WO2020024897A1 - 抗btla抗体 - Google Patents

抗btla抗体 Download PDF

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WO2020024897A1
WO2020024897A1 PCT/CN2019/098150 CN2019098150W WO2020024897A1 WO 2020024897 A1 WO2020024897 A1 WO 2020024897A1 CN 2019098150 W CN2019098150 W CN 2019098150W WO 2020024897 A1 WO2020024897 A1 WO 2020024897A1
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seq
antigen
antibody
binding fragment
amino acid
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PCT/CN2019/098150
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English (en)
French (fr)
Inventor
武海
姚剑
姚盛
冯辉
张静
周岳华
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上海君实生物医药科技股份有限公司
苏州君盟生物医药科技有限公司
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Priority to SG11202100649VA priority Critical patent/SG11202100649VA/en
Priority to AU2019315969A priority patent/AU2019315969A1/en
Priority to KR1020217005959A priority patent/KR20210041585A/ko
Priority to CN201980051548.XA priority patent/CN112638943A/zh
Priority to CA3107144A priority patent/CA3107144A1/en
Priority to EP19843756.8A priority patent/EP3831851A4/en
Application filed by 上海君实生物医药科技股份有限公司, 苏州君盟生物医药科技有限公司 filed Critical 上海君实生物医药科技股份有限公司
Priority to JP2021505851A priority patent/JP7469292B2/ja
Priority to BR112021001530-2A priority patent/BR112021001530A2/pt
Publication of WO2020024897A1 publication Critical patent/WO2020024897A1/zh
Priority to US17/164,597 priority patent/US20210246209A1/en
Priority to PH12021550244A priority patent/PH12021550244A1/en
Priority to ZA2021/01177A priority patent/ZA202101177B/en

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    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6845—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • 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
    • 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
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/54—F(ab')2
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/74—Inducing cell proliferation
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to an antibody or an antigen-binding fragment thereof that binds BTLA, and uses thereof. More specifically, the present invention relates to active antibodies that recognize human BTLA and can be used to treat or prevent tumors, infectious diseases, inflammatory, autoimmune diseases, and the like.
  • the prototype T cell co-stimulatory molecule CD28 When interacting with B7.1 or B7.2 on the surface of antigen-presenting cells (APC), the prototype T cell co-stimulatory molecule CD28 sends signals that promote T cell proliferation and differentiation in response to TCR participation, but the CD28 homolog cytotoxic T Lymphocyte antigen-4 (CTLA-4) mediates the inhibition of T cell proliferation and effector function (Chambers et al., Ann. Rev. Immunol., 19: 565-594, 2001; Egen et al., Nature Immunol, 3: 611- 618, 2002).
  • CTLA-4 cytotoxic T Lymphocyte antigen-4
  • B and T lymphocyte attenuators are members of the CD28 family, which also includes CD28, ICOS, CTLA-4, and PD-1. Based on the functional effect of increasing the proliferation of T cells after the addition of monoclonal antibodies, it was found that the original members of the family, CD28 and ICOS, have immune activating effects (Hutloff et al., 1999). BTLA, CTLA-4, and PD-1 are described as negative regulatory proteins. Several in vivo studies have confirmed the inhibitory role of BTLA in lymphocyte responses. BTLA-deficient mice made by Murphy and colleagues (Washington University St. Louis) showed a 3-fold increase in IgG produced in response to T-dependent antigens.
  • T cells and B cells isolated from BTLA - mouse showed a large proliferative response to antigen-receptor stimulation using CD3- and anti-IgM, respectively (Watanabe, 2003).
  • BTLA was found to associate with B cell receptor complexes and with T cell receptors. Consistent with the results of this study, antigen-receptor independent stimulation using ConA (T cells) or LPS (B cells) in BTLA-deficient lymphocytes is not affected, and the use of anti-BTLA antibodies cannot be regulated.
  • BTLA knockout mice have been shown to develop spontaneous autoimmune disease over time and shorten lifespan (Oya, 2008). BTLA knockout mice show increased disease severity in autoimmune encephalomyelitis (EAE) and allergic airway inflammation models, both models rely on T cell activity (Watanabe, 2005; Deppong, 2006).
  • HVEM Herpesvirus entry mediator
  • BTLA Herpesvirus entry mediator
  • CDRs extracellular cysteine-rich regions
  • BTLA and HVEM mainly regulate the functions of T cells and APC through dynamic expression on the cell surface.
  • the binding of BTLA and ligand not only inhibits T cell proliferation and down-regulates the CD25 T cell activation marker, but also inhibits the production of IFN- ⁇ , IL-2, IL-4, and IL-10, but cannot induce apoptosis.
  • Antibodies can be used as therapeutic drugs. Certain antibodies can cause unwanted antibody immunogenicity when used as therapeutic drugs in the body. Because most monoclonal antibodies are derived from rodents, repeated use in humans results in an immune response against a therapeutic antibody (eg, a human anti-mouse antibody or HAMA). This type of immune response results in at least a loss of therapeutic efficacy, and the highest results in a potentially lethal allergic reaction.
  • a therapeutic antibody eg, a human anti-mouse antibody or HAMA.
  • HAMA human anti-mouse antibody
  • One method of reducing the immunogenicity of rodent antibodies involves the production of chimeric antibodies in which mouse variable regions (Fv) are fused to human constant regions (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84 : 3439-43).
  • mice injected with a hybrid of the human variable region and the mouse constant region developed a strong antibody response against the human variable region, suggesting that retention of the complete rodent Fv region
  • CDR loop exchange still cannot uniformly produce antibodies with the same binding properties as the starting antibody.
  • FRs framework residues
  • sequences of known antibodies are already being used, or more generally the sequences of antibodies with known X-ray crystal structures such as antibodies NEW and KOL. See, for example, Jones et al., Supra; Verhoeyen et al., Supra; and Gorman et al., Supra. Exact sequence information for a few humanized constructs has been reported.
  • anti-BTLA antibodies particularly anti-BTLA monoclonal antibodies
  • Such antibodies may preferably have low immunogenicity in human subjects, allowing repeated administrations without adverse immune responses.
  • the present invention relates to one or more anti-human BTLA antibodies or antigen-binding fragments thereof, and the use of said antibodies or antigen-binding fragments thereof in the treatment of diseases.
  • the present invention relates to human BTLA (B - and T - cell attenuator) specifically binds to an isolated antibody or antigen binding fragment thereof, comprising one or more selected from nature:
  • HVEM herpes virus entry mediator
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human BTLA, which comprises at least one selected from SEQ ID NO: 7, 8, 9, 10, 11, 12, 16, 17 , 18, 22, 23, 24, 31, 32, and 33 light chain CDR domains and at least one selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 13, 14, 15, 19, 20 , 21, 25, 26, 27, 28, 29, and 30 heavy chain CDR domains.
  • the present invention relates to an isolated antibody that binds to human BTLA or an antigen-binding fragment thereof, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain CDRs thereof are shown in any one of the following groups A-E:
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human BTLA, the amino acid sequence of CDR1, CDR2, and CDR3 of the heavy chain CDR and the amino acid sequence of CDR1, CDR2, and CDR3 of the light chain CDR
  • the sequence is shown in any of the following I-IX groups:
  • the present invention relates to an isolated antibody that binds to human BTLA or an antigen-binding fragment thereof, comprising a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the light chain variable region is selected From the amino acid sequence shown in SEQ ID NO: 36, 37, 39, 41, 44, 46, 47 or 48, the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO: 34, 35, 38, 40, 42, The amino acid sequence shown in 43 or 45.
  • the present invention relates to an isolated antibody or an antigen-binding fragment thereof that binds to human BTLA.
  • the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 36;
  • the amino acid sequence is shown in SEQ ID NO: 34.
  • the present invention relates to an isolated antibody or an antigen-binding fragment thereof that binds to human BTLA.
  • the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 36;
  • the amino acid sequence is shown in SEQ ID NO: 35.
  • the present invention relates to an isolated antibody or an antigen-binding fragment thereof that binds to human BTLA.
  • the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 37;
  • the amino acid sequence is shown in SEQ ID NO: 34.
  • the present invention relates to an isolated antibody or an antigen-binding fragment thereof that binds to human BTLA.
  • the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 37;
  • the amino acid sequence is shown in SEQ ID NO: 35.
  • the present invention relates to an isolated antibody or an antigen-binding fragment thereof that binds to human BTLA.
  • the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 39;
  • the amino acid sequence is shown in SEQ ID NO: 38.
  • the present invention relates to an isolated antibody or an antigen-binding fragment thereof that binds to human BTLA.
  • the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 41;
  • the amino acid sequence is shown in SEQ ID NO: 40.
  • the present invention relates to an isolated antibody or an antigen-binding fragment thereof that binds to human BTLA.
  • the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 41;
  • the amino acid sequence is shown in SEQ ID NO: 42.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human BTLA.
  • the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 44;
  • the amino acid sequence is shown in SEQ ID NO: 43.
  • the present invention relates to an isolated antibody or an antigen-binding fragment thereof that binds to human BTLA.
  • the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 46;
  • the amino acid sequence is shown in SEQ ID NO: 45.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human BTLA.
  • the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 47;
  • the amino acid sequence is shown in SEQ ID NO: 45.
  • the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human BTLA.
  • the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 48;
  • the amino acid sequence is shown in SEQ ID NO: 45.
  • the isolated antibody or antigen-binding fragment thereof that binds to human BTLA according to the present invention is of the IgG type, and more preferably of the IgG4 subtype.
  • the invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human BTLA is a single-chain Fv antibody; in some embodiments, the antibody or antigen-binding fragment thereof is a Fab antibody; in some embodiments In an embodiment, the antibody or antigen-binding fragment thereof is a Fab 'antibody; in certain embodiments, the antibody or antigen-binding fragment thereof is a (Fab') 2 antibody.
  • the invention relates to an isolated polypeptide comprising a VL domain or a VH domain of any of the antibodies or antigen-binding fragments thereof described herein.
  • the invention relates to an isolated nucleic acid encoding a VL domain and a VH domain of any antibody or antigen-binding fragment described herein.
  • the invention relates to a composition
  • a composition comprising one or more antibodies or antigen-binding fragments thereof described herein and a pharmaceutically acceptable carrier or diluent.
  • the present invention relates to a method for preventing or treating a disease by eliminating, inhibiting or reducing BTLA activity using one or more antibodies or antigen-binding fragments thereof described herein, comprising providing to a subject in need thereof A therapeutically effective amount of an antibody or antigen-binding fragment, nucleic acid, expression vector, host cell, immunoconjugate, or pharmaceutical composition disclosed herein is administered.
  • the prevention or treatment of said disease or condition benefits from the elimination, inhibition or reduction of BTLA activity; preferably, said disease or lesion is selected from cancer, infectious disease or inflammatory disease.
  • the invention also relates to the use of said antibody or antigen-binding fragment, nucleic acid, expression vector, host cell, immunoconjugate or pharmaceutical composition for the manufacture of a medicament for the treatment or prevention of a disease or disorder
  • the said disease or condition is preferably a BTLA-mediated disease, which can be selected from cancer, infectious disease or inflammatory disease.
  • Figure 1 SDS-PAGE electrophoresis image of human BTLA extracellular domain protein.
  • Figure 2 Flow cytometry to detect the binding capacity of BTLA and HVEM.
  • Figure 3 ELISA experiment of chimeric antibody binding to human BTLA.
  • Figure 4 The ability of chimeric antibodies to block BTLA-HVEM binding.
  • Figure 5 Effect of chimeric antibodies on T cell activity.
  • Figure 6 Specific binding experiments of humanized antibodies to BTLA.
  • Figure 7 Binding experiments of humanized antibodies to hBTLA on 293F cells.
  • Figure 8 Experiment of humanized antibodies blocking BTLA binding to HVEM on the cell surface.
  • Figure 9 Experiment of humanized antibodies promoting T cell activation.
  • Figure 10 Comparative experiment of humanized antibody 17 binding to BTLA of different species.
  • Figure 11 Effect of hu18 on tumor volume of MC38-hHVEM cell transplanted B-hBTLA mice.
  • Figure 12 Effect of test article on body weight of B-hBTLA mice transplanted with MC38-hHVEM cells.
  • the mammalian immune system has developed several pathways to control the potentially harmful activities of T and B lymphocytes. They include various cytokine-receptor pathways and costimulatory pathways involving receptors like CD28, CTLA-4, PD-1, and BTLA. Although the CD28-B7 interaction is an example of a positive co-stimulatory pathway (ie, CD28 triggers an increase in T cell response to antigen-specific triggers), the other three receptors appear to be inhibitory co-stimulatory pathways.
  • CTLA-4, PD-1, and BTLA show overlapping but unique expression profiles and limit the activity of T and B lymphocytes and other immune cells (see Deppong et al., JImmunol 2006; Tao et al., J Immunol 2005).
  • CTLA-4 competes with CD28 for binding to B7.1 and B7.2 (CD80 and CD86) and sets primitive thresholds for naive T cell activation in lymph nodes and spleen
  • PD-1 and BTLA each have their own unique ligands ( PD-L1 / -L2 and HVEM, respectively), and seem to control peripheral T cell homeostasis and reactivation (see Krieg et al., Nat Immunol 2007).
  • BTLA B and T lymphocyte attenuators
  • BTLA B and T lymphocyte attenuators
  • BTLA is a type I transmembrane glycoprotein with a cytoplasmic tail containing several tyrosine-inhibitory motifs (Watanabe, 2003).
  • BTLA has some structural similarity to members of the CD28 / CTLA-4 family, but it has unique properties.
  • BTLA is associated with a variety of immune, inflammatory and proliferative diseases. Therefore, the development of monoclonal antibody drugs capable of blocking human BTLA-HVEM binding is a continuing need for disease treatment.
  • activation As used herein, “activation”, “stimulation”, and “treatment” for a cell or receptor may have the same meaning, such as activation, stimulation, or treatment of a cell or receptor with a ligand, unless the context indicates otherwise.
  • Ligand includes natural and synthetic ligands, such as cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies.
  • Ligand also includes small molecules, such as peptide mimetics of cytokines and peptide mimetics of antibodies.
  • Activation may refer to cell activation regulated by internal mechanisms as well as external or environmental factors.
  • Response / response such as a response of a cell, tissue, organ, or organism, including changes in biochemical or physiological behavior (such as concentration, density, adhesion or migration, rate of gene expression, or differentiation status within a biological zone), where changes Related to activation, stimulation, or processing, or to internal mechanisms such as genetic programming.
  • the "activity" of a molecule can describe or refer to the binding of the molecule to a ligand or receptor; catalytic activity; the ability to stimulate gene expression or cell signal transduction, differentiation, or maturation; antigen activity; to regulate the activity of other molecules, and the like.
  • the "activity” of a molecule may also refer to activity in modulating or maintaining a cell-to-cell interaction (e.g., adhesion), or in maintaining a cell structure (e.g., cell membrane or cytoskeleton).
  • Activity may also refer to specific activity, such as [catalytic activity] / [mg protein] or [immunity] / [mg protein], concentration in a biological compartment, and the like.
  • Activity may refer to the regulation of components of the innate or adaptive immune system.
  • Proliferative activity includes activities that promote, are necessary for, or are specifically related to, for example, normal cell division and cancer, tumors, dysplasia, cell transformation, metastasis, and angiogenesis.
  • administering and “treatment” applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refers to the combination of foreign drugs, therapeutics, diagnostics or compositions with animals, humans, subjects, cells, Tissue, organ or biological fluid contact.
  • administering may refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contacting the agent with the cells and contacting the agent with a fluid (where the fluid is in contact with the cells).
  • administeristering” and “treatment” also mean in vitro and ex vivo treatments, such as in vitro and ex vivo treatment of a cell with an agent, a diagnosis, a binding compound, or with another cell.
  • Treatment also includes internal or external administration of a therapeutic agent, such as a composition containing any antibody or antigen-binding fragment thereof, to a patient in need thereof.
  • a therapeutic agent such as a composition containing any antibody or antigen-binding fragment thereof
  • the antibodies or antigen-binding fragments or corresponding pharmaceutical compositions described herein are administered in an amount effective to reduce the symptoms of one or more diseases in a treated patient or population, whether by inducing the regression or suppression of these symptoms These symptoms progress to any clinically measurable degree.
  • the amount of therapeutic agent also referred to as a "therapeutically effective amount” effective to reduce the symptoms of any particular disease can vary depending on factors such as the patient's disease state, age and weight, and the ability of the drug to cause the desired response in the patient. Symptoms of the disease can be assessed by any clinical measurement commonly used by a physician or other professional health care provider to assess the severity or state of progression of the symptoms.
  • subject or “patient” includes any organism, preferably an animal, more preferably a mammal (eg, rat, mouse, dog, cat, rabbit), and most preferably a human.
  • a mammal eg, rat, mouse, dog, cat, rabbit
  • administration can be achieved by an invasive route, such as by injection administration of any of the anti-BTLA antibodies or antigen-binding fragments thereof or their corresponding pharmaceutical compositions described herein. It is also within the scope of the present invention to administer by a non-invasive route (e.g., orally; for example, as a pill, capsule, or tablet).
  • a non-invasive route e.g., orally; for example, as a pill, capsule, or tablet.
  • the anti-BTLA antibody or antigen binding thereof is administered intravenously, subcutaneously, intramuscularly, intra-arterially, intra-articularly (e.g. in an arthritic joint), by inhalation, aerosol delivery or intratumorally. Fragment or a pharmaceutical composition thereof.
  • any of the anti-BTLA antibodies or antigen-binding fragments thereof or the corresponding compositions described herein can be administered using medical devices known in the art.
  • the pharmaceutical composition of the present invention may be administered by injection using a hypodermic needle; or the pharmaceutical composition of the present invention may be administered by injection using an intravenous needle.
  • any of the anti-BTLA antibodies or antigen-binding fragments thereof or their corresponding pharmaceutical compositions described herein can be used alone or in combination to treat or prevent any of the subjects in need of such treatment or prevention Disease or condition.
  • B and T lymphocyte attenuator and "BTLA” genes / proteins are used interchangeably and include variants, isotypes, homologs, orthologs, and intraspecific isomorphisms.
  • Source paralog
  • human BTLA-specific antibodies can cross-react with BTLA from non-human species.
  • human BTLA-specific antibodies may be fully specific for human BTLA and not have species cross-reactivity or other types of cross-reactivity.
  • the term “human BTLA” or “hBTLA” refers to a human BTLA sequence.
  • human BTLA sequences include all isotypes and BTLA variants, such as the complete amino acid sequence of human BTLA with Genbank accession number AAP44003.
  • Transcript variant 1 encodes a protein that is 289 amino acids in length (GenBank accession number NP_861445) and is nearly 98% identical to the BTLA sequence of accession number AAP44003; transcript variants 2 encodes a protein with a protein length of 241 amino acids (GenBank accession number NP_001078826).
  • BTLA is a negative regulator of immune response, and its C-terminal inhibitory motif is involved in inhibiting IL-2 production and T cell proliferation (Watanabe et al., Nat. Immunol., 4,670-679, 2003; Chemnitz et al., J Immunol., 176, 6603-6614, 2006).
  • human BTLA may be an epitope in the extracellular domain of BTLA that specifically binds to the antibodies of the invention.
  • the specific extracellular nucleotide sequence of a specific BTLA sequence can generally be at least 90% identical to the nucleotide sequence of the extracellular region of human BTLA of SEQ ID NO: 49 or other isotypes, and should be identical to other species (such as When compared with the amino acid sequence of BTLA, it contains amino acid residues identified as human amino acid sequences.
  • the human BTLA extracellular region may be at least 95% or even at least 96%, 97%, 98%, or 99% of the human BTLA extracellular region or other isoform or variant of SEQ ID NO: 49. Identity.
  • immune response refers to, for example, the action of lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complements) produced by the above-mentioned cells or liver, which results in selective action from the human body. Damages, destroys or removes cells or tissues that invade pathogens, infect pathogens, cancer cells, or normal human cells or tissues in the case of autoimmune or pathological inflammation.
  • antibody refers to any form of antibody having the desired biological activity. Therefore, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), humanized antibodies, fully human antibodies, Chimeric antibodies and camelized single domain antibodies.
  • anti-BTLA antibody or "antigen-binding fragment” of an antibody refers to an antibody that binds to BTLA and blocks the binding of BTLA to HVEM, including fragments or derivatives of the antibody, usually including the antigen-binding or variable region of the antibody (E.g., one or more CDRs) at least one fragment that maintains at least some binding specificity of the antibody.
  • antibody-binding fragments include, but are not limited to, Fab, Fab ′, F (ab ′) 2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobody formed from antibody fragments And multispecific antibodies.
  • a binding fragment or derivative When BTLA-binding activity is expressed on a molar basis, a binding fragment or derivative generally retains at least 10% of its BTLA-binding activity. Preferably the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the BTLA binding affinity of the antibody. It is also contemplated that anti-BTLA antigen-binding fragments may include conservative or non-conservative amino acid substitutions (referred to as “conservative variants" or “functionally conservative variants” of antibodies) that do not significantly alter their biological activity.
  • Isolated antibody refers to the purified state of the antibody or its antigen-binding fragment, and in this case means that the molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, sugars or other substances such as cell debris and growth Culture medium.
  • isolated does not mean the absence of such substances at all or the absence of water, buffers or salts, unless they are present in an amount that significantly interferes with the experimental or therapeutic application of the antibodies or antigen-binding fragments thereof described herein.
  • the term "functional fragment” or "antigen-binding fragment” as used herein refers in particular to antibody fragments such as Fv, scFv, Fab, F (ab ') 2, Fab', scFv-Fc fragment or diabody, or by chemical modification Or any fragment that should be able to increase half-life by incorporation into liposomes, such as chemical additions such as the addition of poly (alkylene) glycols such as polyethylene glycol (“PEGylated, PEGylated”) (known as PEGylated fragments of Fv-PEG, scFv-PEG, Fab-PEG, F (ab ') 2-PEG, or Fab'-PEG) (“PEG” is polyethylene glycol), said fragments have EGFR binding activity .
  • poly (alkylene) glycols such as polyethylene glycol (“PEGylated, PEGylated") (known as PEGylated fragments of Fv-PEG, scF
  • the functional fragment is composed of or includes a partial sequence of a heavy or light variable chain of the antibody from which it is derived, which partial sequence is sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived.
  • BTLA it is preferably at least Equal to 1/100 of the affinity of the source antibody, and in a more preferred manner at least 1/10.
  • Such a functional fragment will contain a minimum of 5 amino acids, preferably 10, 15, 25, 50, and 100 consecutive amino acids from the antibody sequence from which it is derived.
  • a "Fab fragment” consists of a light chain and the variable regions of CH1 and a heavy chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • the "Fc" region contains two heavy chain fragments comprising the CH1 and CH2 domains of the antibody.
  • the two heavy chain fragments are held together by two or more disulfide bonds and by a hydrophobic interaction of the CH3 domain.
  • a "Fab 'fragment” contains a light chain and a heavy chain portion or fragment containing a VH domain and a CH1 domain and a region between the CH1 and CH2 domains such that two Fab' fragments An interchain disulfide bond can be formed between the two heavy chains to form an F (ab ′) 2 molecule.
  • the "F (ab ′) 2 fragment” contains 2 light chains and 2 heavy chains containing a portion of the constant region between the CH1 and CH2 domains such that an interchain disulfide bond is formed between the 2 heavy chains . Therefore, the F (ab ′) 2 fragment consists of two Fab ′ fragments held together by disulfide bonds between the two heavy chains.
  • An "Fv region” contains variable regions derived from both heavy and light chains, but lacks a constant region.
  • single-chain Fv or "scFv” antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain.
  • An scFv polypeptide generally also includes a polypeptide linker between the VH and VL domains, which enables scFv to form the desired structure for antigen binding.
  • a “domain antibody” is an immunofunctional immunoglobulin fragment containing only the heavy or light chain variable regions.
  • two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody.
  • the two VH regions of a bivalent domain antibody can target the same or different antigens.
  • bivalent antibody contains two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. However, bivalent antibodies can be bispecific.
  • anti-BTLA antibody refers to an antibody raised against human BTLA or a variant or any antigen fragment thereof.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous antibody population, that is, the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, being directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes.
  • the modifier “monoclonal” indicates the characteristics of an antibody obtained from a substantially homogeneous antibody population and should not be construed as requiring the production of an antibody by any particular method.
  • the monoclonal antibody used in the present invention can be prepared by a hybridoma method, or can be prepared by a recombinant DNA method. "Monoclonal antibodies” can also be isolated using phage antibody libraries.
  • a monoclonal antibody includes a "chimeric" antibody (immunoglobulin) in which a portion of the heavy and / or light chain corresponds to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass Identical or homologous, and the rest of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass and fragments of such antibodies, as long as they have the desired biological activity.
  • chimeric antibody immunoglobulin
  • a "chimeric antibody” is an antibody having a variable domain of a first antibody and a constant domain of a second antibody, wherein the first antibody and the second antibody are from different species.
  • the variable domain is obtained from an antibody to an experimental animal such as a rodent ("parent antibody”) and the constant domain sequence is obtained from a human antibody such that the resulting chimeric antibody is in a human subject compared to the parent rodent antibody The likelihood of inducing an adverse immune response is low.
  • the monoclonal antibodies herein also include camelized single domain antibodies. See, eg, Muyldermans et al. (2001) Trends Biochem. Sci. 26: 230; Reichmann et al. (1999) J. Immunol. Methods 231: 25.
  • diabody refers to a small antibody fragment having two antigen-binding sites, said fragment comprising a light chain variable domain (VL) linked to the same polypeptide chain (VH-VL or VL-VH) Heavy chain variable domain (VH).
  • VL light chain variable domain
  • VH-VL or VL-VH Heavy chain variable domain
  • humanized antibody refers to the form of an antibody containing sequences from human and non-human (e.g., mouse, rat) antibodies.
  • a humanized antibody contains at least one, usually two, variable domains, of which all or substantially all hypervariable regions are equivalent to non-human immunoglobulin hypervariable regions, and all or substantially all A framework (FR) region is a framework region of a human immunoglobulin sequence.
  • a humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (Fc).
  • the basic antibody structural unit comprises a tetramer.
  • Each tetramer includes two identical pairs of polypeptide chains, each pair having one "light” chain (about 25 kDa) and one "heavy” chain (about 50-70 kDa).
  • the amino-terminal portion or fragment of each chain may include a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal portion or fragment of each chain may define a constant region primarily responsible for effector function.
  • Human light chains are generally classified as kappa and lambda light chains.
  • human heavy chains are generally classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are linked by a "J" region of about 12 or more amino acids, where the heavy chain also includes a "D” region of about 10 amino acids. See generally Chapter 7 of Fundamental Immunology (Editor, Paul, W., 2nd edition. Raven Press, N.Y. (1989)).
  • variable region pairs of each light / heavy chain pair form an antibody binding site. Therefore, intact IgG antibodies generally have two binding sites. With the exception of bifunctional or bispecific antibodies, the two binding sites are usually the same.
  • each chain has the same general structure of a relatively conserved framework region (FR) connected by three hypervariable regions (also known as complementarity determining regions or CDRs).
  • the CDRs of the two chains of each pair are usually aligned through the framework regions, enabling them to bind to a particular epitope.
  • both the light and heavy chains contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus to the C-terminus.
  • amino acids of each domain are generally specified according to the definitions in the following documents: Sequences of Proteins of Immunological Interest, Kabat et al .; National Institutes of Health, Bethesda, Md .; 5th edition; NIH Bulletin No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32: 1-75; Kabat et al. (1977) J. Biol. Chem. 252: 6609-6616; Chothia et al. (1987) J. Mol. Biol. 196: 901-917 or Chothia et al. (1989) Nature 342: 878-883.
  • hypervariable region refers to the amino acid residues of an antibody responsible for antigen binding.
  • the hypervariable region contains amino acid residues of "complementarity determining regions” or "CDRs".
  • frame or "FR” residue refers to a variable domain residue other than a hypervariable region residue defined herein as a CDR residue.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent the symptoms or signs of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's general health, the route of administration and dosage, and the severity of the side effects.
  • An effective amount may be the maximum dose or dosage regimen to avoid significant side effects or toxic effects.
  • the results can lead to an improvement in diagnostic measurements or parameters of at least 5%, usually at least 10%, more often at least 20%, most often at least 30%, preferably at least 40%, more preferably at least 50%, most preferably at least 60%, ideally It is at least 70%, more preferably at least 80%, and most preferably at least 90%, of which 100% is defined as the diagnostic parameters displayed by normal subjects (see, for example, Maynard et al. (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, FL; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).
  • “Homology” refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences. When a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit, for example, if a position in each of the two DNA molecules is occupied by adenine, the molecules are homologous at that position of.
  • the percentage homology between two sequences is a function of the number of matching positions or homology positions shared by the two sequences divided by the number of compared positions x 100. For example, if 6 of the 10 positions of the two sequences match or are homologous when the sequences are optimally aligned, the two sequences are 60% homologous. Comparisons are generally made when the two sequences are aligned to obtain the maximum percent homology.
  • immune disorder includes, for example, pathological inflammation, inflammatory disorders, and autoimmune disorders or diseases.
  • Immuno disorder also refers to infections, persistent infections, and proliferative conditions, such as cancer, tumors, and angiogenesis, including infections, tumors, and cancers that fight the eradication of the immune system.
  • Constant conditions include, for example, cancer, cancer cells, tumors, angiogenesis, and precancerous conditions, such as dysplasia.
  • the "immune lesions” and “cancerous conditions” described herein are preferably both mediated by BTLA.
  • Immune system cells include, for example, T cells, B cells, monocytes or macrophages, antigen presenting cells (APC), dendritic cells, microglia, NK cells, NKT cells, neutrophils, eosinophils Cell, mast cell, or any other cell of particular interest to immunology, such as cytokine-producing endothelial or epithelial cells.
  • the "inflammatory condition” described herein is preferably mediated by BTLA.
  • isolated nucleic acid molecule means DNA or RNA of genomic, mRNA, cDNA or synthetic origin, or some combination thereof, which is not associated with all or a portion of a polynucleotide (where the isolated polynucleotide is naturally occurring) or is naturally associated with it Unlinked polynucleotides are linked.
  • a "nucleic acid molecule” that "comprises” a particular nucleotide sequence does not include a complete chromosome.
  • an isolated nucleic acid molecule that "comprises" a specified nucleic acid sequence may include coding sequences for up to 10 or even up to 20 or more other proteins or parts or fragments thereof, or may include operatively linked controls The listed regulatory sequences for expression of the coding region of a nucleic acid sequence, and / or may include a vector sequence.
  • the expressions "cell”, “cell line” and “cell culture” are used interchangeably, and all such names include progeny.
  • the terms “transformants” and “transformed cells” include primary subject cells and cultures derived therefrom, regardless of the number of transfers. It is also understood that the DNA content of all offspring may not be exactly the same due to intentional or inadvertent mutations. Mutant progeny screened for the same function or biological activity in the initially transformed cells are included. Although different names are specified, it will be clear from the context.
  • the host cell according to the present invention may be a prokaryotic host cell, a eukaryotic host cell, or a phage.
  • the prokaryotic host cell may be E. coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis.
  • Eukaryotic host cells can be fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces cerevisiae, Trichoderma and other fungi, insect cells such as grassland armyworm, plant cells such as tobacco, such as BHK cells, CHO cells, and COS cells , Myeloma cells and other mammalian cells.
  • the host cell of the present invention is preferably a mammalian cell, more preferably a BHK cell, a CHO cell, an NSO cell, or a COS cell.
  • PCR polymerase chain reaction
  • oligonucleotide primers can be designed; these primers can be compared to the sequence of the opposite strand of the template to be amplified The same or similar.
  • the 5 ′ end nucleotides of the two primers may coincide with the ends of the amplified material.
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences from whole genomic DNA, and cDNA transcribed from total cellular RNA, phage, or plasmid sequences. See generally Mullis et al. (1987) Cold Spring Harbor Symp. Quant. Biol.
  • PCR as used herein, is considered to be one (but not the only) example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which method involves using known nucleic acids and nucleic acid polymerases as primers to amplify or produce specific Nucleic acid fragment.
  • the present invention relates generally to isolated antibodies or antigen-binding fragments thereof that bind to BTLA and the use of such antibodies or antigen-binding fragments thereof. More specifically, the present invention provides isolated anti-BTLA antibodies and the use of these antibodies or antigen-binding fragments thereof in the treatment and prevention of diseases.
  • anti-BTLA antibodies include, but are not limited to, ch7, ch12, ch17, ch22, ch27, hu17, hu18, and hu19 described herein.
  • the present invention provides human BTLA (B - and T - cell attenuator) binding to isolated antibody or antigen binding fragment, wherein said antibody or antigen binding fragment thereof comprising one or more of the following properties: A) block BTLA binding of HVEM (herpes virus entry mediator); B) with cross-reactivity to cynomolgus BTLA; K D ⁇ 0.28nM C) binding to human BTLA.
  • HVEM herpes virus entry mediator
  • the light chain CDR of the isolated antibody or antigen-binding fragment thereof that binds to human BTLA provided by the present invention comprises at least one selected from SEQ ID NO: 7, 8, 9, 10, 11, 12, 16, 17, 18, 22, 23, 24, 31, 32, and 33 light chain CDRs.
  • the LCDR1 in the light chain CDR of the isolated antibody or antigen-binding fragment thereof that binds to human BTLA provided by the present invention may be selected from any of SEQ ID NOs: 7, 10, 16, 22, and 31 A CDR sequence; in certain embodiments, the LCDR2 in the light chain CDR of the isolated antibody or antigen-binding fragment thereof that binds to human BTLA provided by the present invention may be selected from SEQ ID NOs: 8, 11, 17, 23, and 32 LCDR3 in the light chain CDR of the light chain CDR of the isolated antibody or antigen-binding fragment thereof that binds to human BTLA provided by the present invention may be selected from any of the CDR sequences in SEQ ID NO: 9, 12, 18, 24, and 33 .
  • LCDR1 is selected from any one of the CDRs of SEQ ID NO: 7, 10, 16, 22, and 31 Sequence; LCDR2 is selected from any of the CDR sequences in SEQ ID NO: 8, 11, 17, 23, and 32; LCDR3 is selected from any of the CDR sequences in SEQ ID NO: 9, 12, 18, 24, and 33.
  • the LCDR1, LCDR2, and LCDR3 sequences of the light chain thereof are as shown in any one of the following groups A-E:
  • the heavy chain CDR of the isolated antibody or antigen-binding fragment thereof that binds to human BTLA provided by the present invention includes at least one selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 13, 14, 15, 19, 20, 21, 25, 26, 27, 28, 29 and 30 heavy chain CDRs.
  • the HCDR1 in the heavy chain CDR of the isolated antibody or antigen-binding fragment thereof that binds to human BTLA provided by the present invention may be selected from SEQ ID NO: 1, 4, 13, 19, 25, and 28
  • the HCDR2 in the heavy chain CDR of the isolated antibody or antigen-binding fragment thereof that binds to human BTLA provided by the present invention may be selected from SEQ ID NO: 2, 5, 14, 20, Any of the CDR sequences of 26 and 29;
  • the HCDR3 in the heavy chain CDR of the isolated antibody or antigen-binding fragment thereof that binds to human BTLA provided by the present invention may be selected from SEQ ID NO: 3, 6, A CDR sequence of any of 15, 21, 27, and 30.
  • HCDR1 is selected from any one of SEQ ID NO: 1, 4, 13, 19, 25, and 28 A CDR sequence
  • HCDR2 is selected from any of the CDR sequences of SEQ ID NO: 2, 5, 14, 20, 26, and 29
  • HCDR3 is selected from any of SEQ: ID NO: 3, 6, 15, 21, 27, and 30 A CDR sequence.
  • the amino acid sequence of HCDR1, HCDR2, and HCDR3 of the heavy chain CDR in the isolated antibody or antigen-binding fragment thereof that binds to human BTLA provided by the present invention is as shown in any one of the following F-K groups:
  • LCDR1 is selected from any one of SEQ ID NOs: 7, 10, 16, 22, and 31 A CDR sequence
  • LCDR2 is selected from any one of the CDR sequences of SEQ ID NO: 8, 11, 17, 23, and 32
  • LCDR3 is selected from any one of the CDR sequences of SEQ ID NO: 9, 12, 18, 24, and 33
  • HCDR1 is selected from any of the CDR sequences of SEQ ID NO: 1, 4, 13, 19, 25, and 28
  • HCDR2 is selected from SEQ ID NO: 2, 5, 14, 20, 26, and 29
  • HCDR3 is selected from any of the CDR sequences in SEQ ID NO: 3, 6, 15, 21, 27, and 30.
  • the LCDR1, LCDR2, and LCDR3 sequences of its light chain are as shown in any one of the following groups A-E:
  • HCDR1, HCDR2 and HCDR3 of their heavy chain CDRs are shown in any of the following F-K groups:
  • each CDR sequence of the light chain disclosed herein can be arbitrarily combined.
  • any one of the sequences defined as LCDR1 herein may be any of the sequences defined as LCDR2 herein, any of the sequences of LCDR3, any of the sequences of HCDR1, any of the sequences of HCDR2
  • any one of the sequences of HCDR3 is combined to form the complete 6 CDR domains contained in the isolated antibody or antigen-binding fragment thereof binding to human BTLA described herein.
  • the amino acid sequences of the heavy chain CDRs HCDR1, HCDR2, and HCDR3 and the light chain CDRs thereof are shown in any of the following groups I-IX:
  • the isolated antibody or antigen-binding fragment thereof provided by the present invention that binds to human BTLA comprises any one selected from SEQ ID NO: 36, 37, 39, 41, 44, 46, 47, and 48.
  • an isolated antibody of the invention comprises a heavy chain constant region, preferably a human constant region, such as a ⁇ 1, ⁇ 2, ⁇ 3, or ⁇ 4 human heavy chain constant region or a variant thereof.
  • an isolated antibody of the invention comprises a light chain constant region, preferably a human light chain constant region, such as a lambda or kappa human light chain region or a variant thereof.
  • the human heavy chain constant region may be ⁇ 4
  • the human light chain constant region may be ⁇ .
  • the Fc region of the antibody may be ⁇ 4 with conservative modifications or conservative substitutions or conservative mutations.
  • the terms “conservatively modified variants” or “conservative substitutions” or “conservative mutations” refer to amino acids in a protein having similar properties (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation, and rigidity). And other amino acid substitutions, so that changes can be made frequently without altering the biological activity of the protein. Those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide generally do not significantly alter biological activity (see, eg, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 ( 4th edition)).
  • Antibodies or antigen-binding fragments thereof comprise a polypeptide chain having the following sequence: a specific amino acid sequence disclosed herein (e.g., SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 , 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, and 48)
  • SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 , 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, and 48 When comparing, it includes up to 0 (no change), 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 12, 15, 20, or more conservative amino acid substitutions; for example, may include 0-20 amino acid substitutions, or include 1-15, 1-10, 1- 8. 1-5 substitutions,
  • the present invention also includes functionally conserved variants of the antibody or antigen-binding fragment of the invention, that is, one or more amino acid residues in the antibody or antigen-binding fragment of the invention without changing the overall conformation and function of the antibody Altered variants include, but are not limited to, amino acid substitutions by amino acids with similar properties. Generally, the number of substitutions can be in the range described above, such as 0-20 conservative substitutions.
  • the isolated antibody or antigen-binding fragment thereof of the present invention that binds to human BTLA is a single-chain Fv antibody; in some embodiments, the antibody or antigen-binding fragment thereof is a Fab antibody; in some embodiments, the antibody or antigen-binding fragment thereof is a Fab 'antibody; in certain embodiments, the antibody or antigen-binding fragment thereof is a (Fab') 2 antibody.
  • amino acid sequence of the light chain variable region of the isolated antibody or antigen-binding fragment thereof that binds to human BTLA according to the present invention is shown in SEQ ID NO: 36
  • amino acid sequence of the heavy chain variable region is shown in FIG. SEQ ID NO: 34 or 35.
  • amino acid sequence of the light chain variable region of the isolated antibody or antigen-binding fragment thereof that binds to human BTLA according to the present invention is shown in SEQ ID NO: 37
  • amino acid sequence of the heavy chain variable region is shown in FIG. SEQ ID NO: 34 or 35.
  • amino acid sequence of the light chain variable region of the isolated antibody or antigen-binding fragment thereof that binds to human BTLA according to the present invention is shown in SEQ ID NO: 39, and the amino acid sequence of the heavy chain variable region is shown in FIG. SEQ ID NO: 38.
  • amino acid sequence of the light chain variable region of the isolated antibody or antigen-binding fragment thereof that binds to human BTLA according to the present invention is shown in SEQ ID NO: 41, and the amino acid sequence of the heavy chain variable region is shown in FIG. SEQ ID NO: 40 or 42.
  • amino acid sequence of the light chain variable region of the isolated antibody or antigen-binding fragment thereof that binds to human BTLA according to the present invention is shown in SEQ ID NO: 44, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 43.
  • the amino acid sequence of the light chain variable region of the isolated antibody or antigen-binding fragment thereof that binds to human BTLA according to the present invention is shown in SEQ ID NO: 46, 47, or 48.
  • the heavy chain variable region The amino acid sequence is shown in SEQ ID NO: 45.
  • the invention also provides an isolated nucleic acid, such as DNA, which encodes an isolated antibody or an antigen-binding fragment thereof of the invention.
  • an isolated nucleic acid of the invention encodes an antibody or antigen-binding fragment thereof comprising at least one mature antibody light chain variable (VL) domain and at least one mature antibody heavy chain variable (VH) domain, wherein
  • VL domain contains at least 3 CDRs having a sequence selected from SEQ ID NO: 7-8, 10-12, 16-18, 22-24, and 31-33
  • the VH domain contains at least 3 CDRs NO: CDRs for sequences of 1-3, 4-6, 13-15, 19-21, 25-27 and 28-30.
  • the isolated nucleic acid encodes the mature light and heavy chain variable region sequences of SEQ ID NO: 36 and SEQ ID NO: 34, respectively. In one embodiment, the isolated nucleic acid encodes the mature light and heavy chain variable region sequences of SEQ ID NO: 37 and SEQ ID NO: 35, respectively. In one embodiment, the isolated nucleic acid encodes the mature light and heavy chain variable region sequences of SEQ ID NO: 39 and SEQ ID NO: 38, respectively. In one embodiment, the isolated nucleic acid encodes the mature light and heavy chain variable region sequences of SEQ ID NO: 41 and SEQ ID NO: 40, respectively.
  • the isolated nucleic acid encodes the mature light chain and heavy chain variable region sequences of SEQ ID NO: 41 and SEQ ID NO: 42, respectively. In one embodiment, the isolated nucleic acid encodes the mature light chain and heavy chain variable region sequences of SEQ ID NO: 44 and SEQ ID NO: 43, respectively. In one embodiment, the isolated nucleic acid encodes the mature light and heavy chain variable region sequences of SEQ ID NO: 46 and SEQ ID NO: 45, respectively. In one embodiment, the isolated nucleic acid encodes the mature light and heavy chain variable region sequences of SEQ ID NO: 47 and SEQ ID NO: 45, respectively.
  • the isolated nucleic acid encodes the mature light and heavy chain variable region sequences of SEQ ID NO: 48 and SEQ ID NO: 45, respectively. In one or more embodiments, the isolated nucleic acid encodes both the light and heavy chains on a single nucleic acid molecule, while in other embodiments, the light and heavy chains are encoded on two or more independent nucleic acid molecules.
  • the present invention also provides an expression vector comprising the isolated nucleic acid of the present invention.
  • a host cell comprising the expression vector of the present invention is also provided.
  • the invention also relates to a method for producing an antibody or antigen-binding fragment thereof of the invention.
  • the invention also relates to antibodies or antigen-binding fragments thereof that bind to the same epitope on human BTLA as the antibodies ch7, ch12, ch17, ch22, ch27, hu17, hu18, and hu19 described herein, for example, capable of cross-blocking any of the invention Binding of antibodies.
  • the present invention also provides a method for treating or preventing a subject (including a human subject) in need of treatment with an isolated antibody or antigen-binding fragment thereof with an antibody of the invention or an antigen-binding fragment thereof, preferably a humanized antibody.
  • the method generally includes administering to a subject in need thereof a therapeutically or prophylactically effective amount of an antibody or an antigen-binding fragment thereof according to any one of the embodiments of the present invention, or a pharmaceutical composition containing the antibody or an antigen-binding fragment thereof.
  • the appropriate mode of administration can be selected according to the actual situation, including but not limited to oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (e.g. in arthritic joints), by inhalation, aerosol delivery or intratumor Give etc.
  • BTLA-mediated diseases are all diseases related to immunosuppression, including autoimmune diseases, transplant rejection, tumors, and the like.
  • the tumor includes melanoma, breast cancer, kidney cancer, prostate cancer, colon cancer, lung cancer, pancreatic cancer, bone cancer, skin cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer , Testicular cancer, esophageal cancer, small intestine cancer, cervical cancer, vaginal cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, endocrine system cancer, thyroid cancer, adrenal love, soft tissue cancer, urethral cancer, chronic Or acute leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system burden Biology, Primary central nervous system lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi'
  • the autoimmune diseases include organ-specific autoimmune diseases and systemic autoimmune diseases;
  • the organ-specific autoimmune diseases include chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes, myasthenia gravis, ulcers Colitis, malignant anemia with chronic atrophic gastritis, pulmonary hemorrhagic nephritis syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple cerebral spinal sclerosis, acute idiopathic polyneuritis Etc .
  • the systemic autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, lysosphosis, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia Ulcerative colitis.
  • This method of treatment may also include the administration of one or more other therapeutic drugs, such as tumor vaccines, standard tumor chemotherapy drugs, and other immune-inducing agents.
  • other therapeutic drugs such as tumor vaccines, standard tumor chemotherapy drugs, and other immune-inducing agents.
  • kits of the invention include one or more components, including but not limited to antibodies or antigen-binding fragments (e.g., antibodies ch7, ch12, ch17, ch22, ch27, hu17, hu18, and hu18) that specifically bind to BTLA as described herein. hu19, but not limited to the above antibodies) and one or more other components, including but not limited to pharmaceutically acceptable carriers and / or chemotherapeutic drugs described herein.
  • the antibody or antigen-binding fragment and / or chemotherapeutic agent can be formulated into a pure composition or combined with a pharmaceutically acceptable carrier in a pharmaceutical composition.
  • the kit includes an antibody of the invention or an antigen-binding fragment thereof (e.g. antibodies ch7, ch12, ch17, ch22, ch27, hu17, hu18, and hu19) in a container (e.g., a sterile glass or plastic vial).
  • a container e.g., a sterile glass or plastic vial.
  • the antibody of the present invention or an antigen-binding fragment thereof, or a pharmaceutical composition and / or a chemotherapeutic agent in another container for example, a sterile glass or plastic vial.
  • the kit includes a combination drug of the invention, including antibodies or antigen-binding fragments (e.g. antibodies ch7, ch12, ch17, ch22, ch27, hu17, hu18, and hu19) in a single common container, (But not limited to the above antibodies), together with a pharmaceutically acceptable carrier, optionally in combination with one or more chemotherapeutic drug components formulated together, optionally in a pharmaceutical composition.
  • antibodies or antigen-binding fragments e.g. antibodies ch7, ch12, ch17, ch22, ch27, hu17, hu18, and hu19
  • a pharmaceutically acceptable carrier optionally in combination with one or more chemotherapeutic drug components formulated together, optionally in a pharmaceutical composition.
  • the kit may include a drug package insert, which includes information about the pharmaceutical composition and dosage form in the kit. Such information generally assists patients and physicians in the effective and safe use of the attached pharmaceutical compositions and dosage forms.
  • information about the combination drug of the present invention can be provided in a drug label: pharmacokinetics, pharmacodynamics, clinical studies, effect parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdose Use, proper dosage and administration, supply specifications, suitable storage conditions, references, manufacturer / wholesaler information and patent information.
  • the invention also includes pharmaceutical compositions containing any of the anti-human BTLA antibodies or antigen-binding fragments thereof described herein.
  • pharmaceutical composition as used herein means a combination of at least one drug and optionally a pharmaceutically acceptable carrier or excipient that are combined together to achieve a particular purpose.
  • the pharmaceutical composition includes combinations that are separated in time and / or space as long as they can work together to achieve the purpose of the present invention.
  • the ingredients contained in the pharmaceutical composition (such as an antibody, a nucleic acid molecule, a combination of nucleic acid molecules, and / or a conjugate) according to the present invention may be administered to a subject as a whole or separately.
  • the ingredients contained in the pharmaceutical composition are separately administered to a subject, the ingredients may be administered to the subject simultaneously or sequentially.
  • the pharmaceutically acceptable carrier is water, a buffered aqueous solution, an isotonic saline solution such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, carbonic acid Magnesium, 0.3% glycerol, hyaluronic acid, ethanol or polyalkylene glycols such as polypropylene glycol, triglycerides and the like.
  • PBS phosphate buffered saline
  • glucose mannitol
  • dextrose dextrose
  • lactose lactose
  • starch magnesium stearate
  • cellulose carbonic acid
  • ethanol or polyalkylene glycols such as polypropylene
  • the type of pharmaceutically acceptable carrier used depends in particular on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration.
  • the composition according to the invention may contain as a additive a wetting agent, an emulsifier or a buffer substance.
  • the immunoconjugate of the present invention may contain an antibody or an antigen-binding fragment thereof according to any of the embodiments herein coupled with a therapeutic agent.
  • the therapeutic agent is a well-known and commonly used immune system for preparing immunoconjugates. Toxins, radioisotopes, drugs or cytotoxic agents.
  • the antibody or the antigen-binding fragment thereof may be mixed with a pharmaceutically acceptable carrier or excipient. See, for example, Remington's Pharmaceutical Sciences and U.S. Pharmaceutical Copeia: National Formulary, Mack Publishing Company, Easton, PA (1984).
  • the pharmaceutical composition of the present invention can be prepared into a variety of suitable dosage forms known in the art, including but not prior to lyophilized powder, plaster, aqueous solution or suspension, and the like.
  • Dosage forms of therapeutic and diagnostic agents can be prepared by mixing with an acceptable carrier, excipient, or stabilizer: for example, lyophilized powders, ointments, aqueous solutions, or suspensions (see, for example, Hardman et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis et al.
  • the dosing regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic antibody, the level of symptoms, the immunogenicity of the therapeutic antibody, and the availability of target cells in the biological matrix.
  • the preferred dosing regimen delivers sufficient therapeutic antibodies to achieve improvement in the target disease state while minimizing adverse side effects. Therefore, the amount of biologic delivered depends in part on the specific therapeutic antibody and the severity of the condition to be treated.
  • Guidance on selecting appropriate dosages of therapeutic antibodies is available (see, for example, Wawrzynczak (1996) Antibody Therapy, Bioscientific Pub. Ltd, Oxfordshire, UK; Kresina (Eds.) (1991) Monoclonal Antibodies, Cytokines, and Arthritis, Marcel and Dekker, New York, NY; Bach (ed.
  • the present invention provides the use of anti-BTLA antibodies and antigen-binding fragments thereof according to any of the embodiments herein in the treatment, prevention, and diagnosis of BTLA-mediated diseases.
  • the invention provides a BTLA antibody and antigen-binding fragment thereof according to any of the embodiments herein for use in the treatment, prevention, and diagnosis of a BTLA-mediated disease.
  • a plasmid HG11895-GN containing a human BTLA gene cDNA sequence was purchased from Yiqiao Shenzhou, using a forward primer 5'-gtacGCTCTTCATGTaaagaatcatgtgatgtacagcttta-3 '(SEQ ID ID NO: 50) and a reverse primer 5'-gatcGCTCTTCTAGCatacaggagccagggtctggttgcca-3' (SEQ ID ID) : 51), and the human BTLA extracellular fragment is amplified by PCR (the nucleotide sequence is shown in SEQ ID NO: 49).
  • the amplified fragment was digested with BSPQI and inserted into an autonomously constructed eukaryotic expression plasmid system (HX1-FC) to generate an expression plasmid for human BTLA extracellular domain protein (hBTLA-ECD-FC).
  • HX1-FC eukaryotic expression plasmid system
  • hBTLA-ECD-FC human BTLA extracellular domain protein
  • Figure 1 shows an SDS-PAGE electrophoresis picture of human BTLA extracellular domain protein.
  • HHVEM-293F stable transfected cell line (hHVEM-293F) expressing human HVEM (Gene Bank: NP_003811.2) was constructed.
  • HHVEM-293F cell suspension was mixed with biotin-labeled hBTLA (300ng / mL) and incubated at room temperature for 30 minutes. After washing the cells 3 times with FACS buffer, 5 ⁇ g / ml NA-PE was added and incubated for 30 minutes. After washing the cells 3 times with FACS buffer, the binding of BTLA recombinant protein to HVEM on the surface of 293F cells was verified by flow cytometry. The results are shown in Figure 2.
  • the recombinant protein hBTLA-ECD-FC obtained in Example 1 was mixed with an equivalent amount of immune adjuvant (Freund's adjuvant) as an antigen, and five 6-week-old female Balb / c mice were subcutaneously immunized. After the initial immunization, booster immunization was performed every two weeks for a total of 6 immunizations.
  • immune adjuvant Freund's adjuvant
  • the mouse inguinal lymph nodes were taken, and the lymphocyte-rich suspension was taken after being milled in physiological saline.
  • the suspension was fused with SP2 / 0 cells according to the conventional electroporation method (see the manual of the BTX electroporation instrument). .
  • the fused cells were placed in 8% CO 2 in DMEM complete medium (Corning) containing HAT, and cultured at 37 ° C.
  • 560 strains were screened by ELISA to clone human BTLA protein.
  • 33 strains can bind BTLA expressed on 293F cells; 16 of these 33 clones have the ability to inhibit the binding of biotin-labeled human BTLA to HVEM on 293F.
  • the hybridoma cell line that secreted the antibody and hBTLA was screened by enzyme-labeled reaction (ELISA).
  • ELISA enzyme-labeled reaction
  • a 384-well microtiter plate was coated with 1 ⁇ g / ml hBTLA and incubated overnight at 4 ° C. The solution in the well was then discarded, washed three times with washing buffer, and blocked by adding a PBS solution containing 1% BSA for 60 minutes. After washing three times with the washing buffer, the hybridoma culture supernatant was added, and after incubating at room temperature for 60 minutes, the washing buffer was washed three times. Then add 1: 5000-fold diluted HRP-labeled goat anti-mouse IgG secondary antibody and incubate at room temperature for 30 minutes.
  • a hybridoma cell line that combines the antibodies secreted by hybridomas with cynomolgus monkey BTLA was screened by enzyme-labeled reaction (ELISA).
  • ELISA enzyme-labeled reaction
  • a 96-well microtiter plate was coated with 1 ⁇ g / ml cynomolgus monkey BTLA and incubated for 60 minutes at room temperature. The solution in the well was then discarded, washed three times with washing buffer, and blocked by adding a PBS solution containing 1% BSA for 60 minutes. After washing three times with the washing buffer, the hybridoma culture supernatant was added, and after incubating at room temperature for 60 minutes, the washing buffer was washed three times.
  • the supernatant of 33 antibody cultures was mixed with biotin-labeled human BTLA (300ng / mL), and incubated at room temperature for 30 minutes. The mixture was then incubated with hHVEM-293F stable cell line suspension for 30 minutes at room temperature. After washing the cells 3 times with FACS buffer, 5 ⁇ g / ml NA-PE was added and incubated for 30 minutes. After washing the cells 3 times with FACS buffer, it was verified by flow cytometry that the antibodies secreted by hybridoma cells could block the binding of human BTLA to HVEM on the surface of 293F cells.
  • Example 5 Acquisition of candidate antibody variable region sequences (represented by Kabat or IMGT)
  • the DNA sequence of the variable region of the mouse antibody expressed by the candidate hybridoma was determined using a method based on degenerate primer PCR.
  • the hybridoma cell lines were separately expanded, cells were collected by centrifugation at 1000 rpm, and total RNA was extracted with Trizol.
  • the corresponding variable region DNA sequence is PCR amplified using the first strand cDNA as a subsequent template.
  • the PCR primers used are based on the Ig-primer set.
  • the purified PCR product is recovered and the amplified product is sequenced to obtain the candidate heavy chain variable region and light chain variable region sequences.
  • NCBI Ig-Blast http://www.ncbi.nlm.nih.gov/projects/igblast/ was used to search for consensus sequences in the germline and rearranged Ig variable region sequence databases. Based on Kabat (Wu, TT and Kabat, EA1970J. Exp.Med., 132: 211-250) and IMGT system (Lefranc M.-P.
  • Candidate mouse antibody heavy chain variable region and light chain variable region genes were fused to the N-terminus of the human IgG4 FC and ⁇ chain constant regions by standard PCR techniques, and inserted into the pcDNA3.1 vector to construct a chimeric heavy chain or Light chain expression plasmid. Subsequent chimeric antibodies were transfected with the expression vector plasmids of the heavy chain and light chain in combination, and then purified and extracted to obtain chimeric antibodies ch7, ch12, ch17, ch22, and ch27. The amino acid sequences of the light and heavy chain variable regions and CDRs encoded by the hybridoma cells are shown below.
  • Example 7 Detection of binding of chimeric antibody to hBTLA by ELISA
  • the binding ability of chimeric antibody to hBTLA was detected by enzyme-labeled reaction (ELISA).
  • ELISA enzyme-labeled reaction
  • the 96-well microtiter plate was coated with 0.5 ⁇ g / ml hBTLA and incubated at 37 ° C for 60 minutes. The solution in the well was then discarded, washed three times with washing buffer, and blocked by adding a PBS solution containing 2% BSA for 60 minutes. After washing 3 times with washing buffer, add gradient diluted antibody, incubate at 37 ° C for 60 minutes, and then wash 3 times with washing buffer. Then add 1: 10000-fold diluted HRP-labeled mouse anti-human IgG4 secondary antibody, and incubate at 37 ° C.
  • Table 1 Specific binding data of chimeric antibodies to human BTLA
  • HHVEM-293F stably expressing cells were digested and resuspended in FACS buffer after centrifugation, and the cells were added to a 96-well round bottom plate with 2.5 ⁇ 10 4 cells / 50ul, and pre-biotin-labeled hBTLA (1ug / ml) was added.
  • the results are shown in Figure 4 and Table 2 below.
  • the chimeric antibodies ch12, ch17, ch22, and ch27 can effectively block the binding of human BTLA to HVEM on the cell surface.
  • Example 9 Effect of chimeric antibody on T cell activity by luciferase reporter assay
  • T cell activation is achieved by stimulating T cell receptors (TCRs) that recognize specific peptides presented by the major histocompatibility complex class I or class II proteins on antigen-presenting cells (APC).
  • TCR T cell receptors
  • APC antigen-presenting cells
  • Activated TCR in turn, initiates a cascade of signaling events, which can be through transcription factors such as activator-protein-1 (AP-1), nuclear factor (NFAT) of activated T cells, or nuclear factor of activated B cells ⁇ light chain enhancer (NF ⁇ b)) driven reporter gene to monitor.
  • T cell responses are regulated by the formation or induction of expression of co-receptors on T cells.
  • Programmed cell death protein (PD1) and BTLA are negative regulators of T cell activity.
  • PD-1 and BTLA interact with their ligands (PD-L1) and HVEM expressed on target cells including APC or cancer cells, respectively, and this interaction results in the recruitment of phosphatases to the TCR signalosome To deliver an inhibitory signal, resulting in inhibition of positive signaling.
  • APC and T cells were measured by constructing two engineered stable expression cell lines Jurkat cells (Jurkat / NFAT-Luc / hPD-1-hBTLA) and CHO cells (CHO / hPD-L1-hHVEM) Action-induced T cell signaling.
  • CHO cells stably expressing hPD-L1 / hHVEM were plated into 96-well plates, the cell volume per well was 5 ⁇ 10 4 , cultured at 37 ° C., 7% CO 2 overnight, the cell supernatant was removed, and 40ul of chimeric antibody was added to each well.
  • BTLA antibody dilution starting concentration: 60ug / ml, 3-fold titration
  • adding 40ul of Jurkat reporter cells that can continuously express hPD-1 / hBTLA / NFAT-luciferase the total number of cells is 1 ⁇ 10 5 cells
  • 37 Incubate at 7% CO 2 for 6 hours add luciferase reagent, and check the luminescence value with a microplate reader.
  • Humanized transformation is performed based on the variable region sequence of the antibody secreted by the hybridoma cell obtained above.
  • the humanization transformation process involves the following steps: A. The gene sequence of the antibody secreted by each hybridoma cell is compared with the human embryonic antibody gene sequence to find a sequence with high homology; B. Analysis and investigation HLA-DR affinity, select human embryonic frame framework sequences with low affinity; C. Use computer docking technology to analyze molecular amino acid sequences of the variable region and its surroundings using molecular docking, and investigate its spatial stereoscopic binding mode.
  • Example 11 Detection of binding of humanized antibodies to hBTLA by ELISA
  • the binding specificity of humanized antibodies to hBTLA was detected by conventional ELISA detection methods.
  • the 96-well microtiter plate was coated with 0.5 ⁇ g / ml hBTLA and incubated at 37 ° C for 60 minutes. The solution in the well was then discarded, washed three times with washing buffer, and blocked by adding a PBS solution containing 2% BSA for 60 minutes. After washing 3 times with washing buffer, add gradient diluted antibody, incubate at 37 ° C for 60 minutes, and then wash 3 times with washing buffer.
  • Table 4 Specific binding data of humanized antibody combinations hu17, hu18 and hu19 with hBTLA
  • FACS Cell-based flow cytometry
  • the results are shown in Fig. 7.
  • the humanized antibodies hu17, hu18, hu19 and hBTLA on 293F cells can effectively bind.
  • the EC50 value of each antibody is shown in Table 5 below.
  • FACS flow cytometry
  • Washed cells were resuspended in a 4 ° C buffer containing propidium iodide (PI) and 0.02% sodium azide to prevent receptor internalization, and analyzed by flow cytometry. Based on the exclusion of PI-positive cells from the FSC / SSC gate, viable cells were gated and their geometric mean fluorescence was measured. Data were analyzed using an S-shaped dose-response model in Prism TM software.
  • PI propidium iodide
  • Example 14 Humanized anti-BTLA antibody promotes T cell activation
  • CHO cells stably expressing hPD-L1 / hHVEM were plated into 96-well plates, the cell volume per well was 5 ⁇ 10 4 , cultured at 37 ° C., 7% CO 2 overnight, the cell supernatant was removed, and 40ul humanized was added to each well.
  • Anti-BTLA antibody dilution solution (starting concentration: 60ug / ml, 3-fold concentration dilution), adding 40ul of Jurkat reporter cells that can continuously express hPD-1 / hBTLA / NFAT-luciferase, the total number of cells is 1 ⁇ 10 5
  • the cells were cultured at 37 ° C, 7% CO 2 for 6 hours, and the luciferase reagent was added, and the luminescence value was detected by a microplate reader.
  • Example 15 Affinity of humanized anti-BTLA antibody to hBTLA
  • Biacore T200 instrument from GE Medical Life Sciences was used for detection experiments.
  • the Series CM5 chip was loaded on the instrument.
  • the system buffer used was HBS-EP + (10mM HEPES, pH7.4, 150mM NaCl, 3mM EDTA, 0.05% surface activity Agent P20).
  • the BTLA-Fc antigen was coupled to the chip detection channel.
  • a mixture of 400 mM EDC and 100 mM NHS was injected into the surface of the active chip at 10 ⁇ L / min at 420s.
  • the BTLA-Fc antigen was diluted in 10 mM sodium acetate / acetic acid (pH 5.5) buffer.
  • Antibodies were diluted 2-fold with Biacore System Buffer for a total of 6 concentration points.
  • the concentration gradients were 24nM, 12nM, 6nM, 3nM, 1.5nM, and 0.75nM, of which 24nM was a repeated test.
  • the data analysis used the data analysis software Biacore T200 Evaluation Software version 3.0 of GE Medical Life Sciences.
  • Data fitting uses a model of 1: 1Binding. The kinetic constant binding rate ka (1 / Ms), dissociation rate kd (1 / s), and affinity constant KD (M) were obtained by fitting. The results are shown in Table 8.
  • Example 16 Characterization of the binding kinetics of humanized antibodies to BTLA of different species
  • a Fortebio assay was used to detect cross-reactivity between chimeric antibodies and cynomolgus monkey and mouse BTLA. Briefly, human BTLA, cynomolgus BTLA, or murine BTLA was coupled to an activated CM5 biosensor chip to achieve approximately 100-200 response units (RU), and unreacted groups were blocked with IM ethanolamine. Samples of humanized antibodies from 0.12 nM to 90 nM in increasing concentrations were injected in the SPR running buffer at 30 times / minute, and the binding response of BTLA from different sources was calculated by subtracting RU from the blank flow cell.
  • the MC38-hHVEM plasmid was electrotransformed on MC38 cells (ATCC) to construct the MC38-hHVME cell bank, and then the cells were subcloned by limiting dilution. Monoclonals were selected by flow cytometry to obtain MC38-HVEM cells.
  • Cells were inoculated at the concentration of 1 ⁇ 10 6 cells / 0.1 mL in the right subcutaneous of B-hBTLA humanized female mice. When tumors grew to about 118 mm 3 , they were randomly divided into groups according to tumor volume. There were 8 mice in each group, a total of 5 groups.
  • G1 0.9% sodium chloride injection solvent control group G2KLH (10 mg / kg) negative control group
  • G3hu18 (1 mg / kg) group G4hu18 (3 mg / kg) group
  • G5hu18 (10 mg / kg) group The administration route of all groups was intraperitoneal injection, twice a week, 7 times in a row, and the experiment was finished 4 days after the last dose. Tumor volume and body weight were measured twice a week, and mouse body weight and tumor volume were recorded. At the end of the experiment, the animals were euthanized, the tumors were removed, weighed, and photographed to calculate the relative tumor suppression rate (TGI TW %).
  • Table 9 and Figure 11 show the effects of the test substances in each group on the tumor volume of B-hBTLA mice transplanted with MC38-hHVEM cells. 21 days after the first administration, the average tumor volume of the KLH (10mg / kg) negative control group was 1560 ⁇ 256mm 3 , and the average tumor volumes of the other administration groups were 1073 ⁇ 224mm 3 , 747 ⁇ 268mm 3, and 868 ⁇ 211mm 3 .
  • the TGI TV % of each administration group was 33.7%, 56.4%, and 48.0%, and the P values were 0.175, 0.046, and 0.056, indicating that the test drug hu18 was effective against tumors at a dose level of 3mg / kg. Growth has a certain inhibitory effect.
  • a mean ⁇ standard error
  • b statistical comparison of tumor volume in the administration group and tumor volume in the KLH negative control group 21 days after administration, t-test.
  • Table 10 and Figure 12 show the effects of the test items in each group on the body weight of MC38-hHVEM cell transplanted B-hBTLA mice. All the experimental animals had good movement and eating conditions during the administration, and the weight of the animals in each administration group increased to a certain extent. There was no death of experimental animals during the experiment. 21 days after the administration, compared with the weight of the KLH negative control mice, there was no significant change in the weight of the mice in each administration group (P> 0.05), indicating that the experimental animals were well tolerated by the test product.
  • a mean ⁇ standard error
  • b statistical comparison of body weight of the administration group and KLH negative control group after 21 days of administration, t-test.
  • Example 18 Humanized antibody hu18 has no ADCC effector function
  • ADCC is initiated when the antibody binds to a cell surface target protein and then connects to an Fc ⁇ receptor (Fc ⁇ R) expressed on an effector cell.
  • Fc ⁇ R Fc ⁇ receptor
  • human IgG1 has a significantly higher binding affinity for Fc ⁇ R than IgG4, especially for Fc ⁇ R-I and Fc ⁇ R-IIIA, and this affinity is related to the intensity of ADCC activated by IgG1.
  • CDC is activated.
  • the detection of antibody binding to Fc ⁇ R and C1q can serve as a basic indicator of ADCC and CDC. Therefore, the present invention uses biacore T200 (GE) to assess the kinetic affinity of the monoclonal antibody for the binding of the main Fc ⁇ R.
  • GE biacore T200
  • GE's anti-His antibody is immobilized on the sensor chip.
  • Various Fc receptors including recombinant human Fc ⁇ RIIIA (CD16a) V176, recombinant human Fc ⁇ RIIA (CD32a) R167, recombinant human Fc ⁇ RI (CD64) and recombinant human FcRn were captured and then injected with a series of diluted recombinant human anti-BTLA antibodies (i.e. hu18), to detect and analyze the binding properties of the interaction.
  • hu18 is an IgG4 subtype antibody
  • hu18-IgG1 is an IgG1 subtype, which shares the same Fab as hu18 and serves as a positive control.

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Abstract

涉及抗BTLA抗体或其抗原结合片段，所述抗体或其抗原结合片段包含至少一个选自SEQ ID NO：7、8、9、10、11、12、16、17、18、22、23、24、31、32和33的轻链CDR结构域和/或至少一个选自SEQ ID NO：1、2、3、4、5、6、13、14、15、19、20、21、25、26、27、28、29和30的重链CDR结构域。还涉及编码所述抗体或其抗原结合片段的核酸分子、相应的表达载体和宿主细胞，以及所述抗体或其抗原结合片段、核酸分子、表达载体和宿主细胞的治疗用途。

Description

抗BTLA抗体 技术领域
本发明属于生物技术领域，具体涉及结合BTLA的抗体或其抗原结合片段及其用途。更具体而言，本发明涉及识别人BTLA并可用于治疗或预防肿瘤、感染性疾病、炎性、自身免疫性疾病等的活性抗体。
背景技术
正性和负性共刺激信号在B细胞和T细胞活性的调节中起决定性作用，而且已证实介导这些信号的分子是免疫调节剂的有效靶标。除T细胞受体(TCR)参与外，幼稚T细胞的最佳活化也需要正性共刺激，而负性共刺激则被认为是自身免疫耐受性的获得以及效应T细胞功能的终止所需要的。与抗原呈递细胞(APC)表面上的B7.1或B7.2相互作用时，原型T细胞共刺激分子CD28响应TCR参与而发出促进T细胞增殖和分化的信号，但是CD28同源物细胞毒性T淋巴细胞抗原-4(CTLA-4)介导T细胞增殖和效应子功能的抑制(Chambers等，Ann.Rev.Immunol.，19：565-594，2001；Egen等，Nature Immunol，3：611-618，2002)。已发现与B7家族同源的若干新分子(Abbas等，Nat.Med.，5：1345-6，1999；Coyle等，Nat.Immunol.，2：203-9，2001；Carreno等，Annu.Rev.Immunol.，20：29-53，2002；Liang等，Curr.Opin.Immunol.，14：384-90，2002)，并且刚开始阐明它们在T细胞活化中的作用。
B和T淋巴细胞弱化子(BTLA)是CD28家族的成员，该家族还包括CD28、ICOS、CTLA-4和PD-1。根据加入单抗后对提高T细胞增殖的功能作用，发现了该家族最初的成员CD28和ICOS具有免疫激活作用(Hutloff等，1999)。而BTLA、CTLA-4和PD-1等被描述为负性调节蛋白。若干体内研究证实了BTLA在淋巴细胞应答中的抑制作用。由Murphy与同事(Washington University St.Louis)制备的BTLA缺陷型小鼠在响应T依赖性抗原产生的IgG方面表现出3倍增加。另外，自BTLA -小鼠分离的T细胞和B细胞对分别使用CD3-和抗IgM进行抗原-受体刺激显示较大的增殖应答(Watanabe，2003)。在过量表达研究中，发现BTLA与B细胞受体复合物以及与T细胞受体缔合。与该研究结果一致，在BTLA缺陷型淋巴细胞中，使用ConA(T细胞)或LPS(B细胞)进行抗原-受体非依赖性刺激不受影响，而且使用抗BTLA抗体也不能被调节。已经表明BTLA敲除小鼠随时间推移发生自发性自身免疫病，且寿命缩短(Oya，2008)。BTLA敲除小鼠 显示在自身免疫性脑脊髓炎(EAE)和变应性气道炎症模型中疾病严重程度加剧，这两种模型均有赖于T细胞活性(Watanabe，2005；Deppong，2006)。
已经表明疱疹病毒进入介体(HVEM)是BTLA的配体(Scully等，2005)。HVEM是I型跨膜糖蛋白，属于TNF受体超家族的成员，带有4个胞外半胱氨酸富含区域(CDRs)，包含6个假性重复的半胱氨酸。BTLA及HVEM主要通过在细胞表面的动态表达来调节T细胞和APC的功能。BTLA与配体结合不仅抑制T细胞增殖，下调T细胞活化标志CD25，还可以抑制IFN-γ，IL-2，IL-4，和IL-10等的产生，但不能诱导细胞凋亡。HVEM与BTLA结合导致T细胞活化和增殖的下调(Sedy，2005)。这些研究结果表明，BTLA的表达或BTLA-HVEM结合情况与T细胞的活化与增值密切相关。
抗体可用作治疗药物。某些抗体在体内用作治疗药物时可引起不需要的抗体免疫原性。因为大多数单克隆抗体来源于啮齿动物，所以在人中重复使用导致产生针对治疗性抗体(例如，人抗小鼠抗体或HAMA)的免疫应答。这类免疫应答至少导致丧失治疗功效，而最高则导致潜在致死过敏反应。降低啮齿动物抗体的免疫原性的一种方法包括嵌合抗体的产生，其中将小鼠可变区(Fv)与人恒定区融合(Liu等(1987)Proc.Natl.Acad.Sci.USA 84：3439-43)。然而，用人可变区和小鼠恒定区的杂合体注射的小鼠发生针对人可变区的强抗体应答，这就表明这种嵌合抗体中的完整啮齿动物Fv区的保留仍可在患者中引起有害的免疫原性。
另外，将啮齿动物可变结构域的互补决定区(CDR)环移植到人构架上(即人源化)已被用于进一步将啮齿动物序列减至最低。Jones等(1986)Nature 321：522；Verhoeyen等(1988)Science 239：1534。然而，CDR环交换仍不能均匀产生具有与起始抗体相同的结合性质的抗体。在人源化抗体中，常常还需要构架残基(FR)(参与CDR环支持的残基)改变以保持抗原结合亲和力。Kabat等(1991)J.Immunol.147：1709。虽然已经报道了许多人源化抗体构建体中CDR移植和构架残基保持的使用，但是难以预测特定序列是否可产生具有所需结合性质以及间或具有生物学性质的抗体。参见例如Queen等，(1989)Proc.Natl.Acad.Sci.USA 86：10029；Gorman等，(1991)Proc.Natl.Acad.Sci.USA 88：4181；及Hodgson，(1991)Biotechnology(NY)，9：421-5。此外，大部分现有研究对动物轻链和重链可变序列使用不同人序列，致使这种研究的预测性成问题。已在使用已知抗体的序列，或者更通常使用具有已知X射线晶体结构的抗体例如抗体NEW和KOL的序列。参见例如Jones等，同上；Verhoeyen等，同上；以及Gorman等，同上。已经报道了少数人源化构建体的确切序列信息。
存在对用于治疗人病症(例如炎性病症、自身免疫病症和增殖性病症)的抗BTLA抗体、特别是抗BTLA单克隆抗体的需要。这种抗体可优选在人受试者中具有低免疫原性，允 许重复给予而无不良免疫应答。
发明内容
本发明涉及在一种或多种抗人BTLA抗体或其抗原结合片段，以及所述抗体或其抗原结合片段在治疗疾病中的用途。
在一个或多个实施方式中，本发明涉及与人BTLA(B -和T -淋巴细胞弱化子)特异性结合的分离的抗体或其抗原结合片段，其包括一种或多种选自以下的性质：
A)阻断BTLA与HVEM(疱疹病毒进入介体)结合；
B)与食蟹猴BTLA交叉反应；
C)与人BTLA结合的K D≤0.28nM；或
D)不介导ADCC效应。
在一个或多个实施方式中，本发明涉及与人BTLA结合的分离抗体或其抗原结合片段，其包含至少一个选自SEQ ID NO：7、8、9、10、11、12、16、17、18、22、23、24、31、32和33的轻链CDR结构域和至少一个选自SEQ ID NO：1、2、3、4、5、6、13、14、15、19、20、21、25、26、27、28、29和30的重链CDR结构域。
在一个或多个实施方式中，本发明涉及与人BTLA结合的分离抗体或其抗原结合片段，其轻链CDR的CDR1，CDR2和CDR3的氨基酸序列如以下A-E组中的任一组所示：
组别 LCDR1 LCDR2 LCDR3
A SEQ ID NO：7 SEQ ID NO：8 SEQ ID NO：9
B SEQ ID NO：10 SEQ ID NO：11 SEQ ID NO：12
C SEQ ID NO：16 SEQ ID NO：17 SEQ ID NO：18
D SEQ ID NO：22 SEQ ID NO：23 SEQ ID NO：24
E SEQ ID NO：31 SEQ ID NO：32 SEQ ID NO：33
和/或其重链CDR的CDR1，CDR2和CDR3的氨基酸序列如以下F-K组中的任一组所示：
组别 HCDR1 HCDR2 HCDR3
F SEQ ID NO：1 SEQ ID NO：2 SEQ ID NO：3
G SEQ ID NO：4 SEQ ID NO：5 SEQ ID NO：6
H SEQ ID NO：13 SEQ ID NO：14 SEQ ID NO：15
I SEQ ID NO：19 SEQ ID NO：20 SEQ ID NO：21
G SEQ ID NO：25 SEQ ID NO：26 SEQ ID NO：27
K SEQ ID NO：28 SEQ ID NO：29 SEQ ID NO：30
在一个或多个实施方式中，本发明涉及与人BTLA结合的分离抗体或其抗原结合片 段，其重链CDR的CDR1，CDR2和CDR3的氨基酸序列和轻链CDR的CDR1，CDR2和CDR3的氨基酸序列如以下I-IX组中的任一组所示：
组别 LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3
I SEQ ID NO:7 SEQ ID NO:8 SEQ ID NO:9 SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3
II SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6
III SEQ ID NO:16 SEQ ID NO:17 SEQ ID NO:18 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15
IV SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24 SEQ ID NO:19 SEQ ID NO:20 SEQ ID NO:21
V SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24 SEQ ID NO:25 SEQ ID NO:26 SEQ ID NO:27
VI SEQ ID NO:31 SEQ ID NO:32 SEQ ID NO:33 SEQ ID NO:28 SEQ ID NO:29 SEQ ID NO:30
VII SEQ ID NO:7 SEQ ID NO:8 SEQ ID NO:9 SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6
VIII SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3
IX SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15
在一个或多个实施方式中，本发明涉及与人BTLA结合的分离抗体或其抗原结合片段，其包含轻链可变区和重链可变区，其中，轻链可变区的氨基酸序列选自SEQ ID NO：36、37、39、41、44、46、47或48所示的氨基酸序列，重链可变区的氨基酸序列选自SEQ ID NO：34、35、38、40、42、43或45所示的氨基酸序列。
在一个或多个实施方式中，本发明涉及与人BTLA结合的分离抗体或其抗原结合片段，其轻链可变区的氨基酸序列如SEQ ID NO：36所示；其重链可变区的氨基酸序列如SEQ ID NO：34所示。
在一个或多个实施方式中，本发明涉及与人BTLA结合的分离抗体或其抗原结合片段，其轻链可变区的氨基酸序列如SEQ ID NO：36所示；其重链可变区的氨基酸序列如SEQ ID NO：35所示。
在一个或多个实施方式中，本发明涉及与人BTLA结合的分离抗体或其抗原结合片段，其轻链可变区的氨基酸序列如SEQ ID NO：37所示；其重链可变区的氨基酸序列为SEQ ID NO：34所示。
在一个或多个实施方式中，本发明涉及与人BTLA结合的分离抗体或其抗原结合片段，其轻链可变区的氨基酸序列如SEQ ID NO：37所示；其重链可变区的氨基酸序列如SEQ ID NO：35所示。
在一个或多个实施方式中，本发明涉及与人BTLA结合的分离抗体或其抗原结合片段，其轻链可变区的氨基酸序列如SEQ ID NO：39所示；其重链可变区的氨基酸序列如 SEQ ID NO：38所示。
在一个或多个实施方式中，本发明涉及与人BTLA结合的分离抗体或其抗原结合片段，其轻链可变区的氨基酸序列如SEQ ID NO：41所示；其重链可变区的氨基酸序列如SEQ ID NO：40所示。
在一个或多个实施方式中，本发明涉及与人BTLA结合的分离抗体或其抗原结合片段，其轻链可变区的氨基酸序列如SEQ ID NO：41所示；其重链可变区的氨基酸序列如SEQ ID NO：42所示。
在一个或多个实施方式中，本发明涉及与人BTLA结合的分离抗体或其抗原结合片段，其轻链可变区的氨基酸序列如SEQ ID NO：44所示；其重链可变区的氨基酸序列如SEQ ID NO：43所示。
在一个或多个实施方式中，本发明涉及与人BTLA结合的分离抗体或其抗原结合片段，其轻链可变区的氨基酸序列如SEQ ID NO：46所示；其重链可变区的氨基酸序列如SEQ ID NO：45所示。
在一个或多个实施方式中，本发明涉及与人BTLA结合的分离抗体或其抗原结合片段，其轻链可变区的氨基酸序列如SEQ ID NO：47所示；其重链可变区的氨基酸序列如SEQ ID NO：45所示。
在一个或多个实施方式中，本发明涉及与人BTLA结合的分离抗体或其抗原结合片段，其轻链可变区的氨基酸序列如SEQ ID NO：48所示；其重链可变区的氨基酸序列如SEQ ID NO：45所示。
在一个或多个实施方式中，本发明涉及的与人BTLA结合的分离抗体或其抗原结合片段是IgG类型，更优选为IgG4亚型。
在一个或多个实施方式中，本发明涉及与人BTLA结合的分离抗体或其抗原结合片段是单链Fv抗体；在某些实施方式中，抗体或其抗原结合片段是Fab抗体；在某些实施方式中，抗体或其抗原结合片段是Fab’抗体；在某些实施方式中，抗体或其抗原结合片段是(Fab’) 2抗体。
在另外的实施方式中，本发明涉及包含本文所述的任意抗体或其抗原结合片段的VL结构域或VH结构域的分离多肽。
在另外的实施方式中，本发明涉及编码本文所述任意抗体或抗原结合片段的VL结构域和VH结构域的分离核酸。
在另外的实施方式中，本发明涉及包含本文所述的一种或多种抗体或其抗原结合片段和药学上可接受的载体或稀释剂的组合物。
在另外的实施方式中，本发明涉及利用本文所述的一种或多种抗体或其抗原结合片段 通过消除、抑制或降低BTLA活性来预防或治疗疾病的方法，其包括向有此需要的对象施用治疗有效量的本发明所公开的抗体或其抗原结合片段，核酸，表达载体，宿主细胞，免疫缀合物或药物组合物。优选地，所述疾病或病症的预防或治疗受益于BTLA活性的消除、抑制或降低；优选地，所示疾病或病灶选自癌症、感染性疾病或炎性疾病。
在另外的实施方式中，本发明还涉及所述抗体或抗原结合片段、核酸、表达载体、宿主细胞、免疫缀合物或药物组合物在制备用于治疗或预防疾病或病症的药物中的用途，所述的疾病或病症优选为BTLA介导的疾病，可选自癌症、感染性疾病或炎性疾病。
附图说明
图1：人BTLA胞外区蛋白的SDS-PAGE电泳图。
图2：流式细胞仪检测BTLA与HVEM的结合能力。
图3：嵌合抗体与人BTLA结合的ELISA实验。
图4：嵌合抗体阻断BTLA-HVEM结合的能力。
图5：嵌合抗体对T细胞活性的影响实验。
图6：人源化抗体与BTLA特异性结合实验。
图7：人源化抗体与293F细胞上hBTLA的结合实验。
图8：人源化抗体阻断BTLA与细胞表面的HVEM结合的实验。
图9：人源化抗体促进T细胞的激活实验。
图10：人源化抗体17与不同种属BTLA结合的对比实验。
图11：hu18对MC38-hHVEM细胞移植B-hBTLA小鼠肿瘤体积的影响。
图12：受试品对MC38-hHVEM细胞移植B-hBTLA小鼠体重的影响。
具体实施方式
哺乳动物免疫系统已发展控制T淋巴细胞和B淋巴细胞的潜在有害活性的若干途径。它们包括各种细胞因子-受体途径以及涉及受体(像CD28、CTLA-4、PD-1和BTLA)的共刺激途径。尽管CD28-B7相互作用是正性共刺激途径的例子(即CD28触发提高对抗原特异性触发物的T细胞应答)，但其它3种受体表现为抑制性共刺激途径。CTLA-4、PD-1和BTLA显示重叠但独特的表达概况，并限制T淋巴细胞和B淋巴细胞以及其它免疫细胞的活性(参见Deppong等，JImmunol 2006；Tao等，J Immunol 2005)。虽然CTLA-4与CD28竞争结合B7.1和B7.2(CD80和CD86)并为淋巴结和脾中的幼稚T细胞活化设定原始阈值，但是PD-1和BTLA各有自己独特的配体(分别为PD-L1/-L2和HVEM)，并且似乎控制外周T细胞稳态和再活化(参见Krieg等，Nat Immunol 2007)。
BTLA下调B细胞和T细胞活化。如其名称所表示的一样，B和T淋巴细胞弱化子(BTLA)在静息和活化的B淋巴细胞和T淋巴细胞两者上表达。BTLA是I型跨膜糖蛋白，具有含有若干抑制酪氨酸基序的胞质尾(Watanabe，2003)。BTLA具有CD28/CTLA-4家族成员的某种结构相似性，但其具有独特的性质。BTLA与多种免疫性疾病、炎性疾病和增殖性疾病相关。因此，开发能够阻断人BTLA-HVEM结合的单抗药物是疾病治疗的持续需要。
定义
为了可以更容易的理解本发明，某些科技术语具体定义如下。除非本文其他部分另有明确定义，否则所用的所有其他科技术语都具有本发明所述领域普通技术人员通常理解的含义。
本文中，用于细胞或受体的“活化”、“刺激”和“处理”可具有相同含义，例如细胞或受体用配体活化、刺激或处理，除非上下文另外或明确规定。“配体”包括天然和合成配体，例如细胞因子、细胞因子变体、类似物、突变蛋白和来源于抗体的结合化合物。“配体”还包括小分子，例如细胞因子的肽模拟物和抗体的肽模拟物。“活化”可指通过内部机制以及外部或环境因素调节的细胞活化。“应答/反应”，例如细胞、组织、器官或生物体的应答，包括生化或生理行为(例如生物区室内的浓度、密度、粘附或迁移、基因表达速率或分化状态)的改变，其中改变与活化、刺激或处理有关，或者与例如遗传编程等内部机制有关。
分子的“活性”可描述或指该分子与配体或受体的结合；催化活性；刺激基因表达或细胞信号转导、分化或成熟的能力；抗原活性；调节其它分子的活性等。分子的“活性”还可指在调节或保持细胞-细胞间相互作用(例如粘附)中的活性，或在保持细胞结构(例如细胞膜或细胞骨架)中的活性。“活性”也可指比活，例如[催化活性]/[mg蛋白质]或[免疫活性]/[mg蛋白质]、生物区室中的浓度等。“活性”可指先天免疫系统或适应性免疫系统的组分的调节。“增殖活性”包括促进、对以下方面是必需的或与以下方面明确有关的活性：例如正常的细胞分裂以及癌症、肿瘤、发育异常、细胞转化、转移和血管生成。
应用于动物、人、实验对象、细胞、组织、器官或生物流体的“给予”和“治疗”是指使外源药物、治疗药、诊断药或组合物与动物、人、受试者、细胞、组织、器官或生物流体接触。“给予”和“治疗”可指例如治疗、药代动力学、诊断、研究和实验方法。细胞的治疗包括使试剂与细胞接触以及使试剂与流体(其中该流体与细胞接触)接触。“给予”和“治疗”还意指体外和离体治疗，例如细胞用试剂、诊断、结合化合物或用另一种细胞的体外和离体治疗。“治疗”也包括将治疗药物例如含有本发明的任何抗体或其抗原结合片 段的组合物，经内部或外部给予有需要的患者。通常，以有效减轻受治疗的患者或群体的一种或多种疾病症状的量给予本文所述的抗体或其抗原结合片段或相应的药物组合物，而不论是通过诱导这些症状的消退还是抑制这些症状的进展达到任何临床可测量的程度。有效减轻任何特定疾病症状的治疗药物的量(亦称为“治疗有效量”)可随例如患者的疾病状态、年龄和体重及药物在患者中引起所需反应的能力等因素而变化。可通过医师或其他专业保健提供者常用于评价疾病症状的严重程度或进展状态的任何临床测量来评价该症状是否减轻。
术语“受试者”或“患者”包括任何生物体，优选动物，更优选哺乳动物(例如大鼠、小鼠、狗、猫、兔)，最优选人。
本文中，“给予”或“治疗”可通过侵入性途径例如通过注射给予本文所述的任一抗BTLA抗体或其抗原结合片段或其相应的药物组合物而得以实现。通过非侵入性途径(例如口服；例如以丸剂、胶囊剂或片剂口服)给药也落入本发明的范围内。在本发明的一个实施方式中，经静脉内、皮下、肌内、动脉内、关节内(例如在关节炎关节中)、通过吸入、气雾剂递送或肿瘤内给予抗BTLA抗体或其抗原结合片段或其药物组合物。
可用本领域已知的医疗装置给予本文所述的任一抗BTLA抗体或其抗原结合片段或其相应的组合物。例如，可用皮下注射针经注射给予本发明的药物组合物；或可用静脉注射针经注射给与本发明的药物组合物。
在某些实施方式中，可单独或组合使用本文所述的任一抗BTLA抗体或其抗原结合片段或其相应的药物组合物，以治疗或预防需要这种治疗或预防的受试者的任何疾病或病况。
本文中，术语“B和T淋巴细胞弱化子”和“BTLA”基因/蛋白质可互换使用，其包括变体、同种型、同源物、直向同源物(ortholog)和种内同源物(paralog)。例如，在某些实施方式中，人BTLA特异性抗体可与来自非人物种的BTLA交叉反应。在其它实施方式中，人BTLA特异性抗体可以是人BTLA完全特异性的，并且不具有物种交叉反应性或其它类型的交叉反应性。除非另有说明，否则术语“人BTLA”或“hBTLA”是指人BTLA序列。除非另有说明，否则人BTLA序列包括所有人同种型和BTLA变体，例如Genbank登记号为AAP44003的人BTLA的完整氨基酸序列。还存在至少两种人BTLA转录物变体，转录物变体1编码蛋白长度为289个氨基酸(GenBank登记号NP_861445)，并且与登记号AAP44003的BTLA序列有近98％同一性；转录物变体2编码蛋白长度为241个氨基酸的蛋白质(GenBank登记号NP_001078826)。
BTLA是一种免疫应答负性调节因子，其C端抑制基序具有参与抑制IL-2产生和T细胞扩繁(Watanabe等，Nat.Immunol.，4，670-679，2003；Chemnitz等，J.Immunol.， 176，6603-6614，2006)。另外，人BTLA可为与本发明所述抗体特异性结合的BTLA的胞外结构域中的表位。
具体的BTLA序列其胞外核苷酸序列一般可与SEQ ID NO：49的人BTLA的胞外区或其它同种型的核苷酸序列有至少90％同一性，并且当与其它物种(例如鼠)的BTLA氨基酸序列相比时，含有鉴定为人的氨基酸序列的氨基酸残基。在某些情况下，人BTLA胞外区可与SEQ ID NO：49的人BTLA胞外区或其它同种型或变体有至少95％或甚至至少96％、97％、98％或99％同一性。
术语“免疫应答”是指例如淋巴细胞、抗原呈递细胞、吞噬细胞、粒细胞和由上述细胞或肝产生的可溶性大分子(包括抗体、细胞因子和补体)的作用，该作用导致从人体选择性损害、破坏或清除侵入病原体、感染病原体的细胞或组织、癌细胞或者在自体免疫或病理性炎症的情况下的正常人细胞或组织。
本文所用术语“抗体”是指具有所需生物活性的任何形式的抗体。因此，其以最广义使用，具体包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、人源化抗体、完全人抗体、嵌合抗体和骆驼源化(camelized)单结构域抗体。本文所用术语“抗BTLA抗体”或抗体的“抗原结合片段”是指与BTLA结合并阻断BTLA与HVEM结合的抗体，包括抗体的片段或衍生物，通常包括抗体的抗原结合区或可变区(例如一个或多个CDR)的至少一个片段，其保持抗体的至少一些结合特异性。抗体结合片段的实例包括但不限于Fab、Fab′、F(ab′) 2和Fv片段；双抗体；线性抗体；单链抗体分子，例如sc-Fv；由抗体片段形成的纳米抗体(nanobody)和多特异性抗体。当BTLA结合活性在摩尔浓度基础上表示时，结合片段或衍生物通常保持其BTLA结合活性的至少10％。优选结合片段或衍生物保持抗体的BTLA结合亲和力的至少20％、50％、70％、80％、90％、95％或100％或更高。还预期抗BTLA抗原结合片段可包括不明显改变其生物活性的保守或非保守氨基酸取代(称为抗体的“保守变体”或“功能保守变体”)。
“分离抗体”是指抗体或其抗原结合片段的纯化状态，且在这种情况下意指该分子基本不含其它生物分子，例如核酸、蛋白质、脂质、糖或其它物质例如细胞碎片和生长培养基。术语“分离(的)”并非意指完全不存在这类物质或不存在水、缓冲液或盐，除非它们以明显干扰本文所述抗体或其抗原结合片段的实验或治疗应用的量存在。
本文所使用的术语“功能性片段”或“抗原结合片段”尤其是指抗体片段如Fv、scFv、Fab、F(ab’)2、Fab’、scFv-Fc片段或者双抗体、或者通过化学修饰或通过掺入脂质体中应能够增加半衰期的任何片段，所述化学修饰例如添加聚(亚烷基)二醇如聚乙二醇(“聚乙二醇化，PEG化”)(被称为Fv-PEG、scFv-PEG、Fab-PEG、F(ab')2-PEG或Fab'-PEG的聚乙二醇化片段)(“PEG”为聚乙二醇)，所述片段具有EGFR结合活性。优选地， 所述功能片段由其来源抗体的重或轻可变链的部分序列构成或者包含它们，所述部分序列足以保留与其来源抗体相同的结合特异性和充分的亲和力，对于BTLA，优选至少等于其来源抗体亲和力的1/100，在更优选方式中至少等于1/10。这种功能片段将包含最少5个氨基酸，优选其来源的抗体序列的10、15、25、50和100个连续氨基酸。
“Fab片段”由一条轻链及CH1和一条重链的可变区组成。Fab分子的重链不能与另一重链分子形成二硫键。
“Fc”区含有包含抗体的CH1和CH2结构域的2个重链片段。该2个重链片段通过两个或更多个二硫键以及通过CH3结构域的疏水相互作用保持在一起。
“Fab′片段”含有一条轻链和一条重链的部分或片段，该重链的部分或片段含有VH结构域和CH1结构域以及CH1和CH2结构域之间的区域，使得2个Fab′片段的2条重链之间可形成链间二硫键以形成F(ab′) 2分子。
“F(ab′)2片段”含有2条轻链和2条含有介于CH1和CH2结构域之间的恒定区的一部分的重链，使得在2条重链之间形成链间二硫键。因此，F(ab′) 2片段由通过2条重链间的二硫键保持在一起的2个Fab′片段组成。
“Fv区”包含来源于重链和轻链两者的可变区，但缺乏恒定区。
术语“单链Fv”或“scFv”抗体是指包含抗体的VH和VL结构域的抗体片段，其中这些结构域存在于单条多肽链中。scFv多肽一般还包含VH和VL结构域之间的多肽接头，其使scFv能够形成用于抗原结合的所需结构。
“结构域抗体”是只含有重链可变区或轻链可变区的免疫功能性免疫球蛋白片段。在某些情况下，两个或更多个VH区与肽接头共价连接形成二价结构域抗体。二价结构域抗体的2个VH区可靶向相同或不同的抗原。
“二价抗体”包含2个抗原结合部位。在某些情况下，2个结合部位具有相同的抗原特异性。然而，二价抗体可以是双特异性的。
除非另有规定，否则本文所用“抗BTLA抗体”是指针对人BTLA或其变体或其任何抗原片段产生的抗体。
本文所用术语“单克隆抗体”是指获自基本均质抗体群的抗体，即组成该群的各个抗体除可少量存在的可能天然存在的突变之外是相同的。单克隆抗体是高度特异性的，针对单一抗原表位。相比之下，常规(多克隆)抗体制备物通常包括大量针对不同表位(或对不同表位有特异性)的抗体。修饰语“单克隆”表明获自基本均质抗体群的抗体的特征，且不得解释为需要通过任何特定方法产生抗体。例如，本发明使用的单克隆抗体可通过杂交瘤方法制备，或可通过重组DNA方法制备。还可采用噬菌体抗体文库分离“单克隆抗体”。
在某些实施方式中，单克隆抗体包括“嵌合”抗体(免疫球蛋白)，其中重链和/或轻链 的一部分与来源于特定物种或属于特定抗体类别或亚类的抗体的相应序列相同或同源，而该链的其余部分与来源于另一物种或属于另一抗体类别或亚类的抗体以及这类抗体的片段的相应序列相同或同源，只要它们具有所需生物活性即可。
本文所用“嵌合抗体”是具有第一抗体的可变结构域和第二抗体的恒定结构域的抗体，其中第一抗体和第二抗体来自不同物种。通常可变结构域获自啮齿动物等实验动物的抗体(“亲代抗体”)，而恒定结构域序列获自人抗体，使得与亲代啮齿动物抗体相比，所得嵌合抗体在人受试者中诱导不良免疫应答的可能性较低。
在某些实施方式中，本文的单克隆抗体还包括骆驼源化单结构域抗体。参见例如Muyldermans等(2001)Trends Biochem.Sci.26：230；Reichmann等(1999)J.Immunol.Methods231：25。
本文所用术语“双抗体”是指具有两个抗原结合部位的小抗体片段，所述片段包含在同一多肽链(VH-VL或VL-VH)中与轻链可变结构域(VL)连接的重链可变结构域(VH)。通过使用短得不允许在同一链的两个结构域之间配对的接头，迫使该结构域与另一链的互补结构域配对并产生两个抗原结合部位。
本文所用术语“人源化抗体”是指含有来自人和非人(例如鼠、大鼠)抗体的序列的抗体形式。一般而言，人源化抗体包含基本所有的至少一个、通常两个可变结构域，其中所有或基本所有的高变区相当于非人免疫球蛋白的高变区，而所有或基本所有的构架(FR)区是人免疫球蛋白序列的构架区。人源化抗体任选可包含至少一部分的人免疫球蛋白恒定区(Fc)。
总的来说，已知基本的抗体结构单位包含四聚体。每个四聚体包括两个相同的多肽链对，每对具有一条“轻”链(约25kDa)和一条“重”链(约50-70kDa)。每条链的氨基端部分或片段可包括主要负责抗原识别的约100-110个或更多个氨基酸的可变区。每条链的羧基端部分或片段可限定主要负责效应子功能的恒定区。通常将人轻链归类为κ和λ轻链。此外，通常将人重链归类为μ、δ、γ、α或ε，并将抗体的同种型分别定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过约12个或更多个氨基酸的“J”区连接，其中重链还包括约10多个氨基酸的“D”区。一般参见Fundamental Immunology第7章(Paul，W.主编，第2版。Raven Press，N.Y.(1989))。
每个轻链/重链对的可变区配对形成抗体结合部位。因此，完整IgG抗体一般具有2个结合部位。除双功能或双特异性抗体以外，2个结合部位通常相同。
通常，各链均具有相同的通过3个高变区(亦称为互补决定区或CDR)连接的相对保守构架区(FR)的通用结构。每对的2条链的CDR通常通过构架区对准，使之能与特定表位结合。总的来说，轻链和重链两者从N端到C端包含结构域FR1、CDR1、FR2、CDR2、 FR3、CDR3和FR4。一般按照以下文献中的定义指定每个结构域的氨基酸：Sequences of Proteins of Immunological Interest，Kabat等；National Institutes of Health，Bethesda，Md.；第5版；NIH公告号91-3242(1991)；Kabat(1978)Adv.Prot.Chem.32：1-75；Kabat等(1977)J.Biol.Chem.252：6609-6616；Chothia等(1987)J Mol.Biol.196：901-917或Chothia等(1989)Nature 342：878-883。
本文所用术语“高变区”是指负责抗原结合的抗体氨基酸残基。高变区包含“互补决定区”或“CDR”的氨基酸残基。本文所用术语“构架”或“FR”残基是指本文定义为CDR残基的超变区残基以外的可变结构域残基。
“有效量”包括足以改善或预防医学病症的症状或体征的量。有效量还意指足以允许或便于诊断的量。特定患者或兽医对象的有效量可根据例如待治疗的病况、患者的总体健康状况、给药的方法途径和剂量及副作用的严重程度等因素而变化。有效量可以是避免明显副作用或毒性作用的最大剂量或给药方案。结果可导致诊断测量或参数改进至少5％、通常为至少10％、更常为至少20％、最常为至少30％、优选至少40％、更优选至少50％、最优选至少60％、理想为至少70％、更理想为至少80％、最理想为至少90％，其中100％定义为正常受试者显示的诊断参数(参见例如Maynard等(1996)A Handbook of SOPs for Good Clinical Practice，Interpharm Press，Boca Raton，FL；Dent(2001)Good Laboratory and Good ClinicalPractice，Urch Publ.，London，UK)。
“同源性”是指两个多核苷酸序列之间或两个多肽序列之间的序列相似性。当所比较的两个序列中某一位置被相同碱基或氨基酸单体亚单元占据时，例如如果两个DNA分子每个中某一位置均被腺嘌呤占据，则分子在该位置上是同源的。两个序列间的同源性百分比是两个序列共有的匹配位置或同源位置数除以所比较的位置数×100的函数。例如，如果在对序列进行最佳比对时两个序列10个位置中有6个匹配或同源，则两个序列是60％同源的。一般在比对两个序列时进行比较以得到最大百分比同源性。
本文中，“免疫病症”包括例如病理性炎症、炎性病症和自身免疫病症或疾病。“免疫病症”还指感染、持续感染和增殖性病况，例如癌症、肿瘤和血管生成，包括抵抗免疫系统根除的感染、肿瘤和癌症。“癌性病况”包括例如癌症、癌细胞、肿瘤、血管生成和癌前病况，例如发育异常。本文所述的“免疫病灶”和“癌性病况”优选均为BTLA所介导。
“炎性病症”意指其中病理全部或部分由例如免疫系统细胞的数目改变、迁移速率改变或活化改变引起的病症或病理状况。免疫系统细胞包括例如T细胞、B细胞、单核细胞或巨噬细胞、抗原呈递细胞(APC)、树突细胞、小胶质细胞、NK细胞、NKT细胞、嗜中性粒细胞、嗜酸性粒细胞、肥大细胞或与免疫学特别有关的任何其它细胞，例如产细 胞因子的内皮或上皮细胞。本文所述的“炎性病症”优选为BTLA所介导。
“分离核酸分子”意指基因组、mRNA、cDNA或合成来源或其某些组合的DNA或RNA，其不与多核苷酸的全部或部分缔合(其中分离多核苷酸天然存在)，或者与其天然不连接的多核苷酸连接。对于本公开内容的目的，应当理解的是，“包含”特定核苷酸序列的“核酸分子”不包括完整的染色体。除规定序列外，“包含”规定核酸序列的分离核酸分子还可包括多达10个或甚至多达20个或更多个其它蛋白质或其部分或片段的编码序列，或者可包括有效连接的控制所列举的核酸序列编码区表达的调节序列，和/或可包括载体序列。
本文所用表述“细胞”、“细胞系”和“细胞培养物”可互换使用，所有这类名称包括子代。因此，术语“转化体”和“转化细胞”包括原代主题细胞和由其得到的培养物而不论传递次数。还要理解的是，由于有意的或不经意的突变所致，所有子代的DNA内容物可能并不完全相同。包括了在最初转化的细胞中筛选的具有相同功能或生物活性的突变型子代。虽然指定不同名称，但从上下文看将是清楚的。
本发明所述的宿主细胞可以为原核宿主细胞、真核宿主细胞或噬菌体。原核宿主细胞可以为大肠杆菌、枯草杆菌、链霉菌或奇异变形菌等。真核宿主细胞，可以为如巴斯德毕赤酵母、酿酒酵母、裂殖酵母、木霉等真菌，如草地粘虫等昆虫细胞，如烟草等植物细胞，如BHK细胞、CHO细胞、COS细胞、骨髓瘤细胞等哺乳动物细胞。在一些实施方案中，本发明所述宿主细胞优选为哺乳动物细胞，更优选BHK细胞、CHO细胞、NSO细胞或COS细胞。
本文所用“聚合酶链式反应”或“PCR”一般目标区末端或以外的序列信息必须是可得的，使得可设计寡核苷酸引物；这些引物可与待扩增模板的相反链的序列相同或相似。2个引物的5′端核苷酸可与扩增材料的末端一致。可采用PCR扩增特定的RNA序列、来自全基因组DNA的特定DNA序列和自细胞总RNA、噬菌体或质粒序列转录的cDNA等。一般参见Mullis等(1987)Cold Spring Harbor Symp.Quant.Biol.51：263；Erlich主编(1989)PCR TECHNOLOGY(Stockton Press，N.Y)。本文所用PCR被视为是一个(但不是唯一的)用于扩增核酸试验样品的核酸聚合酶反应方法的实例，该方法包括使用作为引物的已知核酸和核酸聚合酶以扩增或产生特定的核酸片段。
人BTLA特异性抗体
本发明总的来讲涉及与BTLA结合的分离抗体或其抗原结合片段和这类抗体或其抗原结合片段的用途。更具体而言，本发明提供分离的抗BTLA抗体以及这些抗体或其抗原结合片段在疾病治疗和预防中的应用。抗BTLA抗体的实例包括但不限于本文所述的 ch7、ch12、ch17、ch22、ch27、hu17、hu18和hu19。
本发明提供与人BTLA(B -和T -淋巴细胞弱化子)结合的分离抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包括以下一种或多种性质：A)阻断BTLA与HVEM(疱疹病毒进入介体)结合；B)与食蟹猴BTLA交叉反应；C)与人BTLA结合的K D≤0.28nM。
在一个或多个实施方式中，本发明提供的与人BTLA结合的分离抗体或其抗原结合片段的轻链CDR至少包含一个选自SEQ ID NO：7、8、9、10、11、12、16、17、18、22、23、24、31、32和33的轻链CDR。例如，在某些实施方式中，本发明提供的与人BTLA结合的分离抗体或其抗原结合片段的轻链CDR中LCDR1可选自SEQ ID NO:7、10、16、22和31中的任一CDR序列；在某些实施方式中，本发明提供的与人BTLA结合的分离抗体或其抗原结合片段的轻链CDR中LCDR2可选自SEQ ID NO:8、11、17、23和32中的任一CDR序列；本发明提供的与人BTLA结合的分离抗体或其抗原结合片段的轻链CDR中LCDR3可选自SEQ ID NO:9、12、18、24和33中的任一CDR序列。
在某些实施方式中，本发明提供的与人BTLA结合的分离抗体或其抗原结合片段的轻链CDR中，LCDR1选自SEQ ID NO:7、10、16、22和31中的任一CDR序列；LCDR2选自SEQ ID NO:8、11、17、23和32中的任一CDR序列；LCDR3选自SEQ ID NO:9、12、18、24和33中的任一CDR序列。
在某些实施方式中，本发明提供的与人BTLA结合的分离抗体或其抗原结合片段中，其轻链的LCDR1、LCDR2和LCDR3序列如以下A-E组中的任一组所示：
组别 LCDR1 LCDR2 LCDR3
A SEQ ID NO：7 SEQ ID NO：8 SEQ ID NO：9
B SEQ ID NO：10 SEQ ID NO：11 SEQ ID NO：12
C SEQ ID NO：16 SEQ ID NO：17 SEQ ID NO：18
D SEQ ID NO：22 SEQ ID NO：23 SEQ ID NO：24
E SEQ ID NO：31 SEQ ID NO：32 SEQ ID NO：33
在一个或多个实施方式中，本发明提供的与人BTLA结合的分离抗体或其抗原结合片段的重链CDR至少包括一个选自SEQ ID NO：1、2、3、4、5、6、13、14、15、19、20、21、25、26、27、28、29和30的重链CDR。例如，在某些实施方式中，本发明提供的与人BTLA结合的分离抗体或其抗原结合片段的重链CDR中HCDR1可选自SEQ ID NO:1、4、13、19、25和28中的任一CDR序列；在某些实施方式中，本发明提供的与人BTLA结合的分离抗体或其抗原结合片段的重链CDR中HCDR2可选自SEQ ID NO:2、 5、14、20、26和29中的任一CDR序列；在某些实施方式中，本发明提供的与人BTLA结合的分离抗体或其抗原结合片段的重链CDR中HCDR3可选自SEQ ID NO:3、6、15、21、27和30中的任一CDR序列。
在某些实施方式中，本发明提供的与人BTLA结合的分离抗体或其抗原结合片段的重链CDR中，HCDR1选自SEQ ID NO:1、4、13、19、25和28中的任一CDR序列；HCDR2选自SEQ ID NO:2、5、14、20、26和29中的任一CDR序列；HCDR3选自SEQ ID NO:3、6、15、21、27和30中的任一CDR序列。
在某些实施方式中，本发明提供的与人BTLA结合的分离抗体或其抗原结合片段中，其重链CDR的HCDR1，HCDR2和HCDR3的氨基酸序列如以下F-K组中的任一组所示：
组别 HCDR1 HCDR2 HCDR3
F SEQ ID NO：1 SEQ ID NO：2 SEQ ID NO：3
G SEQ ID NO：4 SEQ ID NO：5 SEQ ID NO：6
H SEQ ID NO：13 SEQ ID NO：14 SEQ ID NO：15
I SEQ ID NO：19 SEQ ID NO：20 SEQ ID NO：21
G SEQ ID NO：25 SEQ ID NO：26 SEQ ID NO：27
K SEQ ID NO：28 SEQ ID NO：29 SEQ ID NO：30
在一个或多个实施方式中，本发明提供的与人BTLA结合的分离抗体或其抗原结合片段的轻链CDR中，LCDR1选自SEQ ID NO:7、10、16、22和31中的任一CDR序列；LCDR2选自SEQ ID NO:8、11、17、23和32中的任一CDR序列；LCDR3选自SEQ ID NO:9、12、18、24和33中的任一CDR序列；且其重链CDR中，HCDR1选自SEQ ID NO:1、4、13、19、25和28中的任一CDR序列；HCDR2选自SEQ ID NO:2、5、14、20、26和29中的任一CDR序列；HCDR3选自SEQ ID NO:3、6、15、21、27和30中的任一CDR序列。
在一个或多个实施方式中，本发明提供的与人BTLA结合的分离抗体或其抗原结合片段中，其轻链的LCDR1、LCDR2和LCDR3序列如以下A-E组中的任一组所示：
组别 LCDR1 LCDR2 LCDR3
A SEQ ID NO：7 SEQ ID NO：8 SEQ ID NO：9
B SEQ ID NO：10 SEQ ID NO：11 SEQ ID NO：12
C SEQ ID NO：16 SEQ ID NO：17 SEQ ID NO：18
D SEQ ID NO：22 SEQ ID NO：23 SEQ ID NO：24
E SEQ ID NO：31 SEQ ID NO：32 SEQ ID NO：33
和/或其重链CDR的HCDR1，HCDR2和HCDR3的氨基酸序列如以下F-K组中的任一组所示：
组别 HCDR1 HCDR2 HCDR3
F SEQ ID NO：1 SEQ ID NO：2 SEQ ID NO：3
G SEQ ID NO：4 SEQ ID NO：5 SEQ ID NO：6
H SEQ ID NO：13 SEQ ID NO：14 SEQ ID NO：15
I SEQ ID NO：19 SEQ ID NO：20 SEQ ID NO：21
G SEQ ID NO：25 SEQ ID NO：26 SEQ ID NO：27
K SEQ ID NO：28 SEQ ID NO：29 SEQ ID NO：30
应理解的是，本文公开的轻链的各CDR序列，尤其是各LCDR1、LCDR2和LCDR3序列，与重链的各CDR序列，尤其是各HCDR1、HCDR2和HCDR3序列，可任意地组合。例如，由本文定义为LCDR1的序列中的任意一条序列可与本文定义为LCDR2的序列中的任意一条、LCDR3的序列中的任意一条、HCDR1的序列中的任意一条、HCDR2的序列中的任意一条以及HCDR3的序列中的任意一条组合，形成本文所述的与人BTLA结合的分离抗体或其抗原结合片段所包含的完整的6个CDR结构域。
因此，举例而言，在一个或多个实施方式中，本发明提供的与人BTLA结合的分离抗体或其抗原结合片段中，其重链CDR的HCDR1，HCDR2和HCDR3的氨基酸序列和轻链CDR的LCDR1，LCDR2和LCDR3的氨基酸序列如以下I-IX组中的任一组所示：
组别 LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3
I SEQ ID NO:7 SEQ ID NO:8 SEQ ID NO:9 SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3
II SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6
III SEQ ID NO:16 SEQ ID NO:17 SEQ ID NO:18 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15
IV SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24 SEQ ID NO:19 SEQ ID NO:20 SEQ ID NO:21
V SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24 SEQ ID NO:25 SEQ ID NO:26 SEQ ID NO:27
VI SEQ ID NO:31 SEQ ID NO:32 SEQ ID NO:33 SEQ ID NO:28 SEQ ID NO:29 SEQ ID NO:30
VII SEQ ID NO:7 SEQ ID NO:8 SEQ ID NO:9 SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6
VIII SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3
IX SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15
在一个或多个实施方式中，本发明提供的与人BTLA结合的分离抗体或其抗原结合片段包含选自SEQ ID NO：36、37、39、41、44、46、47和48中任一所示的轻链可变区的氨基酸序列；和/或选自SEQ ID NO：34、35、38、40、42、43和45中任一所示的重链可变区的氨基酸序列。
在一个或多个实施方式中，本发明的分离抗体包含重链恒定区，优选人恒定区，例如γ1、γ2、γ3或γ4人重链恒定区或其变体。在另一个实施方式中，本发明的分离抗体包含轻链恒定区，优选人轻链恒定区，例如λ或κ人轻链区或其变体。通过举例而非限制，人重链恒定区可为γ4，人轻链恒定区可为κ。在一个实施方式中，抗体的Fc区可以是具有保守修饰或保守取代或保守突变的γ4。
本文中，术语“保守修饰变体”或“保守取代”或“保守突变”是指蛋白质中的氨基酸被具有相似性质(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸取代，使得在不改变蛋白质的生物活性的情况下可频繁进行变化。本领域技术人员认识到，多肽非必要区的单个氨基酸取代一般不会显著改变生物活性(参见例如Watson等，(1987)Molecular Biology of the Gene，The Benjamin/Cummings Pub.Co.，第224页(第4版))。另外，结构或功能上相似的氨基酸的取代不太可能破坏生物活性。本发明各种实施方式的抗体或其抗原结合片段包含具有下述序列的多肽链：与本文公开的具体氨基酸序列(例如SEQ ID NO：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47和48)比较时，其包括至多0(无变化)、1、2、3、4、5、6、7、8、9、10、12、15、20个或更多个保守氨基酸取代；例如，可包括0-20个氨基酸取代，或包括1-15、1-10、1-8、1-5个取代，或取代的数量在上述任意两个数值所组成的范围内。
因此，本发明也包括本发明抗体或其抗原结合片段的功能保守变体，即本发明抗体或其抗原结合片段中的一个或多个氨基酸残基在不改变抗体的整体构象和功能的情况下发生改变的变体，包括但不限于氨基酸被具有类似性质的氨基酸置换。通常，置换的数量可在前文所述的范围内，如0-20个保守取代。
在一个或多个实施方式中，本发明与人BTLA结合的分离抗体或其抗原结合片段是单链Fv抗体；在某些实施方式中，该抗体或其抗原结合片段是Fab抗体；在某些实施方式中，该抗体或其抗原结合片段是Fab’抗体；在某些实施方式中，该抗体或其抗原结合 片段是(Fab’) 2抗体。
在一个或多个实施方式中，本发明与人BTLA结合的分离抗体或其抗原结合片段的轻链可变区的氨基酸序列如SEQ ID NO：36所示，重链可变区的氨基酸序列如SEQ ID NO：34或35所示。
在一个或多个实施方式中，本发明与人BTLA结合的分离抗体或其抗原结合片段的轻链可变区的氨基酸序列如SEQ ID NO：37所示，重链可变区的氨基酸序列如SEQ ID NO：34或35所示。
在一个或多个实施方式中，本发明与人BTLA结合的分离抗体或其抗原结合片段的轻链可变区的氨基酸序列如SEQ ID NO：39所示，重链可变区的氨基酸序列如SEQ ID NO：38所示。
在一个或多个实施方式中，本发明与人BTLA结合的分离抗体或其抗原结合片段的轻链可变区的氨基酸序列如SEQ ID NO：41所示，重链可变区的氨基酸序列如SEQ ID NO：40或42所示。
在一个或多个实施方式中，本发明与人BTLA结合的分离抗体或其抗原结合片段的轻链可变区的氨基酸序列如SEQ ID NO：44所示，重链可变区的氨基酸序列如SEQ ID NO：43所示。
在一个或多个实施方式中，本发明与人BTLA结合的分离抗体或其抗原结合片段的轻链可变区的氨基酸序列如SEQ ID NO：46、47或48所示，重链可变区的氨基酸序列如SEQ ID NO：45所示。
本发明还提供分离的核酸，例如DNA，其编码本发明的分离抗体或其抗原结合片段。在某些实施方式中，本发明分离的核酸编码包含至少一个成熟抗体轻链可变(VL)结构域和至少一个成熟抗体重链可变(VH)结构域的抗体或其抗原结合片段，其中VL结构域包含至少3个具有选自SEQ ID NO：7-8、10-12、16-18、22-24和31-33的序列的CDR，VH结构域包含至少3个具有选自SEQ ID NO：1-3、4-6、13-15、19-21、25-27和28-30的序列的CDR。在一个实施方式中，分离核酸分别编码SEQ ID NO：36和SEQ ID NO：34的成熟轻链和重链可变区序列。在一个实施方式中，分离核酸分别编码SEQ ID NO：37和SEQ ID NO：35的成熟轻链和重链可变区序列。在一个实施方式中，分离核酸分别编码SEQ ID NO：39和SEQ ID NO：38的成熟轻链和重链可变区序列。在一个实施方式中，分离核酸分别编码SEQ ID NO：41和SEQ ID NO：40的成熟轻链和重链可变区序列。在一个实施方式中，分离核酸分别编码SEQ ID NO：41和SEQ ID NO：42的成熟轻链和重链可变区序列。在一个实施方式中，分离核酸分别编码SEQ ID NO：44和SEQ ID NO：43的成熟轻链和重链可变区序列。在一个实施方式中，分离核酸分别编码SEQ ID NO： 46和SEQ ID NO：45的成熟轻链和重链可变区序列。在一个实施方式中，分离核酸分别编码SEQ ID NO：47和SEQ ID NO：45的成熟轻链和重链可变区序列。在一个实施方式中，分离核酸分别编码SEQ ID NO：48和SEQ ID NO：45的成熟轻链和重链可变区序列。在一个或多个实施方式中，分离核酸编码单个核酸分子上的轻链和重链两者，而在其它实施方式中，在两个或更多个独立核酸分子上编码轻链和重链。
本发明还提供包含本发明的分离核酸的表达载体。还提供包含本发明的表达载体的宿主细胞。本发明还涉及产生本发明的抗体或其抗原结合片段的方法。
本发明还涉及与本文所述的抗体ch7、ch12、ch17、ch22、ch27、hu17、hu18和hu19一样结合人BTLA上的相同表位的抗体或其抗原结合片段，例如能够交叉阻断任何本发明抗体的结合的抗体。
疾病及其治疗或预防
本发明还提供用本发明的抗体或其抗原结合片段(优选人源化抗体)治疗或预防需要用分离抗体或其抗原结合片段治疗的受试者(包括人受试者)的方法。该方法通常包括给予需要的对象治疗或预防有效量的本发明任一实施方式所述的抗体或其抗原结合片段，或含有所述抗体或其抗原结合片段的药物组合物。可根据实际情况选择合适的给药方式，包括但不限于口服、、静脉内、皮下、肌内、动脉内、关节内(例如在关节炎关节中)、通过吸入、气雾剂递送或肿瘤内给予等。
受试者通常患有BTLA介导的疾病，即受益于BTLA活性的消除、抑制或降低的疾病。通常，BTLA介导的疾病为与免疫抑制相关所有疾病，包括自身免疫性疾病、移植排斥反应、肿瘤等。所述的肿瘤包括黑色素瘤、乳腺癌、肾癌、前列腺癌、结肠癌、肺癌、胰腺癌、骨癌、皮肤癌、头或颈癌、子宫癌、卵巢癌、直肠癌、肛区癌、胃癌、睾丸癌、食道癌、小肠癌、宫颈癌、阴道癌、何杰金氏病、非何杰金氏淋巴瘤、食道癌、内分泌系统癌、甲状腺癌、肾上腺爱、软组织癌、尿道癌、慢性或急性白血病、慢性髓细胞样白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病、儿童期实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统的赘生物、原发性中枢神经系统淋巴瘤、肿瘤血管发生、脊髓轴肿瘤、脑干胶质瘤、垂体腺瘤、卡波西氏肉瘤、表皮样癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症、其包括石棉所诱发的那些，和所述癌症的组合。所述自身免疫性疾病包括器官特异性自身免疫病和系统性自身免疫病；所述器官特异性自身免疫病包括慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症、急性特发性多神经炎等；所述系 统性自身免疫病包括系统性红斑狼疮、类分湿关节炎、系统性血管炎、硬皮病、天胞疮、皮肌炎、混合性结缔组织病、自身免疫性溶血性贫血、溃疡性结肠炎等。
这种治疗方法还可包括给予一种或多种其它的治疗药物，例如肿瘤疫苗、标准肿瘤化学疗法治疗药物、其它免疫诱导剂。
药盒
本发明还提供呈药盒形式的包括本发明的联合药物的组分的药盒。本发明的药盒包括一种或多种组分，包括但不限于本文所述的与BTLA特异性结合的抗体或抗原结合片段(例如抗体ch7、ch12、ch17、ch22、ch27、hu17、hu18和hu19，但不限于上述抗体)以及一种或多种其它组分，包括但不限于本文所述药学上可接受的载体和/或化疗药物。可将所述抗体或抗原结合片段和/或化疗药物配成纯的组合物或在药物组合物中与药学上可接受的载体组合。
在一个实施方式中，药盒包括在一个容器(例如无菌玻璃或塑料小瓶)中的本发明的抗体或其抗原结合片段(例如抗体ch7、ch12、ch17、ch22、ch27、hu17、hu18和hu19，但不限于上述抗体)或其药物组合物，以及在另一个容器(例如无菌玻璃或塑料小瓶)中的本发明的抗体或其抗原结合片段或其药物组合物和/或化疗药物。
在本发明的另一个实施方式中，药盒包括本发明的联合药物，包括在单个公用容器中的抗体或抗原结合片段(例如抗体ch7、ch12、ch17、ch22、ch27、hu17、hu18和hu19，但不限于上述抗体)连同药学上可接受的载体，任选与配制在一起的一种或多种化疗药物组分联合，任选在药物组合物中。
药盒可包括药品说明书，其包括有关药盒中的药物组合物和剂型的信息。这类信息一般帮助患者和医师有效安全地使用所附药物组合物和剂型。例如，药品说明书中可提供有关本发明的联合药物的下列信息：药代动力学、药效学、临床研究、效果参数、适应证和用法、禁忌证、警告事项、预防措施、不良反应、过量使用、适当剂量和给药、供应规格、合适的贮存条件、参考、生产商/批发商信息和专利信息。
药物组合物和给药
本发明也包括含有本文所述任一抗人BTLA抗体或其抗原结合片段的药物组合物。本文使用的术语“药物组合物”表示组合在一起以实现某种特定目的的至少一种药物以及任选地可药用载体或辅料的组合。在某些实施方案中，所述药物组合物包括在时间和/或空间上分开的组合，只要其能够共同作用以实现本发明的目的。例如，所述药物组合物中所含的成分(例如根据本发明的抗体、核酸分子、核酸分子组合和/或缀合物)可以以整 体施用于对象，或者分开施用于对象。当所述药物组合物中所含的成分分开地施用于对象时，所述成分可以同时或依次施用于对象。优选地，所述可药用载体是水、缓冲水溶液、等渗盐溶液如PBS(磷酸盐缓冲液)、葡萄糖、甘露醇、右旋葡萄糖、乳糖、淀粉、硬脂酸镁、纤维素、碳酸镁、0.3％甘油、透明质酸、乙醇或聚亚烷基二醇如聚丙二醇、甘油三酯等。所用可药用载体的类型尤其依赖于根据本发明的组合物是否配制为用于口服、鼻、皮内、皮下、肌内或静脉施用。根据本发明的组合物可包含润湿剂、乳化剂或缓冲液物质作为添加剂。本发明的免疫缀合物可含有与治疗剂偶联的本文任一实施方式所述的抗体或其抗原结合片段，优选地所述治疗剂为本领域周知和常用于制备免疫缀合物的免疫毒素、放射性同位素、药物或细胞毒剂。
为了制备本发明的抗人BTLA抗体或其抗原结合片段的药物组合物或无菌组合物，可将该抗体或其抗原结合片段与药学上可接受的载体或赋形剂混合。参见例如Remington’s Pharmaceutical Sciences and U.S.Pharmacopeia：National Formulary，Mack Publishing Company，Easton，PA(1984)。
可将本发明的药物组合物制备成本领域周知的各种合适的给药剂型，包括但不先于冻干粉、膏剂、水溶液计或混悬剂等。可通过与可接受的载体、赋形剂或稳定剂混合来制备以下形式的治疗剂和诊断剂的剂型：例如冻干粉、膏剂、水溶液剂或混悬剂(参见例如Hardman等(2001)Goodman and Gilman’s The Pharmacological Basis of Therapeutics，McGraw-Hill，New York，NY；Gennaro(2000)Remington：The Science and Practice of Pharmacy，Lippincott，Williams，and Wilkins，New York，NY；Avis等(主编)(1993)Pharmaceutical Dosage Forms：Parenteral Medications，Marcel Dekker，NY；Lieberman等(主编)(1990)Pharmaceutical Dosage Forms：Tablets，Marcel Dekker，NY；Lieberman等(主编)(1990)Pharmaceutical Dosage Forms：Disperse Systems，Marcel Dekker，NY；Weiner和Kotkoskie(2000)Excipient Toxicity and Safety，Marcel Dekker，Inc.，New York，NY)。
给药方案取决于若干因素，包括治疗性抗体的血清或组织周转率、症状水平、治疗性抗体的免疫原性和生物基质中靶细胞的可及性。优选给药方案递送足够的治疗性抗体以实现靶疾病状态的改善，同时使不良副作用降到最低。因此，递送的生物剂(biologic)的量部分取决于具体的治疗性抗体和待治疗病况的严重程度。可获得有关选择适当剂量的治疗性抗体方面的指引(参见例如Wawrzynczak(1996)Antibody Therapy，Bios Scientific Pub.Ltd，Oxfordshire，UK；Kresina(主编)(1991)Monoclonal Antibodies，Cytokines and Arthritis，Marcel Dekker，New York，NY；Bach(主编)(1993)Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases，Marcel Dekker，New York，NY；Baert等(2003)New  Engl.J.Med.348：601-608；Milgrom等(1999)New Engl.J.Med.341：1966-1973；Slamon等(2001)New Engl.J.Med.344：783-792；Beniaminovitz等(2000)New Engl.J.Med.342：613-619；Ghosh等(2003)New Engl.J.Med.348：24-32；Lipsky等(2000)New Engl.J.Med.343：1594-1602)。
应用
本发明提供抗本文任一实施方式所述的BTLA抗体及其抗原结合片段在治疗、预防和诊断BTLA介导的疾病中的应用。在某些实施方案中，本发明提供用于治疗、预防和诊断BTLA介导的疾病的本文任一实施方式所述的BTLA抗体及其抗原结合片段。
实施例
提供以下实施例以证明并进一步解释本发明的一些优选的实施方式和方面，不应被解释为限制其范围。
实施例1、克隆人BTLA胞外结构区入真核表达质粒
从义翘神州购买含有人BTLA基因cDNA序列的质粒HG11895-G-N，利用正向引物5’-gtacGCTCTTCATGTaaagaatcatgtgatgtacagcttta-3’(SEQ ID NO:50)和反向引物5’-gatcGCTCTTCTAGCatacaggagccagggtctgcttgcca-3’(SEQ ID NO:51)，PCR扩增人BTLA胞外片段(核苷酸序列如SEQ ID NO:49所示)。扩增片段经BSPQI酶切后，插入到自主构建的真核表达质粒系统(HX1-FC)中，产生人BTLA胞外区蛋白(hBTLA-ECD-FC)的表达质粒。以此质粒通过PEI转染293E细胞，6天后，收集培养基上清液，通过亲和层析纯化人BTLA胞外区重组蛋白(hBTLA-ECD-FC)。
图1显示人BTLA胞外区蛋白的SDS-PAGE电泳图。
实施例2、FACS检测重组蛋白hBTLA-ECD-FC与细胞上人HVEM的结合
构建表达人HVEM(Gene Bank：NP_003811.2)的293F稳转细胞株(hHVEM-293F)。将hHVEM-293F细胞悬液与生物素标记的hBTLA(300ng/mL)混合，室温孵育30分钟。以FACS缓冲液洗涤细胞3次后，加入5μg/ml的NA-PE并孵育30分钟。FACS缓冲液洗涤细胞3次后，通过流式细胞仪检测验证BTLA重组蛋白与293F细胞表面的HVEM的结合。结果如图2所示。
实施例3、抗BTLA鼠源抗体的制备
3.1、免疫动物
将实施例1获得的重组蛋白hBTLA-ECD-FC作为抗原与等量免疫佐剂(福氏佐剂)混合，取5只6周大雌性Balb/c小鼠进行皮下免疫。在初次免疫以后，每两周进行一次加强免疫，共6次免疫。
3.2、细胞融合
在最后一针加强免疫后，取小鼠腹股沟淋巴结，在生理盐水中碾磨后取富含淋巴细胞的悬浮液，按常规电转方法(参见BTX公司电转仪手册)将其与SP2/0细胞融合。将融合细胞在含有HAT的DMEM完全培养基(Corning)中置于8％CO 2，37℃条件下培养。
实施例4、杂交瘤细胞的筛选实验
在11500株不同多克隆杂交瘤细胞中，通过酶标(ELISA)反应，筛选出560株所分泌的抗体可结合人BTLA蛋白的克隆。在这560株克隆中，有33株可以结合293F细胞上表达的BTLA；在这33株克隆中，有16株具备抑制生物素标记的人BTLA与293F上HVEM的结合的能力。我们集中对这16株克隆中的前4株(2D12、3B3、3E3和6A7)进行后续实验，具体筛选的实验方法如下所示。
4.1、ELISA检测杂交瘤抗体与hBTLA的结合
通过酶标反应(ELISA)，筛选所分泌的抗体与hBTLA结合的杂交瘤细胞株。384孔酶标板，用1μg/ml的hBTLA包被，4摄氏度孵育过夜。然后弃去孔内溶液，用洗涤缓冲液洗3次，加入含有1％BSA的PBS溶液封闭60分钟。用洗涤缓冲液洗3次后加入杂交瘤培养上清，室温孵育60分钟后用洗涤缓冲液冲洗3次。然后加入1：5000倍稀释的HRP标记的山羊抗小鼠IgG二抗，室温孵育30分钟。经洗涤缓冲液冲洗三次后，加入30μl TMB底物溶液显色，室温反应10分钟后，以30μl的盐酸溶液(2M)终止反应并在450nm处读出吸光度。
4.2、ELISA检测杂交瘤抗体结合hBTLA的种属特异性
通过酶标反应(ELISA)筛选杂交瘤分泌的抗体与食蟹猴BTLA结合的杂交瘤细胞株。96孔酶标板，用1μg/ml的食蟹猴BTLA包被，室温孵育60分钟。然后弃去孔内溶液，用洗涤缓冲液洗3次，加入含有1％BSA的PBS溶液封闭60分钟。用洗涤缓冲液洗3次后加入杂交瘤培养上清，室温孵育60分钟后用洗涤缓冲液冲洗3次。然后加入1：5000倍稀释的HRP标记的山羊抗小鼠IgG二抗，室温孵育30分钟。经洗涤缓冲液冲洗三次后，加入100μl TMB底物溶液显色，室温反应10分钟后，以100μl的盐酸溶液(2M)终止反应并在450nm处读出吸光度。
4.3、FACS检测杂交瘤抗体阻断BTLA与HVEM结合作用
将33株抗体培养液上清与生物素标记的人BTLA(300ng/mL)混合，室温孵育30分钟。然后将混合物与hHVEM-293F稳转细胞株悬液在室温孵育30分钟。以FACS缓冲液洗涤细胞3次后，加入5μg/ml的NA-PE并孵育30分钟。FACS缓冲液洗涤细胞3次后，通过流式细胞仪检测验证杂交瘤细胞分泌的抗体能够阻断人BTLA与293F细胞表面的HVEM的结合作用。
实施例5、候选抗体可变区序列的获得(Kabat或IMGT表示)
用基于简并引物PCR的方法，测定由候选杂交瘤表达的小鼠抗体可变区的DNA序列。简言之，将杂交瘤细胞株分别扩大培养，1000rpm离心收集细胞，并以Trizol提取总RNA。以此为模板，合成第一链cDNA后，以第一链cDNA为后续模板PCR扩增对应的可变区DNA序列，所用PCR引物基于Ig-引物组。回收纯化PCR产物，将扩增产物测序后，得到候选杂交瘤重链可变区和轻链可变区序列。
用NCBI Ig-Blast(http://www.ncbi.nlm.nih.gov/projects/igblast/)在种系和重排Ig可变区序列数据库中搜索共有序列。基于Kabat(Wu,T.T及Kabat,E.A.1970J.Exp.Med.，132:211-250)及IMGT系统(Lefranc M.-P.等人，1999Nucleic Acids Research,27,209-212)，藉由序列批注及藉由基于因特网的序列分析(http://www.Imgt.org/IMGT_vquest/share/textes/index.html与http://www.ncbi.nlm.nih.gov/igblast/)鉴定互补决定区(CDR)。
杂交瘤细胞编码的轻链和重链可变区及CDR的氨基酸序列如下所示(其中 加粗下划 线为基于Kabat系统划分的CDRs，而斜体加粗为基于IMGT系统划分的CDRs)：
2D12 HC1
EVQLQQSGAELVKPGASVNLSCTASGFAIR DTYLHWVKQRPEQVLEWTG RIDPANGNTKYDPRFQGKATLTADTSSNTAYLHLSSLTSEDTAVYYCVA DYYGSSLFDYWGQGTTLTVSS(SEQ ID NO:34，3个HCDR的序列分别对应于SEQ ID NO:1－3)
2D12 HC2
EVQLQQSGAELVKPGASVKLSCTVSGFNIK DSYIHWVKQRPEQGLEWIG RIDPANGNTKYDPKFRGKATITADASSNTAALQVSSLTSEDTAVYFCVG DHYGSSLFDYWGHGTTLTVSS(SEQ ID NO:35，3个HCDR的序列分别对应于SEQ ID NO:4－6)
2D12 LC1
DVVMTQTPLTLSVTIGQPASISC KSSQSLLDSDGKTYLNWLLQRPGQSPKRLIY VLSKLESGVPDRFTGVGSGTDFTLKISRVEAVDLGVYYC WQGTHFPYTFGGGTKLEIK(SEQ ID NO:36，3个LCDR的序列分别对应于SEQ ID NO:7－9)
2D12 LC2
EVVMTQTPLTLSVTIGQSASISC KSSQSLLDSDGKTYLNWFLQRPGQSPKRLMH LVSKLDSGVPDRFTGSGSGTDFTLKISGVEAEDLGVYYC WQGTYFPYTFGGGTKLETK(SEQ ID NO:37，3个LCDR的序列分别对应于SEQ ID NO:10－12)
3B3 HC
EVQLQQSGADLVKPGASVKLSCTASGFNFK HTYAHWVKQRPEQGLEWIG RIDPANGNTKYDPKFQGKATMTADTASNAAFLQLSSLTSEDTAVYYCVA DHYGSSLLDYWGQGTSLTVSS(SEQ ID NO:38，3个HCDR的序列分别对应于SEQ ID NO:13－15)
3B3 LC
DIQMTQSPSSLSASLGGKVTITC KASQDINKYIAWYQHKPGKGPRLLIH YTSTLQPGIPSRFSGSGSGSDYSLTISSLESEDSANYYC LQYATYAPPAVSIFGAGTKLELK(SEQ ID NO:39，3个LCDR的序列分别对应于SEQ ID NO:16－18)
3E3 HC1
EVQLQQSGADLVKPGASVKLSCTASGFNIK DTYVHWVKQRPEQGLEWIG RIDPANGHTKFDPKFQGKATITADTSSNTANLQISSLTSEDTAVYYCVS DYYGSSLLDYWGQGTTLTVSS(SEQ ID NO:40，3个HCDR的序列分别对应于SEQ ID NO:19－21)
3E3 LC
DPDSTHFVDYHWTTCLHLLQVQSEPPSEDGKTYLSWIFQMFHLSPKRLIYVVSKLNSGVPVRLSANHSRTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKLEIK(SEQ ID NO:41，3个LCDR的序列分别对应于SEQ ID NO:22－24)
3E3 HC2
EVQLQQSGAELVRPGASVKLSCTVSGFNIK DTYVHWVKQRPEQGLEWIG RIDPANGHTKYDPKLQGTATITADTSSNTAYLQLSSLTSEDTAVYYCAT DYYGSSLLDYWGQGTTLTVSS(SEQ ID NO:42，3个HCDR的 序列分别对应于SEQ ID NO:25－27)
6A7 HC
EVHLQQSGTELMKPGASVKLSCTASGLNIR DTYMHWVKQRPEQGLEWIG RIDPANGNTKFDPKFQGKATLTSDTSSNTAYLHFSSLTSEDAAVYYCVS DHYGSSLLDYWGQGTSLTVSS(SEQ ID NO:43，3个HCDR的序列分别对应于SEQ ID NO:28－30)
6A7 LC
DIKMTQSPSSMYASLGERVTITC KASQDIKSYLSWYQQKPWKSPKTLIY YATSLADGVPSRFSGSGSGQDYSLTITSLESDDTATYYC LQHGADAAPTVSIFGGGTKLEIK(SEQ ID NO:44，3个LCDR的序列分别对应于SEQ ID NO:31－33)
实施例6、构建嵌合抗体表达载体
通过标准PCR技术将候选鼠源抗体重链可变区和轻链可变区基因分别融合至人类IgG4的FC及κ链恒定区的N末端，插入pcDNA3.1载体来构建嵌合型重链或轻链的表达质粒。后续的嵌合抗体均由上述重链和轻链的表达载体质粒两两组合转染表达细胞后，纯化提取获得，获得嵌合抗体ch7、ch12、ch17、ch22和ch27。杂交瘤细胞编码的轻链和重链可变区及CDR的氨基酸序列如下所示。
Figure PCTCN2019098150-appb-000001
Figure PCTCN2019098150-appb-000002
实施例7、ELISA检测嵌合抗体与hBTLA的结合
通过酶标反应(ELISA)检测嵌合抗体与hBTLA的结合能力。用0.5μg/ml的hBTLA包被96孔酶标板，37摄氏度恒温孵育60分钟。然后弃去孔内溶液，用洗涤缓冲液洗3次，加入含有2％BSA的PBS溶液封闭60分钟。用洗涤缓冲液洗3次后加入梯度稀释的 抗体，37摄氏度孵育60分钟后用洗涤缓冲液冲洗3次，然后加入1：10000倍稀释的HRP标记的小鼠抗人IgG4二抗，37摄氏度孵育1小时，经洗涤缓冲液冲洗三次后，加入100μl TMB底物溶液显色，室温反应30分钟后，以100μl 2M的盐酸溶液终止反应并在450nm处读出吸光度。
结果如图3所示，嵌合抗体ch7，ch12，ch17，ch22和ch27可以与hBTLA特异性结合。其EC 50值如下表1所示。
表1：嵌合抗体与人BTLA特异性结合数据
嵌合抗体 ch7 ch12 ch17 ch22 ch27
EC 50(ng/mL) 0.066 0.094 0.298 0.373 0.052
实施例8、FACS检测嵌合抗体对阻断BTLA与HVEM结合作用
采用FACS检测嵌合抗体阻断hBTLA结合表达于293F细胞上hHVEM的能力。将hHVEM-293F稳定表达细胞消化后离心重悬到FACS缓冲液中，按细胞量为2.5×10 4个/50ul加入到96孔圆底板孔中，加入预先生物素标记的hBTLA(1ug/ml)蛋白混合，4℃孵育15分钟；再加入50ul不同浓度的嵌合抗体稀释液(起始5ug/ml，3倍滴定)混匀，室温孵育30min；用FACS缓冲液洗涤细胞两次后加入100ul山羊抗人IgG-PE抗体，避光孵育30min；用FACS缓冲液洗涤两次后进行FACS检测。将经洗涤的细胞重悬于含有碘化丙啶(PI)以及防止受体内化的0.02％叠氮化钠的4℃缓冲剂中后，通过流式细胞术分析。根据从FSC/SSC门排除PI阳性细胞，对活细胞进行门控，并测量其几何平均荧光强度(MFI)。使用Prism TM软件内的S形剂量-响应模型进行分析数据。
结果如图4和下表2所示，嵌合抗体ch12，ch17，ch22和ch27能有效阻断人BTLA与细胞表面的HVEM结合。
表2
嵌合抗体 ch12 ch17 ch22 ch27
FACS，IC 50(ng/ml) 77.17 76.07 75.67 69.54
实施例9、荧光素酶报告基因实验检测嵌合抗体对T细胞活性的影响
通过刺激识别抗原呈递细胞(APC)上由主要组织相容性复合体I类或II类蛋白呈递的特异性肽的T细胞受体(TCR)来实现T细胞活化。活化的TCR转而启动信号传导事件的级联，其可通过转录因子(例如活化子-蛋白-l(AP-l)、活化的T细胞的核因子(NFAT)或活 化的B细胞的核因子κ轻链增强子(NFκb))驱动的报道基因来监测。通过在T细胞上组成或诱导表达的共同受体的接合(engagement)来调整T细胞应答。程序性细胞死亡蛋白(PD1)和BTLA是T细胞活性的负调节物。PD-1和BTLA分别与在包括APC或癌症细胞的靶细胞上表达的其配体(PD-L1)和HVEM相互作用，这种相互作用导致通过将磷酸酶募集到TCR信号小体(signalosome)来递送抑制信号，从而产生正信号传导的抑制。通过构建两个工程化改造的稳定表达细胞系Jurkat细胞(Jurkat/NFAT-Luc/hPD-1-hBTLA)和CHO细胞(CHO/hPD-L1-hHVEM)来测量通过APC和T细胞之间的相互作用诱导的T细胞信号传导。
将稳定表达hPD-L1/hHVEM的CHO细胞铺到96孔板，每孔细胞量为5×10 4，37℃，7％CO 2培养过夜，去除细胞上清，每孔中加入40ul嵌合抗BTLA抗体稀释液(起始浓度为60ug/ml，3倍滴定)，加入40ul可以持续表达hPD-1/hBTLA/NFAT-荧光素酶的Jurkat报告细胞，总细胞数为1×10 5细胞，37℃，7％CO 2培养6小时，加入荧光素酶试剂，酶标仪检测发光值。
结果如图5和下表3所示，嵌合抗体ch12，ch17，ch22和ch27能显著抑制BTLA和PD-1介导的T细胞活性抑制作用。
表3：嵌合抗体有效促进T细胞活性
嵌合抗体 ch12 ch17 ch22 ch27
荧光素酶，EC 50(ng/mL) 300.6 370.6 912.4 278.6
实施例10、抗体的人源化改造
根据上述获得的杂交瘤细胞分泌的抗体的可变区序列，进行人源化改造。简言之，人源化改造过程涉及以下步骤：A、把各杂交瘤细胞分泌的抗体的基因序列与人胚胎系抗体基因序列进行比对，找出同源性高的序列；B、分析考察HLA-DR亲和性，选出亲和力低的人胚胎系框架序列；C、利用计算机模拟技术，应用分子对接分析可变区及其周边的框架氨基酸序列，考察其空间立体结合方式。通过计算静电力，范德华力，亲疏水性和熵值，分析各杂交瘤细胞分泌的抗体基因序列中可与hBTLA作用以及维护空间构架的关键氨基酸个体，将其嫁接回已经选择的人胚胎系基因框架，并在此基础上标配出必须保留的框架区氨基酸位点，合成人源化抗体(Pini,A.等,et al.(1998).design and use of a phage display library:human antibodies with 10subnanomolar affinity against a marker of angiogenesis eluted from a two-dimensional gel.,Journal of Biological Chemistry,273(34):21769-21776)。在此基础上我们得到了以下多个人源化抗体。其中包括以下克隆，hu17、 hu18和hu19。
hu17：
重链氨基酸序列SEQ ID NO:45；
轻链氨基酸序列SEQ ID NO:46；
hu18：
重链氨基酸序列SEQ ID NO:45；
轻链氨基酸序列SEQ ID NO:47；
hu19：
重链氨基酸序列SEQ ID NO:45；
轻链氨基酸序列SEQ ID NO:48。
实施例11、ELISA检测人源化抗体与hBTLA的结合
利用常规ELISA检测方法检测人源化抗体与hBTLA结合特异性。用0.5μg/ml的hBTLA包被96孔酶标板，37摄氏度恒温孵育60分钟。然后弃去孔内溶液，用洗涤缓冲液洗3次，加入含有2％BSA的PBS溶液封闭60分钟。用洗涤缓冲液洗3次后加入梯度稀释的抗体，37摄氏度孵育60分钟后用洗涤缓冲液冲洗3次，然后加入1：10000倍稀释的HRP标记的小鼠抗人IgG4二抗，37摄氏度孵育1小时，经洗涤缓冲液冲洗三次后，加入100μl TMB底物溶液显色，室温反应30分钟后，以100μl 2M的盐酸溶液终止反应并在450nm处读出吸光度。
结果如图6所示，人源化抗体hu17，hu18和hu19可以与hBTLA特异性结合。其EC 50值如下表4所示。
表4：人源化抗体组合hu17，hu18和hu19与hBTLA特异性结合数据
人源化抗体 hu17 hu18 hu19
EC 50(ng/mL) 6.3 5.4 6.6
实施例12、FACS检测人源化抗体与293F细胞上hBTLA的结合
采用基于细胞的流式细胞(FACS)测定人源化抗BTLA抗体与细胞上表达的hBTLA结合能力。将表达hBTLA的293F细胞消化后离心重悬到FACS缓冲液中，按细胞量为2.5×10 4，体积为50ul加入到96孔圆底板孔中，加入50ul不同浓度的抗体稀释液(起始浓度10ug/ml，3倍滴定)混匀，室温孵育30min；用FACS缓冲液洗涤细胞两次后加入100ul山羊抗人IgG-PE抗体，避光孵育30min；用FACS buffer洗涤两次后进行FACS检测。 将经洗涤的细胞重悬于含有碘化丙啶(PI)以及防止受体内化的0.02％叠氮化钠的4℃缓冲剂中后，通过流式细胞术分析。根据从FSC/SSC门排除PI阳性细胞，对活细胞进行门控，并测量其几何平均荧光。使用Prism TM软件内的S形剂量-响应模型进行分析数据。
结果如图7所示，人源化抗体hu17、hu18、hu19与293F细胞上hBTLA能有效的结合。各抗体的EC50值如下表5所示。
表5
  hu17 hu18 hu19 ch12
EC50(ng/mL) 96.74 89.34 95.06 88.12
实施例13、FACS检测人源化抗体对阻断BTLA与HVEM结合的阻断作用
采用基于细胞的流式细胞(FACS)测定，检测人源化抗体阻断hBTLA结合表达于293F细胞上hHVEM的能力。将hHVEM-293F稳定表达细胞消化后离心重悬到FACS缓冲液中，按细胞量为2.5×10 4个，体积为50ul加入到96孔圆底板孔中，加入预先生物素标记的hBTLA(1ug/ml)蛋白混合，4℃孵育15分钟；再加入50ul不同浓度的人源化抗体稀释液(起始5ug/ml，3倍滴定)混匀，室温孵育30min；用FACS缓冲液洗涤细胞两次后加入100ul山羊抗人IgG-PE抗体，避光孵育30min；用FACS缓冲液洗涤两次后进行FACS检测。将经洗涤的细胞重悬于含有碘化丙啶(PI)以及防止受体内化的0.02％叠氮化钠的4℃缓冲剂中后，通过流式细胞术分析。根据从FSC/SSC门排除PI阳性细胞，对活细胞进行门控，并测量其几何平均荧光。使用Prism TM软件内的S形剂量-响应模型进行分析数据。
结果如图8所示，人源化抗体hu17、hu18、hu19能有效阻断BTLA与细胞表面的HVEM结合。各抗体的IC50值如下表6所示。
表6
  hu17 hu18 hu19 ch12
IC50(ng/mL) 142.7 172.2 161.5 156.9
实施例14、人源化抗BTLA抗体促进T细胞的激活
将稳定表达hPD-L1/hHVEM的CHO细胞铺到96孔板，每孔细胞量为5×10 4，37℃，7％CO 2培养过夜，去除细胞上清，每孔中加入40ul人源化抗BTLA抗体稀释液(起始浓度为60ug/ml，3倍浓度梯度稀释)，加入40ul可以持续表达hPD-1/hBTLA/NFAT-荧光 素酶的Jurkat报告细胞，总细胞数为1×10 5细胞，37℃，7％CO 2培养6小时，加入荧光素酶试剂，酶标仪检测发光值。
结果如图9所示，人源化抗BTLA抗体hu17、hu18能有效促进T细胞活化。各抗体的EC50值如下表7所示。
表7
  hu17 hu18 hu19 ch12
EC50(ng/mL) 256.7 277.3 290.4 138.7
实施例15、人源化抗BTLA抗体与hBTLA的亲和力
使用GE医疗生命科学公司的Biacore T200仪器进行检测实验，将Series S CM5芯片装载到仪器上，使用的系统缓冲液为HBS-EP+(10mM HEPES,pH 7.4,150mM NaCl,3mM EDTA,0.05％表面活性剂P20)。进行BTLA-Fc抗原在芯片检测通道的偶联，用400mM EDC与100mM NHS的混合液以10μL/min注入420s活化芯片表面，将BTLA-Fc抗原稀释在10mM醋酸钠/醋酸(pH 5.5)缓冲液中至终浓度20μg/mL，并以10μL/min注入进行偶联，将1M乙醇胺-盐酸溶液(pH 8.5)以10μL/min注入420s进行封闭。
用Biacore系统缓冲液将抗体进行2倍梯度稀释，共6个浓度点。浓度梯度为24nM、12nM、6nM、3nM、1.5nM和0.75nM，其中24nM为重复测试。数据分析使用GE医疗生命科学公司的数据分析软件Biacore T200 Evaluation Software版本号3.0。数据拟合使用模型为1:1Binding。拟合得到抗体与抗原间结合的动力学常数结合速率ka(1/Ms)，解离速率kd(1/s)，亲和力常数KD(M)，结果如表8所示。
表8：人源化抗体的亲和力数据
  ka(1/Ms) kd(1/s) KD(M)
hu17 6.24E+05 1.16E-04 1.86E-10
hu18 6.39E+05 8.67E-05 1.36E-10
实施例16、人源化抗体与不同种属BTLA结合动力学的表征
为了检测嵌合抗体与食蟹猴源和鼠源BTLA之间的交叉反应，使用Fortebio检定。简而言之，将人BTLA、食蟹猴BTLA或鼠BTLA偶联至已活化的CM5生物传感器芯片以实现大约100-200个响应单位(RU)，然后用IM乙醇胺封闭未反应基团。以30次/分钟在SPR运行缓冲液中注射自0.12nM至90nM递增浓度的人源化抗体样品，且藉由减去来自 空白流动室的RU来计算在不同种源BTLA的结合响应。
结果如图10所示。hu17与不同种属BTLA结合对比结果显示：其不仅与人BTLA具有高亲和力，并且与食蟹猴源BTLA有相近的亲和力，但其与鼠BTLA基本不发生结合。
实施例17、人源化抗体对小鼠肿瘤生长的抑制作用
在MC38细胞(ATCC)上通过电转Hxp-hHVEM质粒，构建MC38-hHVME细胞库，然后通过有限稀释法进行细胞亚克隆，通过流式筛选出单克隆，得到MC38-HVEM细胞；接着将MC38-hHVEM细胞以1×10 6个/0.1mL浓度接种于B-hBTLA人源化雌性小鼠的右侧皮下，待肿瘤生长到约118mm 3时按肿瘤体积随机分组，每组8只，共5组，分别为：G1 0.9％氯化钠注射液溶剂对照组、G2KLH(10mg/kg)阴性对照组、G3hu18(1mg/kg)组、G4hu18(3mg/kg)组和G5hu18(10mg/kg)组。所有组给药途径均为腹腔注射，每周给药2次，连续给药7次，末次给药4天后结束实验。每周测量肿瘤体积及体重2次，记录小鼠体重和肿瘤体积。实验结束时，动物安乐死，剥取肿瘤称重、拍照，计算相对肿瘤抑制率(TGI TW％)。
各组受试品对MC38-hHVEM细胞移植B-hBTLA小鼠肿瘤体积的影响结果如表9和图11所示。首次给药后21天，KLH(10mg/kg)阴性对照组平均肿瘤体积为1560±256mm 3，其他给药组平均肿瘤体积分别为1073±224mm 3、747±268mm 3和868±211mm 3。各给药组与KLH阴性对照组相比，TGI TV％分别为33.7％、56.4％和48.0％，P值分别为0.175、0.046和0.056，表明受试药物hu18在3mg/kg剂量水平下对肿瘤生长有一定抑制作用。
表9：hu18对MC38-hHVEM细胞移植B-hBTLA小鼠肿瘤体积的影响
Figure PCTCN2019098150-appb-000003
注：a：平均数±标准误；b：给药组肿瘤体积与KLH阴性对照组肿瘤体积在给药21天后统计学比较，t-test。
各组受试品对MC38-hHVEM细胞移植B-hBTLA小鼠体重的影响结果如表10和图12所示。所有实验动物在给药期间活动和进食状态良好，各给药组动物体重均有一定程度的上升。实验期间未出现实验动物死亡情况。给药后21天，与KLH阴性对照组小鼠体重相比，各给药组小鼠体重无显著变化(P＞0.05)，表明实验动物对受试品耐受性良好。
表10：受试品对MC38-hHVEM细胞移植B-hBTLA小鼠体重的影响
Figure PCTCN2019098150-appb-000004
注：a：平均数±标准误；b：给药组体重与KLH阴性对照组体重在给药21天后统计学比较，t-test。
实施例18、人源化抗体hu18没有ADCC效应功能
当抗体结合至细胞表面靶蛋白质，然后连接至效应细胞上所表达的Fcγ受体(FcγR)时，启动ADCC。很清楚地记载了人类IgG1对FcγR具有比IgG4明显更高的结合亲和力，尤其是结合FcγR-I及FcγR-IIIA，该亲和力与IgG1激活ADCC的强度相关联。联想到ADCC，当抗体交联细胞表面靶点及C1q蛋白质，继之以补体复合物形成及靶细胞裂解的级联反应时，激活CDC。作为ADCC和CDC的代理，抗体结合至FcγR及C1q的检测就可充当ADCC及CDC的基本指标。因此，本发明利用biacore T200(GE)评估了单抗对主要FcγR结合的动力学亲和力。
GE的抗-His抗体固定在传感器晶片上。各种Fc受体，包括重组人FcγRIIIA(CD16a)V176，重组人FcγRIIA(CD32a)R167，重组人FcγRI(CD64)和重组人FcRn被捕获，然后注射一系列稀释的重组人抗-BTLA抗体(即hu18)，检测并分析相互作用的结合性质。hu18是IgG4亚型抗体，hu18-IgG1是IgG1亚型，其与hu18共享相同的Fab，并作为阳性对照。
结果如表11所示，相比于IgG1亚型的对照抗体，IgG4亚型的重组人抗-BTLA抗体与Fc受体的结合相对较弱，hu18抗体与IgG1亚型对照抗体相比，其与FcγRIIIA(CD16a) V176的相互作用弱400倍。这显示hu18具有较弱或无ADCC效应。如表12所示，hu18抗体不结合C1q，而hu18-IgG1抗体能够结合C1q。
表11：抗-BTLA抗体与Fc受体之间相互作用的结合亲和性
Figure PCTCN2019098150-appb-000005
表12.抗-BTLA抗体与人C1q之间相互作用的结合亲和力
Figure PCTCN2019098150-appb-000006

Claims (13)

  1. 分离的抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包含至少一个选自SEQ ID NO：7、8、9、10、11、12、16、17、18、22、23、24、31、32和33的轻链CDR结构域和/或至少一个选自SEQ ID NO：1、2、3、4、5、6、13、14、15、19、20、21、25、26、27、28、29和30的重链CDR结构域。
  2. 如权利要求1所述的分离的抗体或其抗原结合片段，其特征在于，所述分离的抗体或其抗原结合片段的轻链CDR中LCDR1选自SEQ ID NO:7、10、16、22和31中的任一CDR序列；和/或其LCDR2选自SEQ ID NO:8、11、17、23和32中的任一CDR序列；和/或其LCDR3选自SEQ ID NO:9、12、18、24和33中的任一CDR序列；和/或
    所述分离的抗体或其抗原结合片段的重链CDR中HCDR1选自SEQ ID NO:1、4、13、19、25和28中的任一CDR序列；和/或其HCDR2选自SEQ ID NO:2、5、14、20、26和29中的任一CDR序列；和/或其HCDR3选自SEQ ID NO:3、6、15、21、27和30中的任一CDR序列。
  3. 如权利要求2所述的分离的抗体或其抗原结合片段，其特征在于，
    所述分离的抗体或其抗原结合片段的轻链CDR中，LCDR1选自SEQ ID NO:7、10、16、22和31中的任一CDR序列；LCDR2选自SEQ ID NO:8、11、17、23和32中的任一CDR序列；和LCDR3选自SEQ ID NO:9、12、18、24和33中的任一CDR序列；和/或
    所述分离的抗体或其抗原结合片段的重链CDR中，HCDR1选自SEQ ID NO:1、4、13、19、25和28中的任一CDR序列；HCDR2选自SEQ ID NO:2、5、14、20、26和29中的任一CDR序列；和HCDR3选自SEQ ID NO:3、6、15、21、27和30中的任一CDR序列。
  4. 如权利要求2所述的分离的抗体或其抗原结合片段，其特征在于，
    所述分离的抗体或其抗原结合片段的轻链的LCDR1、LCDR2和LCDR3序列如以下A-E组中的任一组所示：
    组别 LCDR1 LCDR2 LCDR3 A SEQ ID NO：7 SEQ ID NO：8 SEQ ID NO：9 B SEQ ID NO：10 SEQ ID NO：11 SEQ ID NO：12 C SEQ ID NO：16 SEQ ID NO：17 SEQ ID NO：18 D SEQ ID NO：22 SEQ ID NO：23 SEQ ID NO：24
    E SEQ ID NO：31 SEQ ID NO：32 SEQ ID NO：33
    和/或
    所述分离的抗体或其抗原结合片段的重链CDR的HCDR1，HCDR2和HCDR3的氨基酸序列如以下F-K组中的任一组所示：
    组别 HCDR1 HCDR2 HCDR3 F SEQ ID NO：1 SEQ ID NO：2 SEQ ID NO：3 G SEQ ID NO：4 SEQ ID NO：5 SEQ ID NO：6 H SEQ ID NO：13 SEQ ID NO：14 SEQ ID NO：15 I SEQ ID NO：19 SEQ ID NO：20 SEQ ID NO：21 G SEQ ID NO：25 SEQ ID NO：26 SEQ ID NO：27 K SEQ ID NO：28 SEQ ID NO：29 SEQ ID NO：30
    。
  5. 如权利要求1所述的分离的抗体或其抗原结合片段，其特征在于，所述分离的抗体或其抗原结合片段的重链CDR的HCDR1，HCDR2和HCDR3的氨基酸序列和轻链CDR的LCDR1，LCDR2和LCDR3的氨基酸序列如以下I-IX组中的任一组所示：
    组别 LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 I SEQ ID NO:7 SEQ ID NO:8 SEQ ID NO:9 SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 II SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6 III SEQ ID NO:16 SEQ ID NO:17 SEQ ID NO:18 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15 IV SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24 SEQ ID NO:19 SEQ ID NO:20 SEQ ID NO:21 V SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24 SEQ ID NO:25 SEQ ID NO:26 SEQ ID NO:27 VI SEQ ID NO:31 SEQ ID NO:32 SEQ ID NO:33 SEQ ID NO:28 SEQ ID NO:29 SEQ ID NO:30 VII SEQ ID NO:7 SEQ ID NO:8 SEQ ID NO:9 SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6 VIII SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 IX SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15
    。
  6. 如权利要求1所述的分离的抗体或其抗原结合片段，其特征在于，
    所述分离的抗体或其抗原结合片段的轻链可变区的氨基酸序列选自SEQ ID NO：36、37、39、41、44、46、47和48中任一所示的氨基酸序列；和/或
    所述分离的抗体或其抗原结合片段的重链可变区的氨基酸序列选自SEQ ID NO：34、 35、38、40、42、43和45中任一所示的氨基酸序列。
  7. 如权利要求6所述的分离的抗体或其抗原结合片段，其特征在于，
    所述分离的抗体或其抗原结合片段的轻链可变区的氨基酸序列如SEQ ID NO：36所示，重链可变区的氨基酸序列如SEQ ID NO：34或35所示；或
    所述分离的抗体或其抗原结合片段的轻链可变区的氨基酸序列如SEQ ID NO：37所示，重链可变区的氨基酸序列如SEQ ID NO：34或35所示；或
    所述分离的抗体或其抗原结合片段的轻链可变区的氨基酸序列如SEQ ID NO：39所示，重链可变区的氨基酸序列如SEQ ID NO：38所示；或
    所述分离的抗体或其抗原结合片段的轻链可变区的氨基酸序列如SEQ ID NO：41所示，重链可变区的氨基酸序列如SEQ ID NO：40或42所示；或
    所述分离的抗体或其抗原结合片段的轻链可变区的氨基酸序列如SEQ ID NO：44所示，重链可变区的氨基酸序列如SEQ ID NO：43所示；或
    所述分离的抗体或其抗原结合片段的轻链可变区的氨基酸序列如SEQ ID NO：46、47或48所示，重链可变区的氨基酸序列如SEQ ID NO：45所示。
  8. 如权利要求1－7中任一项所述的抗体或其抗原结合片段，其特征在于，所述抗体是嵌合抗体、人源化抗体或全人抗体。
  9. 分离的核酸，选自：
    (1)编码权利要求1－7中任一项所述的分离的抗体或其抗原结合片段的多核苷酸序列；和
    (2)(1)所述多核苷酸序列的互补序列。
  10. 表达载体或含该表达载体的宿主细胞，其特征在于，所述表达载体含有权利要求9所述的分离的核酸。
  11. 药物组合物，其特征在于，所述药物组合物含有权利要求1－7中任一项所述的分离的抗体或其抗原结合片段、权利要求9所述的核酸、权利要求10所述的表达载体或宿主细胞，或它们的任意组合。
  12. 权利要求1－7中任一项所述的分离的抗体或其抗原结合片段、权利要求9所述的核酸、权利要求10所述的表达载体或宿主细胞在制备用于治疗或预防BTLA介导的疾病的药物中的应用。
  13. 免疫缀合物，其特征在于，所述免疫缀合物含有与治疗剂偶联的权利要求1－7中任一项所述的抗体或其抗原结合片段，优选地所述治疗剂为毒素、放射性同位素、药物或细胞毒剂。
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CA3107144A1 (en) 2020-02-06
KR20210041585A (ko) 2021-04-15
AU2019315969A1 (en) 2021-03-25
US20210246209A1 (en) 2021-08-12
SG11202100649VA (en) 2021-03-30
JP7469292B2 (ja) 2024-04-16
CN112638943A (zh) 2021-04-09

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