for further molecular cloning by ligase and ligase-free methods.
The heavy chain variable domain was cloned into the vector pEE-HC using Sall/Nhel restriction sites. The assembly quality was assessed by Sanger sequencing. The resulting genetic constructs comprised all the elements necessary for protein expression in eukaryotes.
Fragments of genetic constructs from Table 1 encoding the heavy chain of antibody 01-008 with mutations in the Fc region were produced by overlap extension PCR (OE-PCR).
Two primers were constructed for each fragment of the DNA nanomatrix: an ordinary one on one side, and, on the other side, one carrying a sequence (18-25 nucleotides) at the 5' end, complementary to the end of the other fragment. DNA fragments in an equimolar amount and 2 pairs of primers were mixed in the reaction. Amplification was performed in 20-25 cycles. DNA molecules consisting of two fragments were detected by electrophoretic analysis in agarose gel. Then, DNA fragments were isolated from the gel. The resulting fragment was then used in a molecular cloning reaction. The fragments were cloned into the expression vector pEE-HC using Sall/BamHI sites. Genetic constructs were verified by Sanger sequencing.
Example 4. Affinity maturation of antibodies against BDCA-2.
To create mutant antibodies specific for (human) BDCA-2, we conducted a structural analysis based on 3D modeling. PDB 4ZET was selected as the crystal structure of the target (https://www.rcsb.org/structure/4ZET ). Based on the 3D structure of the modeled complex, we produced antibodies with increased antibody affinity and stability of the antigen-antibody complex and generated a library, including variants in which substitutions led to increased stability of the complex and to increased antibody affinity (Table 2).
Table 2 - CDR sequences of antibodies against BDCA-2 produced by affinity maturation
and reverse strands of the DNA site of interest, overlapping each other. The assembly was carried out in 3 stages (rounds). At the first round of assembly, oligonucleotides are hybridized onto each other and combined in a polymerase chain reaction into an entire fragment. At the second round, the fragment produced in the first round is amplified using flanking primers (22 cycles). In the course of the second round, in addition to correctly assembled DNA fragments, amplified are also sequences comprising mutations, i.e. substitutions, insertions and deletions of one or more nucleotides. To reduce the number of mutant sequences, T7 Endonuclease I (NEB) enzyme treatment is performed. Then, a third round is carried out, in which the target sequence is once again amplified using flanking oligonucleotides (20 cycles); then, the fragment can be used in a molecular cloning reaction. The third round is necessary to increase the number of correct sequences.
The heavy chain variable domain was cloned into the vector pEE-HC comprising the sequence of the IgGl constant domain using Sall/Nhel restriction sites. The light chain variable domain was cloned into the vector pEE-CK using Sall/BsiWI restriction sites. The assembly quality was verified by Sanger sequencing. The resulting genetic constructs comprised all the elements necessary for protein expression in eukaryotes.
Example 5. Generation and purification of antibodies against BDCA-2.
Selected sequences of variable domains of antibodies were cloned into expression vectors comprising sequences of constant domains of human IgGl according to the standard procedure. To this end, we generated PCR products containing the genes of the heavy and light chain variable domains of the antibodies. In the course of PCR, the desired overlaps (sequence regions identical for insertion and vector) of 20-30 nucleotide long were added to the 5’ and 3’ ends of the molecule for further molecular cloning by ligase-free methods. The heavy chain variable domain was cloned into the vector pEE-HC comprising the sequence of the IgGl constant domain using Sall/Nhel restriction sites. In some cases, the resulting vector comprised all the elements necessary for generation of protein in eukaryotic cells (for example, in Chinese hamster ovary (CHO) cells). Figure 1 is a schematic map of such vector.
The light chain variable domain was cloned by SLIC into the vector pEE-CK using Sall/BsiWI restriction sites. In some cases, the resulting vector comprised all the elements necessary for generation of protein in eukaryotic cells (for example, in Chinese hamster ovary (CHO) cells). Figure 2 is a schematic map of such vector.
The assembly quality was verified by Sanger sequencing. The resulting genetic constructs comprised all the elements necessary for protein expression in eukaryotes.
Antibodies were generated in the CHO-T suspension cell line by transient transfection using the above genetic constructs. Preparation of a cell culture prior to transient transfection and the culturing of transfected cell culture has been described in Example 1.
Antibody purification for screening analytical purposes was carried out on a Freedom EVO 150 robotic station (Tecan) using dedicated Protein A columns. The sorbent in the columns was equilibrated with a buffer solution of 50 mmol tris-HCl, 150 mmol NaCl, pH 7.5; clarified culture liquid was applied; the sorbent was washed with a buffer solution of 20 mmol tris-HCl, pH 7.5 and the target fraction was eluted using a buffer solution of 100 mmol glycine, pH 3.5.
Purity of resulting molecules was measured using vertical polyacrylamide gel electrophoresis (VEP) according to a conventional technique (Table 3).
Purity of resulting molecules was further measured using the conventional technique of sizeexclusion high-performance liquid chromatography (SE HPLC) (Table 3).
Table 3 - Monomer content in antibody products by VEP and SE HPLC
Example 6. Binding kinetics of antibodies against BDCA-2.
Antibodies 01-008, 05-001, 05-002, 05-003, 05-004, 05-005, 05-006, 05-007, 05-008, 05-009, 05-010, 05-011, 05-012, 05-016, 05-017, 05-018, 05-019, 05-020, 05-021, 05-022, 05-023, 05-024, 05-025, 05-026 were checked on the Octet (ForteBio) using ProA sensors (ForteBio), AHC (ForteBio) and Anti-hlgG Fc Capture (AHC) (Sartorius). Resutling antibodies were checked using the following antigens: human BDCA-2, cynomolgus monkey BDCA-2, and rhesus macaque BDCA- 2. Experiment stages: baseline recording, protein immobilization on the sensor surface, baseline recording, analyte association recording, dissociation recording. The baseline, as well as all other stages of the analysis, was recorded in a kinetic buffer solution (4.3 mmol Na2HPO4, 136.9 mmol NaCl, 1.5 mmol KH2PO4, 2.7 mmol KC1, 0.1% Tween 20, 0.1% BSA, pH 7.4). Concentration of immobilization protein was 10 pg/ml. Concentration of antigens and antibodies was 10 pg/ml.
The resulting data was processed using the Octet Data Analysis (Version 9.0) software and using the 1 : 1 interaction model. Processing results are shown in Tables 4, 5, 6, 7, 8 and 9.
Table 4 - Kinetic constants of interaction of antibody 01-008 with human BDCA-2 obtained using ProA sensors (ForteBio).
Antibody 01-008 specifically interacts with human BDCA-2 wit i kD of 4.37 nM.
Table 5 - Kinetic constants for the interaction of antibody 01-008 with cynomolgus BDCA-2
Antibody 01-008 specifically interacts with cynomolgus BDCA-2 with kD of 28 nM.
Table 6 - Kinetic constants for the interaction of antibodies 05-016, 05-017, 05-018, 05-019, 05-020, 05-021, 05-022, 05-023, 05-024, 05-025, 05-026 with human BDCA-2 obtained using AHC sensors (ForteBio).
Antibodies 05-001, 05-002, 05-003, 05-004, 05-005, 05-006, 05-007, 05-008, 05-009, 05-010, 05-011, 05-012 specifically interact with human BDCA-2 with nanomolar affinity.
Table 7 - Kinetic constants for interaction of antibody 05-008 with rhesus macaque BDCA-2 obtained using AHC sensors (ForteBio).
Antibody 05-008 specifically interacts with human BDCA-2 with affinity of 74 nM. Table 8 - Kinetic constants for the interaction of antibodies 05-016, 05-017, 05-018, 05-019, 05-020, 05-021, 05-022, 05-023, 05-024, 05-025, 05-026 with human BDCA-2 obtained using Anti- hlgG Fc Capture (AHC) sensors (Sartorius).
Antibodies 05-016, 05-017, 05-018, 05-019, 05-020, 05-021, 05-022, 05-023, 05-024, 05-025, 05-026 specifically interact with human BDCA-2 with nanomolar affinity.
Table 9 - Kinetic constants for the interaction of antibodies with rhesus macaque BDCA-2
Test antibody specifically interacts with rhesus macaque BDCA-2.
Example 7. Evaluation of biological activity of antibodies against BDCA-2 through BDCA-2+ cell line/human pDC binding.
The product's ability to bind to BDCA-2-positive cell line (BDCA-2+) or to human plasmocytoid dendritic cells (pDCs) is due to the interaction of the antigen-binding antibody fragment with BDCA-2 receptors on the surface of the cell plasma membrane.
A suspension of BDCA-2+ or BDCA-2-negative cell line (BDCA-2") or pDC cells isolated by negative magnetic separation from human peripheral blood mononuclear cells (PBMCs) at a concentration of 0.5* 106 cells/ml was incubated in a staining buffer (PBS+0.5% BSA) for 20 minutes at +4°C with serial dilutions of test antibodies. After the specified period of time, the cells were washed with a cold (+4°C) staining buffer; bound antibodies were detected by staining with antihuman Fc secondary antibodies labelled with Phycoerythrin for 20 minutes at +4°C. Antibody -free wells and wells with only secondary antibodies were used as controls. After that period of time, the cells were washed with a cold (+4°C) staining buffer and resuspended in 100 pl of a cold (+4°C) staining buffer. The samples were then analyzed on a flow cytometer. Forward and side scatter (FSC and SSC) gating was performed to distinguish the target cell population; thereafter, forward scatter height and forward scatter area (FSC-H and FSC-A) gating was performed to identify single (singlet) cells in the population. The median fluorescence intensity was determined for singlet cells gate events.
The half-maximal effective concentration (EC50) was calculated using a four-parameter logistic regression model, the data are shown in Tables 10, 11, 12.
Table 10 - EC50 values for anti-BDCA-2 antibodies following binding to BDCA-2 on the surface of BDCA-2+ cell line.
Table 11 - EC50 values for anti-BDCA-2 antibodies following binding to BDCA-2 on the surface of BDCA-2" cell line.
Table 12 - EC50 values for anti-BDCA-2 antibodies following binding to BDCA-2 on the surface of human pDCs.
It has been shown that antibodies 01-008 and 05-008 specifically interact with human BDCA- 2 on the surface of the BDCA-2+ cell line with EC50 of 0.06 and 0.08 pg/ml, respectively, and 01- 008 has also been shown to interact with BDCA-2 on the surface of human pDCs with EC50 of 0.43 pg/ml. Taking into account the activity assessment mechanism of Example 7, the EC50 values from Tables 11 and 12 can be mutually extrapolated to other antibodies against BDCA-2 according to the invention, hence identical antigen-binding fragment sequences of said antibodies.
Example 8. Study of ability of antibodies to BDCA-2 to induce cell death mediated by activation of FcyRIIIa receptor (CD16 allele variants V/V176 and F/F176) with respect to BDCA-2+ cell line and study of ability of antibodies to BDCA-2 to induce antibody-dependent cellular phagocytosis mediated by activation of FcyRIIA receptor (CD32 allele variant R/R131).
Ability of the product to inhibit IFN-I production is due to the internalization of the BDCA- 2 and FcyRIIA receptors, which is achieved through the interaction of a crystallizable fragment of IgGl immunoglobulin with FcyRIIA. Ability of the product to induce antibody-dependent cell death is due to the interaction of the crystallizable fragment of IgGl immunoglobulin with FcyRIIIa.
Killing of BDCA-2-positive plasmocytoid dendritic cells (pDCs) by human immune cells is undesirable, since it can theoretically increase the susceptibility of patients receiving anti-BDCA-2 therapy to viral infections due to elimination of pDCs, being one of the main antiviral units of the immune system. However, creation of an effectorless antibody to BDCA-2, which has a low affinity for Fey receptors, in order to reduce adverse events, will lead to a decrease in efficacy of the product also due to decreased affinity for FcyRIIA (CD32) [Pellerin A. et al. Anti-BDCA 2 monoclonal antibody inhibits plasmacytoid dendritic cell activation through Fc-dependent and Fc-independent mechanisms //EMBO molecular medicine, 2015, Vol.7, No.4, P.464-476.].
In humans, there are two allelic isoforms of FCGR3 A gene differing in amino acid substitution at position 176. The allelic variant VI 76, characterized by the presence of valine at position 176 in the second immunoglobulin-like domain, determines the high sensitivity of the receptor to IgG, compared to the variant Fl 76, characterized by the presence of phenylalanine at position 176 [Castro- Dopico T., Clatworthy M.R. IgG and Fey receptors in intestinal immunity and inflammation//Frontiers in Immunology, 2019, Vol. 10],
Ability of the antibody to interact with FcyRIIIa and induce antibody-dependent cell death of pDCs can be investigated in a reporter assay by assessing the activation of the FcyRIIIa receptor signaling pathway mediated by the interaction of the antibody with BDCA-2 on the surface of a BDCA-2+ cell line and with FcyRIIIa on the surface of a modified cell line with surface presentation of FcyRIIIa and the luciferase gene under the promoter of the nuclear factor of activated T-cells (NFAT).
The modified Jurkat FcyRIIIa line was produced based on the Jurkat line (TIB-152, American Type Culture Collection) by transfection with target plasmids using the standard protocol from the Neon system (Neon Electroporation System, Thermo Fisher Scientific). The Jurkat FcyRIIIa cell line carries a luciferase-encoding gene under the control of NFAT-sensitive elements, and has on its surface the CD16 receptor allele variant V176 (Jurkat FcyRIIIa V176), or the CD16 receptor allele variant F176 (Jurkat FcyRIIIa F176). Activation of the FcyRIIIa receptor leads to increased concentration of intracellular calcium and translocation of the NFAT nuclear transcription factor into the nucleus, where NFAT interacts with promoter regions of genes related to the induction of antibody-dependent death of target cells [Oh-hora M., Rao A. The calcium/NFAT pathway: role in development and function of regulatory T cells//Microbes and Infection, 2009, Vol. 11, No. 5, P. 612- 619.]. Thus, activation of the NFAT-dependent signaling pathway leads to the production of luciferase in the cell. Addition of a luciferase-specific substrate, luciferin, leads to the cleavage of the substrate and the appearance of luminescence, the signal intensity is directly proportional to the level of activation of the receptor. A cell line without surface presentation of BDCA-2 (BDCA-2" cell line) is used as a negative control.
Thus, the activation of the FcyRIIIa receptor signaling pathway was measured using luminescence detection using the BDCA-2+ cell line or BDCA-2" cell line and the Jurkat FcyRIIIa line (VI 76 or Fl 76).
A suspension of BDCA-2+ or BDCA-2" cell lines at a concentration of 0.6* 106 cells/ml was introduced into a 96-well white-sided clear plate with flat bottom using a growth medium supplemented with bovine serum with a low content of IgG immunoglobulins (DMEM/F12, PanEco; 2 mM L-glutamine, PanEco; Fetal Bovine Serum ultra-low IgG, Gibco). The plate was incubated at 37°C, 5% CO2 for 12 hours. After incubation, serial dilutions of antibodies and a suspension of Jurkat FcyRIIIa (V176 or F176) cells at a concentration of 6*106 cells/ml were added to the wells of the plate. The plate was incubated at 37°C, 5% CO2 for 22 hours.
Activation of the FcyRIIIa signaling pathway was measured using a commercial luciferase detection kit in accordance with the manufacturer's protocol (BioGio, Promega). Changes in signal intensity were detected using a spectrophotometer. The half-maximal effective concentration (EC50) was calculated using a four-parameter logistic regression model, the data are shown in Tables 13, 14, 15, 16.
Table 13. EC 50 of the antibody against BDCA-2 following induction of antibody-dependent cell death against BDCA-2+ cell line mediated by activation of the FcyRIIIa receptor signaling pathway (CD 16 allele variant V/V176)
Table 14. EC so of the antibody against BDCA-2 following induction of antibody-dependent cell death against BDCA-2+ cell line mediated by activation of the FcyRIIIa receptor signaling pathway (CD 16 allele variant F/F176) Table 15. EC 50 of the antibody against BDCA-2 following induction of antibody-dependent cell death against BDCA-2" cell line mediated by activation of the FcyRIIIa receptor signaling pathway (CD 16 allele variant V/V176)
Table 16. EC so of the antibody against BDCA-2 following induction of antibody-dependent cell death against BDCA-2" cell line mediated by activation of the FcyRIIIa receptor signaling pathway (CD 16 allele variant F/F176)
As a result, it was shown that test antibody does not activate the NF AT signaling pathway through interaction with the FcyRIIIa receptor on the surface of lurkat FcyRIIIa V176/F176 cells. Accordingly, test antibody does not interact with FcyRIIIa (CD16 allele variant V/V176), which means it reduces the likelihood of adverse events associated with possible pDC depletion.
In humans, there are two allelic isoforms of FCGR2A gene differing in amino acid substitution at position 131. The allelic variant H131, characterized by the presence of histidine at position 131 in the second immunoglobulin-like domain, determines the high sensitivity of the receptor to IgGl and IgG2, compared to the variant R131 , characterized by the presence of arginine at position 131 [Castro- Dopico T., Clatworthy M.R. IgG and Fey receptors in intestinal immunity and inflammation//Frontiers in Immunology, 2019, Vol. 10.].
Ability of the antibody to interact with FcyRIIa and induce antibody-dependent phagocytosis of pDCs can be investigated in a reporter assay by assessing the activation of the FcyRIIa receptor signaling pathway mediated by the interaction of the antibody with BDCA-2 on the surface of a BDCA-2+ cell line and with FcyRIIa on the surface of a modified cell line with surface presentation of FcyRIIa and the luciferase gene under the promoter of the nuclear factor of activated T-cells (NFAT).
The modified urkat FcyRIIa line was produced based on the urkat line (TIB-152, American Type Culture Collection) by transfection with target plasmids using the standard protocol from the Neon system (Neon Electroporation System, Thermo Fisher Scientific). The urkat FcyRIIa cell line carries a luciferase-encoding gene under the control of NFAT response elements and has on the surface thereof the allelic variant R131 of the FcyRIIa receptor ( urkat FcyRIIIa R131). Activation of the FcyRIIa receptor leads to increased concentration of intracellular calcium and translocation of the NF AT nuclear transcription factor into the nucleus, where NF AT interacts with promoter regions of genes related to the induction of antibody-dependent phagocytosis of target cells [Oh-hora M., Rao A. The calcium/NFAT pathway: role in development and function of regulatory T cells//Microbes and Infection, 2009, Vol. 11, No. 5, P. 612-619.]. Thus, activation of the NFAT-dependent signaling pathway leads to the production of luciferase in the cell. Addition of a luciferase-specific substrate, luciferin, leads to the cleavage of the substrate and the appearance of luminescence, the signal intensity is directly proportional to the level of activation of the receptor. A cell line without surface presentation of BDCA-2 (BDCA-2" cell line) is used as a negative control.
Thus, the activation of the FcyRIIa receptor signaling pathway was measured using luminescence detection using the BDCA-2+ cell line or BDCA-2" cell line and the Jurkat FcyRIIa line (R131).
A cell suspension of BDCA-2+ cell line at a concentration of 0.6* 106 cells/ml was introduced into a 96-well white-sided clear plate with flat bottom using a growth medium supplemented with bovine serum with a low content of IgG immunoglobulins (DMEM/F12, PanEco; 2 mM L-glutamine, PanEco; Fetal Bovine Serum ultra-low IgG, Gibco). The plate was incubated at 37°C, 5% CO2 for 12 hours. After incubation, serial dilutions of antibodies and a suspension of Jurkat FcyRIIa (R131) cells at a concentration of 6*106 cells/ml were added to the wells of the plate. The plate was incubated at 37°C, 5% CO2 for 22 hours.
Activation of the FcyRIIa signaling pathway was measured using a commercial luciferase detection kit in accordance with the manufacturer's protocol (BioGio, Promega). Changes in signal intensity were detected using a spectrophotometer. The half-maximal effective concentration (EC50) was calculated using a four-parameter logistic regression model, the data are shown in Tables 17, 18.
Table 17. EC50 of anti -BDCA-2 antibodies following induction of antibody-dependent phagocytosis against BDCA-2+ cell line mediated by activation of FcyRIIa receptor (allelic variant R131) Table 18. EC50 of anti-BDCA-2 antibodies following induction of antibody-dependent phagocytosis against BDCA-2" cell line mediated by activation of FcyRIIa receptor (allelic variant R131)
It has been shown that 05-003, 05-004, 05-005, 05-006, 05-008 specifically activate the NF AT signaling pathway through interaction with FcyRIIA on the surface of Jurkat FcyRIIA (R131), which will potentially lead to increased functional activity of said antibodies in patients carrying the FcyRIIA R/R131 allotype.
Taking into account the mechanism for assessing activity described in Example 8, EC50 from Table 18 can be extrapolated to other anti-BDCA-2 antibodies of the invention, in particular 01-008, 05-001, 05-002, 05-003, 05-004, 05-005, 05-006, 05-007, 05-009, 05-010, 05-011, 05-012, since the antigen-binding fragment sequences of antibodies 01-008, 05-001, 05-002, 05-003, 05-004, 05-005, 05-006, 05-007, 05-009, 05-010, 05-011, 05-012 are identical to that of 05-008.
Example 9. Measurement of biological activity of antibodies against BDCA-2 due to inhibition of IFN-a by plasmocytoid dendritic cells.
Oligonucleotide CpG-A (ODN 2216, Invivogen) is an agonist of Toll -like receptor 9 (TLR9 agonist) and activates plasmocytoid dendritic cells (pDCs) isolated by negative magnetic separation or pDCs being part of peripheral blood mononuclear cells (PBMCs), leading to type I IFN production. Activation using CpG-A reflects the activation mechanism of the TLR9 signaling pathway, independent of the antibody Fc fragment, since the TLR9 agonist is able to enter the cell without internalizing the FcyRIIA Fc receptor (CD32a). Also, pDCs can be activated by immune complexes (IC), which are a complex of anti-Sm/RNP antibodies with Sm/RNP antigen, a ligand associated with the disease, which reflects the mechanism of Fc-dependent activation of Toll-like receptor 7 (TLR7) through the interaction of the Fc fragment of IC with FcyRIIa followed by internalization of the IC complex with FcyRIIa.
The biological activity of antibody 01-008 was measured in a system with isolated pDCs from PBMCs. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of donors. Next, plasmacytoid dendritic cells (pDCs) were isolated by negative magnetic separation. A suspension of pDCs with a concentration of 8* 105 cells/ml was incubated in serum-free AIM-V medium (ThermoFisher) with the CpG-A activator at a concentration of 1 pM for 10 minutes at 20°C. Test antibodies were diluted in AIM-V medium to a concentration of 10 pg/ml and added to the cell suspension. The resulting solutions were incubated in a plate for 18-20 hours at 37°C in a CO2 incubator, during which time IFN-a was released from the pDCs. The supernatant was collected and enzyme-linked immunosorbent assay (ELISA) was performed.
The antibody against BDCA-2 according to the invention is able to block IFN-a production. When the antibody was added to pDCs activated by CpG-A, IFN-a production was reduced 17-fold (Figure 3).
The biological activity of antibody 05-008 was measured in a system with pDCs being part of PBMCs.
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy donors by density gradient centrifugation of a polysucrose solution. A suspension of PBMCs at a concentration of 20* 106 cells/ml was incubated in AIM-V medium with the CpG-A activator at a concentration of 1 pM for 10 minutes at 20°C, or a suspension of PBMCs at a concentration of 48* 106 cells/ml was incubated in AIM-V medium with the immune complexes of Table 16 for 90 minutes at 37°C. Test antibodies were serially diluted in AIM-V medium and added to the cell suspension. The resulting solutions were incubated in a plate for 18-20 hours at 37°C in a CO2 incubator, during which time IFN-a was released from pDCs being part of PBMCs. The supernatant was collected and ELISA was performed. The half-maximal inhibitory concentration (IC50) was calculated using a four- parameter logistic regression model. The data are shown in Table 19.
Table 19. IC50 of antibody 05-008 for inhibition of human pDC IFN-a release following activation via Fc-dependent and Fc-independent pathways
Test antibody against BDCA-2 is able to effectively block IFN-a production of different allotypes of the FcyRIIa receptor (CD32a) on the surface of pDCs.
Example 10. Ability of antibody to induce BDCA-2 internalization from the surface of human pDCs.
Ability of anti-BDCA-2 antibodies to induce internalization of the BDCA-2 receptor presented on the surface of plasmacytoid dendritic cells (pDCs) was measured by flow cytometry. PDCs were isolated from whole blood of donors by negative magnetic separation. A suspension of pDCs at a concentration of 0.36* 106 cells/ml was incubated in — AIM-V medium for 60 minutes at +37°C with serial dilutions of test antibodies. After the specified time, the cells were washed with cold (+4°C) staining buffer (PBS + 0.5% BSA). The cell precipitation procedure was repeated and then the cells were resuspended in a solution with secondary antibodies against human Fc fragment with a phycoerythrin tag for 15 minutes at +4°C. Antibody -free wells and wells with only secondary antibodies were used as controls. After the time, the cells were washed 2 times with cold (+4°C) staining buffer. The samples were then analyzed on a flow cytometer. Forward and side scatter (FSC and SSC) gating was performed to distinguish the target cell population; thereafter, forward scatter height and forward scatter area (FSC-H and FSC-A) gating was performed to identify single (singlet) cells in the population. The median fluorescence intensity was determined for singlet cells gate events.
The half-maximal inhibitory concentration (IC50) was calculated using a four-parameter logistic regression model.
According to the resulting experimental data, antibody 05-008 induces the internalization of BDCA-2 following binding to the target on the surface of human pDCs (Figure 4). The median fluorescence intensity value is inversely related to the concentration of antibody 05-008.
Example 11. Ability of antibodies to BDCA-2 to inhibit IL-6 release by human pDCs.
Interleukin-6 (IL-6) is central to the survival and differentiation of plasmablasts, the precursors of plasma cells [Jourdan M. et al. IL-6 supports the generation of human long-lived plasma cells in combination with either APRIL or stromal cell-soluble factors //Leukemia, 2014, Vol.28., No.8., P.1647-1656], In vivo studies in animal models have shown that anti-IL-6 antibodies can lead to a decrease in autoantibody levels.
Immune complexes in the human body, formed by the interaction of autoantibodies with the human cognate antigens, can activate IL-6 production by pDCs, which, in turn, maintains the level of autoreactive B cells and the expansion of plasmablasts, which then leads to the differentiation and accumulation of autoantibody-producing plasma cells [Davison L. M., Jorgensen T. N. New treatments for systemic lupus erythematosus on the horizon: targeting plasmacytoid dendritic cells to inhibit cytokine production //Journal of clinical & cellular immunology, 2017, Vol.8, No. 6] .
If the antibody against BDCA-2 can suppress IL-6 production, it could potentially influence plasma cell differentiation as well as autoreactive antibody production [Jego G. et al. Plasmacytoid dendritic cells induce plasma cell differentiation through type I interferon and interleukin 6 //Immunity, 2003, Vol.19, No.2, P.225-234], Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy donors by density gradient centrifugation of a polysucrose solution (Ficoll-Plaque 1.077 g/ml, GE Healthcare). Next, pDCs were isolated from the PBMCs by negative magnetic separation. A suspension of pDCs at a concentration of 0.36* 106 cells/ml was incubated in AIM-V medium (ThermoFisher) with a CpG-A activator (ODN 2216, Invivogen) at a concentration of 1 pM for 10 minutes at 20°C. Test antibodies were serially diluted in AIM-V medium and added to the cell suspension. The resulting solutions were incubated in a plate for 18-20 hours at 37°C in a CO2 incubator, during which time IL-6 was released from the pDCs. The supernatant was collected and ELISA was performed.
According to the resulting experimental data, antibody 05-008 inhibits the release of interleukin-6 (IL-6) by human pDCs following activation via Fc-independent pathway (Figure 5). Concentration of IL-6 released by human pDCs is inversely related to the concentration of the product. Figure 5 is the results of one-way ANOVA followed by pairwise comparisons of means using Tukey's test (*p-value < 0.05).
Example 12. Results of assessing the ability of antibodies to BDCA-2 to induce cell death mediated by human mononuclear cells carrying the FcyRIIIa receptor (allelic variant F/F176) on surface against BDCA-2+ or BDCA-2" cell lines.
Cell death assessment was performed using human PBMCs and BDCA-2+ cell line or BDCA- 2" cell line as negative control.
PBMCs were isolated from whole blood of healthy donors by density gradient centrifugation of a polysucrose solution. A suspension of BDCA-2+ or BDCA-2" cell lines at a concentration of 0.6* 106 cells/ml using AIM-V medium was added to a 96-well flat-bottom culture plate. The plate was incubated at 37°C in a CO2 incubator for 12 hours. After incubation, serial dilutions of antibodies and a suspension of human PBMCs at a concentration of 20* 106 cells/ml were added to the wells of the plate. The plate was incubated at 37°C in a CO2 incubator for 22 hours.
Lactate dehydrogenase (LDH) release was measured. Change in optical density was detected using a spectrophotometer at 492 nm and 690 nm, the main and reference wavelengths, respectively. Cytotoxicity was measured using the formula given in the manufacturer's protocol. The half-maximal effective concentration (EC50) was calculated using a four-parameter logistic regression model.
According to the resulting experimental data, the antibody 05-008 does not cause antibodydependent cell death due to interaction with FcyRIIIa (CD16 allele variant F/F176) on the surface of PBMCs against the BDCA-2+ cell line, hence reduced likelihood of adverse events due to possible depletion of pDCs. Antibody 05-008 does not induce antibody-dependent cell death of the BDCA-2" cell line (Figure 6). Example 13. Specific activity of antibodies to BDCA-2 in a model of imiquimod-induced psoriasis-like skin inflammation in mice.
Anti-inflammatory activity was measured on immunodeficient NSIG mice humanized with human plasmacytoid dendritic cells.
To induce psoriasis-like inflammation, a cream containing 5% imiquimod was applied three times to a shaved area of the back skin of mice. The antibody 05-008 product was administered intraperitoneally at a dose of 20 mg/kg 12 hours following the first application of the cream. Animals in the control group received a placebo (buffer solution of 05-008 product). Human plasmacytoid dendritic cells isolated from leukapheresis material were injected into the tail vein of mice at a concentration of 3 x io5 cells/mouse the day after the first application of the cream.
Animals were euthanized and skin samples were collected for histological analysis 12 hours following the administration of plasmacytoid dendritic cells.
The results of the histological assessment are shown in Figure 7.
The results of histological analysis showed that the degree of inflammatory infiltration of the skin in the group of animals that received the antibody 05-008 product was significantly lower than that in the control group.

Claims

Claims
1. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to BDCA-2, comprising:
(a) a heavy chain variable domain comprising:
(i) CDR1 with the amino acid sequence of SEQ ID NO: 1;
(ii) CDR2 with an amino acid sequence that is selected from the group: SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; and
(iii) CDR3 with an amino acid sequence that is selected from the group: SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7; and
(b) a light chain variable domain comprising:
(i) CDR1 with an amino acid sequence that is selected from the group: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11;
(ii) CDR2 with the amino acid sequence of SEQ ID NO: 12; and
(iii) CDR3 with an amino acid sequence that is selected from the group: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.
2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising:
(i) (a) a heavy chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 1,
CDR2 with the amino acid sequence of SEQ ID NO: 2 and
CDR3 with the amino acid sequence of SEQ ID NO: 5; and
(b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 8,
CDR2 with the amino acid sequence of SEQ ID NO: 12 and
CDR3 with the amino acid sequence of SEQ ID NO: 13; or
(ii) (a) a heavy chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 1,
CDR2 with the amino acid sequence of SEQ ID NO: 2 and
CDR3 with the amino acid sequence of SEQ ID NO: 6; and
(b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 8, CDR2 with the amino acid sequence of SEQ ID NO: 12 and CDR3 with the amino acid sequence of SEQ ID NO: 13; or
(iii) (a) a heavy chain variable domain comprising: CDR1 with the amino acid sequence of SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 2 and CDR3 with the amino acid sequence of SEQ ID NO: 7; and (b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 8, CDR2 with the amino acid sequence of SEQ ID NO: 12 and CDR3 with the amino acid sequence of SEQ ID NO: 13; or
(iv) (a) a heavy chain variable domain comprising: CDR1 with the amino acid sequence of SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 3 and CDR3 with the amino acid sequence of SEQ ID NO: 5; and (b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 8, CDR2 with the amino acid sequence of SEQ ID NO: 12 and CDR3 with the amino acid sequence of SEQ ID NO: 13; or
(v) (a) a heavy chain variable domain comprising: CDR1 with the amino acid sequence of SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 4 and CDR3 with the amino acid sequence of SEQ ID NO: 5; and (b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 8, CDR2 with the amino acid sequence of SEQ ID NO: 12 and CDR3 with the amino acid sequence of SEQ ID NO: 13; or
(vi) (a) a heavy chain variable domain comprising: CDR1 with the amino acid sequence of SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 2 and CDR3 with the amino acid sequence of SEQ ID NO: 5; and (b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 8, CDR2 with the amino acid sequence of SEQ ID NO: 12 and CDR3 with the amino acid sequence of SEQ ID NO: 14 or (vii) (a) a heavy chain variable domain comprising: CDR1 with the amino acid sequence of SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 2 and CDR3 with the amino acid sequence of SEQ ID NO: 5; and (b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 8, CDR2 with the amino acid sequence of SEQ ID NO: 12 and CDR3 with the amino acid sequence of SEQ ID NO: 15; or
(viii) (a) a heavy chain variable domain comprising: CDR1 with the amino acid sequence of SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 2 and CDR3 with the amino acid sequence of SEQ ID NO: 5; and (b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 8, CDR2 with the amino acid sequence of SEQ ID NO: 12 and CDR3 with the amino acid sequence of SEQ ID NO: 16; or
(ix) (a) a heavy chain variable domain comprising: CDR1 with the amino acid sequence of SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 2 and CDR3 with the amino acid sequence of SEQ ID NO: 5; and (b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 8, CDR2 with the amino acid sequence of SEQ ID NO: 12 and CDR3 with the amino acid sequence of SEQ ID NO: 17; or
(x) (a) a heavy chain variable domain comprising: CDR1 with the amino acid sequence of SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 2 and CDR3 with the amino acid sequence of SEQ ID NO: 5; and (b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 9, CDR2 with the amino acid sequence of SEQ ID NO: 12 and CDR3 with the amino acid sequence of SEQ ID NO: 13; or
(xi) (a) a heavy chain variable domain comprising: CDR1 with the amino acid sequence of SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 2 and
CDR3 with the amino acid sequence of SEQ ID NO: 5; and
(b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 10,
CDR2 with the amino acid sequence of SEQ ID NO: 12 and
CDR3 with the amino acid sequence of SEQ ID NO: 13; or
(xii) (a) a heavy chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 1,
CDR2 with the amino acid sequence of SEQ ID NO: 2 and
CDR3 with the amino acid sequence of SEQ ID NO: 5; and
(b) a light chain variable domain comprising:
CDR1 with the amino acid sequence of SEQ ID NO: 11,
CDR2 with the amino acid sequence of SEQ ID NO: 12 and
CDR3 with the amino acid sequence of SEQ ID NO: 13.
3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable domain comprises an amino acid sequence that is selected from the group: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23.
4. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable domain comprises an amino acid sequence selected from the group: SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31.
5. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein:
(a) the heavy chain variable domain comprises an amino acid sequence that is selected from the group: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23; and
(b) a light chain variable domain comprises an amino acid sequence that is selected from the group: SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31.
6. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein:
(i) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 18 and (b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 24; or
(ii) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 19 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 24; or
(iii) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:
20 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 24; or
(iv) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:
21 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 24; or
(v) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 22 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 24; or
(vi) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 23 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 24; or
(vii) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 19 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 25; or
(viii) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 19 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 26; or
(ix) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 19 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 27; or
(x) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 19 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 28; or
(xi) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 19 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 29; or
(xii) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 19 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 30; or (xiii) (a) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:
19 and
(b) the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 31.
7. The monoclonal antibody according to any one of claims 1-6, wherein the antibody is a full-length IgG antibody, wherein the full-length IgG antibody is of human IgGl, IgG2, IgG3 or IgG4 isotype.
8. The monoclonal antibody according to claim 7, wherein the full-length IgG antibody is of human IgGl isotype.
9. The monoclonal antibody according to claim 8, wherein the antibody comprises mutations that are selected from the group: i) Q1E; or ii) M252Y, S254T and T256E; or iii) G236A; or iv) I332E and G236A; or v) S239D, I332E and G236A; or vi) Y300L; or vii) D270E; or viii) N325S and L328F; or ix) L235M; or x) S298M; or xi) N325W; or xii) S239F; or xiii) deletion 446G and 447K; or xiv) a combination of i) and any mutation selected from the group comprising ii)-xiii); or xv) a combination of i), any mutation selected from the group comprising ii)-xii) and xiii), according to EU numbering scheme of amino acids of antibodies.
10. The monoclonal antibody according to claim 1, comprising a heavy chain comprising an amino acid sequence that is selected from the group: SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 49.
11. The monoclonal antibody according to claim 1, comprising a light chain comprising an amino acid sequence that is selected from the group: SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 or SEQ ID NO: 57.
12. The monoclonal antibody according to claim 1, comprising:
(a) a heavy chain comprising an amino acid sequence that is selected from the group: SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 49, and
(b) a light chain comprising the amino acid sequence that is selected from the group: SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 or SEQ ID NO: 57.
13. The monoclonal antibody according to claim 1, comprising:
(i) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 32 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(ii) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(iii) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(iv) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 35 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(v) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(vi) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 37 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(vii) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(viii) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 39 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(ix) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 40 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(x) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 41 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(xi) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 42 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(xii) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 43 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or (xiii) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 44 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(xiv) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 45 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(xv) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 46 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(xvi) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 47 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(xvii) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 48 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
(xviii) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 51; or
(xix) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 52; or
(xx) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 53; or
(xxi) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 54; or
(xxii) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 55; or
(xxiii) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 56; or
(xxiv) (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49 and
(b) a light chain comprising the amino acid sequence of SEQ ID NO: 57.
14. A nucleic acid that encodes the antibody or antigen-binding fragment thereof according to any one of claims 1-13.
15. An expression vector comprising the nucleic acid according to claim 14.
16. A host cell for producing the antibody or antigen-binding fragment thereof according to any one of claims 1-13, comprising the nucleic acid according to claim 14.
17. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1-13, comprising culturing the host cell according to claim 16 in a growth medium.
18. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-13 in a therapeutically effective amount in combination with one or more pharmaceutically acceptable excipients.
19. The pharmaceutical composition according to claim 18 for treating a BDCA-2-mediated disease or syndrome, wherein the BDCA-2-mediated disease or syndrome is selected from the group: systemic lupus erythematosus, cutaneous systemic lupus erythematosus, discoid lupus erythematosus, chronic cutaneous lupus erythematosus, blast tumor of plasmacytoid dendritic cells, acute myeloid dendritic cell leukemia, blastic plasmacytoid dendritic cell neoplasm, progressive systemic sclerosis, systemic scleroderma, dermatomyositis, Wagner disease, psoriasis, hidradenitis suppurativa, lichen planus, lichen linearis, lichen striatum, parapsoriasis lichenoides, tinea versicolor, atopic dermatitis, polymorphic light eruption, polymorphic photodermatitis, senile keratosis, actinic keratosis, graft- versus-host disease, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, melanoma, Kaposi's sarcoma, mycosis, chromoblastomycosis, lobomycosis, paracoccidioidomycosis, leprosy or Hansen disease.
20. A pharmaceutical composition, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-13 and at least one other therapeutically active compound.
21. The pharmaceutical composition according to claim 20 for treating a BDCA-2-mediated disease or syndrome, wherein the BDCA-2-mediated disease or syndrome is selected from the group: systemic lupus erythematosus, cutaneous systemic lupus erythematosus, discoid lupus erythematosus, chronic cutaneous lupus erythematosus, blast tumor of plasmacytoid dendritic cells, acute myeloid dendritic cell leukemia, blastic plasmacytoid dendritic cell neoplasm, progressive systemic sclerosis, systemic scleroderma, dermatomyositis, Wagner disease, psoriasis, hidradenitis suppurativa, lichen planus, lichen linearis, lichen striatum, parapsoriasis lichenoides, tinea versicolor, atopic dermatitis, polymorphic light eruption, polymorphic photodermatitis, senile keratosis, actinic keratosis, graft- versus-host disease, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, melanoma, Kaposi's sarcoma, mycosis, chromoblastomycosis, lobomycosis, paracoccidioidomycosis, leprosy or Hansen disease.
22. The pharmaceutical composition according to claim 20, wherein the other therapeutically active compound is i) an antibody, a small molecule, a hormone therapy agent or any combination thereof, or ii) an anti-CD20 antibody, or iii) the anti-CD20 antibody that is selected from the group: divosilimab, rutiximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab, tositumomab, ublituximab, ocaratuzumab, ripertamab, veltuzumab, or zuberitamab.
23. Use of the antibody or antigen-binding fragment thereof according to any one of claims
1-13 or the pharmaceutical composition according to any one of claims 18-22 for treating a BDCA-
2-mediated disease or syndrome in a subject in need of such treatment, wherein the BDCA-2 -mediated disease or syndrome is selected from the group: systemic lupus erythematosus, cutaneous systemic lupus erythematosus, discoid lupus erythematosus, chronic cutaneous lupus erythematosus, blast tumor of plasmacytoid dendritic cells, acute myeloid dendritic cell leukemia, blastic plasmacytoid dendritic cell neoplasm, progressive systemic sclerosis, systemic scleroderma, dermatomyositis, Wagner disease, psoriasis, hidradenitis suppurativa, lichen planus, lichen linearis, lichen striatum, parapsoriasis lichenoides, tinea versicolor, atopic dermatitis, polymorphic light eruption, polymorphic photodermatitis, senile keratosis, actinic keratosis, graft-versus-host disease, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, melanoma, Kaposi's sarcoma, mycosis, chromoblastomycosis, lobomycosis, paracoccidioidomycosis, leprosy or Hansen disease.
24. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-13 or the pharmaceutical composition according to any one of claims 18-22 and at least one other therapeutically active compound for treating a BDCA-2-mediated disease or syndrome in a subject in need of such treatment, wherein the BDCA-2-mediated disease or syndrome is selected from the group: systemic lupus erythematosus, cutaneous systemic lupus erythematosus, discoid lupus erythematosus, chronic cutaneous lupus erythematosus, blast tumor of plasmacytoid dendritic cells, acute myeloid dendritic cell leukemia, blastic plasmacytoid dendritic cell neoplasm, progressive systemic sclerosis, systemic scleroderma, dermatomyositis, Wagner disease, psoriasis, hidradenitis suppurativa, lichen planus, lichen linearis, lichen striatum, parapsoriasis lichenoides, tinea versicolor, atopic dermatitis, polymorphic light eruption, polymorphic photodermatitis, senile keratosis, actinic keratosis, graft- versus-host disease, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, melanoma, Kaposi's sarcoma, mycosis, chromoblastomycosis, lobomycosis, paracoccidioidomycosis, leprosy or Hansen disease.
25. The use according to claim 24, wherein the other therapeutically active compound is i) an antibody, a small molecule, a hormone therapy agent or a combination thereof, or ii) an anti-CD20 antibody, or iii) the anti-CD20 antibody that is selected from the group: divosilimab, rutiximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab, tositumomab, ublituximab, ocaratuzumab, ripertamab, veltuzumab, or zuberitamab.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3253413A2 (en) * 2015-02-06 2017-12-13 Kadmon Corporation, LLC Immunomodulatory agents
WO2018227018A1 (en) * 2017-06-07 2018-12-13 Silverback Therapeutics, Inc. Antibody conjugates of immune-modulatory compounds and uses thereof
WO2021023793A1 (en) * 2019-08-05 2021-02-11 Capella Bioscience Ltd Anti bdca-2 antibodies
WO2021142276A1 (en) * 2020-01-12 2021-07-15 Vanderbilt University Human antibodies to rift valley fever virus

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3253413A2 (en) * 2015-02-06 2017-12-13 Kadmon Corporation, LLC Immunomodulatory agents
WO2018227018A1 (en) * 2017-06-07 2018-12-13 Silverback Therapeutics, Inc. Antibody conjugates of immune-modulatory compounds and uses thereof
WO2021023793A1 (en) * 2019-08-05 2021-02-11 Capella Bioscience Ltd Anti bdca-2 antibodies
WO2021142276A1 (en) * 2020-01-12 2021-07-15 Vanderbilt University Human antibodies to rift valley fever virus

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