WO2022184162A1 - 针对NKp46的抗体及其应用 - Google Patents

针对NKp46的抗体及其应用 Download PDF

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WO2022184162A1
WO2022184162A1 PCT/CN2022/079236 CN2022079236W WO2022184162A1 WO 2022184162 A1 WO2022184162 A1 WO 2022184162A1 CN 2022079236 W CN2022079236 W CN 2022079236W WO 2022184162 A1 WO2022184162 A1 WO 2022184162A1
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seq
antibody
nkp46
antigen
binding fragment
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PCT/CN2022/079236
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English (en)
French (fr)
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杨柳青
顾津明
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上海齐鲁制药研究中心有限公司
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Priority to KR1020237033784A priority Critical patent/KR20230154235A/ko
Priority to JP2023553738A priority patent/JP2024509557A/ja
Priority to BR112023017556A priority patent/BR112023017556A2/pt
Priority to CA3210582A priority patent/CA3210582A1/en
Priority to EP22762624.9A priority patent/EP4303234A1/en
Priority to AU2022230011A priority patent/AU2022230011A1/en
Priority to CN202280019014.0A priority patent/CN116964099A/zh
Priority to US18/280,248 priority patent/US20240141038A1/en
Publication of WO2022184162A1 publication Critical patent/WO2022184162A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A—HUMAN NECESSITIES
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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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
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    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
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    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1027—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants
    • A—HUMAN NECESSITIES
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
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    • 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
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • 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
    • C07K—PEPTIDES
    • 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
    • C07K—PEPTIDES
    • 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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • the present disclosure belongs to the field of immunology, and more particularly, the present disclosure relates to an antibody or antigen-binding fragment thereof directed against the NKp46 receptor, derivatives comprising the antibody or antigen-binding fragment thereof, pharmaceutical compositions, and their relevance in the treatment of cancer application.
  • NK cells are effector lymphocytes in the innate immune system and are the body's first line of defense against viral infections and tumorigenesis. It plays a similar role as cytotoxic T cells in the acquired immune system, but they differ in that: under normal circumstances, cytotoxic T cells need to detect MHC on the surface of viral cells to cause cytokines. Released, leading to lysis or apoptosis of target cells; NK cells can recognize these cells and carry out a rapid immune response in the absence of antibodies or MHC. For those cells that have lost their self-marked MHC-I type, NK cells can kill without activation, and these cells are usually harmful but cannot be detected and destroyed by other immune cells.
  • NK cells regulate the activity of NK cells through the balance of surface activating receptors and inhibitory receptors.
  • Activating receptors on the surface of NK cells are divided into human leukocyte antigen-I (human leukocyte antigen-I, HLA-I) molecule-related receptors and non-related receptors.
  • the former includes the second domain of the killer cell immunoglobulin-like receptor.
  • NK cell receptor 2C natural killer cell group 2C, CD94/NKG2C
  • the latter mainly includes NK cell receptor 2 region (natural killer cell group 2D, NKG2D) and natural cytotoxicity receptors (natural cytotoxicity receptors, NCR; including NKp46, NKp30, NKp44) and DNAX accessory molecule 1 ( DNAX accessory molecule-1, DNAM-1); the corresponding inhibitory receptors are KIR2DL, KIR3DL and CD94/NKG2A, etc.
  • NKp46 the NKp46 receptor in 1997, which is the earliest discovered natural cytotoxicity receptor and the most important natural cytotoxicity receptor, which is expressed on the surface of all NK cells (including mature, immature, quiescent and activated NK cells). Plays a key role in natural killing. Because it is specifically expressed on the surface of NK cells and its molecular weight is 46kD, it is named NKp46.
  • the NKp46 gene is located on chromosome 19 and is a type I transmembrane glycoprotein.
  • the extracellular region of NKp46 contains two Ig-like domains, namely D1 and D2.
  • D2 the region near the cell membrane is the region where tumor and virus-infected cells bind ligands.
  • the transmembrane region has a positively charged arginine and a cytoplasmic tail that lacks an immunoreceptor tyrosine-based activation sequence that can establish a salt bridge with aspartate residues in the transmembrane region of CD3 ⁇ and Fc ⁇ R, which are mediated by receptors.
  • Body binding results in tyrosine phosphorylation and thus mediates NKp46 signaling.
  • the only ligands of NKp46 identified so far are influenza virus hemagglutinin and parainfluenza virus hemagglutinin, and cellular ligands are unknown.
  • NK cells kill target cells, the toxic particles will reach the serosa and fuse with the cell membrane, causing the release of particle contents and eventually the death of the target cells.
  • CD107a molecules are transported to the cell membrane surface, and the upregulation of CD107a molecules is consistent with the secretion of perforin. Therefore, NK cells with positive expression of CD107a molecule can represent NK cells with killing activity.
  • NKp46 Activated NK cells have a strong killing effect on tumor cells, and the NKp46 receptor is necessary for the transmission of activating stimuli.
  • Reduced expression of NKp46 is known to be involved in immune escape in cervical cancer and its precursor lesions, and loss of NKp46/NCR1 expression increases lymphoma growth.
  • Yasser et al demonstrated that NKp46 is required for killing of all MM cell lines.
  • NKp46 also plays an important role in the clearance of microglia in the central nervous system. Lunemann et al. confirmed that infiltrating NK cells in the brain are mediated by NKp46 and NKG2D, and activated NK cells form synapses with microglia. Contact junctions, the latter's perforin polarizes to the cell interface to play a killing role.
  • the present disclosure provides an anti-NKp46 antibody or antigen-binding fragment thereof that specifically binds to human and cynomolgus NKp46.
  • an anti-NKp46 antibody or antigen-binding fragment thereof is provided, the antibody or antigen-binding fragment thereof can specifically bind to NKp46, comprising 3 proteins selected from the group consisting of SEQ ID NOs: 44-133 CDRs.
  • an anti-NKp46 antibody or antigen-binding fragment thereof comprising the group consisting of SEQ ID NOs: 44, 47, 50, 53, 56 , 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131 and, selected from SEQ ID NOs: 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99 , HCDR2 shown in 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132; and, selected from SEQ ID NO: 46, 49, 52, 55, 58, 61,
  • an anti-NKp46 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a variable region selected from the group consisting of HCDR1, HCDR2 and HCDR3 of the following group: SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46; or SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49; or SEQ ID NO:49 :50, SEQ ID NO:51 and SEQ ID NO:52; or SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55; or SEQ ID NO:56, SEQ ID NO:57 and SEQ ID NO: : 58; or SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61; or SEQ ID NO:62, SEQ ID NO:63, and SEQ ID NO:64; or SEQ ID NO:65, SEQ ID NO:
  • an anti-NKp46 antibody or antigen-binding fragment thereof capable of specifically binding NKp46 comprising HCDR1, HCDR2 and HCDR3 derived from Heavy chain variable regions shown in SEQ ID NOs: 14-43, 136-152.
  • an anti-NKp46 antibody or antigen-binding fragment thereof capable of specifically binding NKp46, comprising a heavy chain variable region having the same ID NO: 14-43, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the anti-NKp46 antibody or antigen-binding fragment thereof is provided, wherein the monoclonal antibody is a recombinant antibody, preferably an alpaca-derived antibody, a chimeric antibody or a humanized antibody.
  • an anti-NKp46 antibody or antigen-binding fragment thereof according to the present disclosure, wherein the monoclonal antibody is a Nanobody, preferably a humanized camelid VHH.
  • the anti-NKp46 antibody or antigen-binding fragment thereof described in the present disclosure is provided, which further includes a heavy chain constant region and/or a light chain constant region, preferably, the heavy chain constant region includes Fc or variable Body Fc, Fc derived from murine or human.
  • the anti-NKp46 antibodies or antigen-binding fragments thereof described in the present disclosure are provided in the form of IgGl, IgG2, IgG3, or IgG4.
  • a conjugate described in the present disclosure is provided, which is formed by coupling any of the aforementioned anti-NKp46 antibodies or antigen-binding fragments thereof to a capture label or a detection label, and the detection label includes Radionuclides, luminescent substances, coloured substances or enzymes.
  • a bispecific antibody wherein one antigen-binding domain comprises any of the foregoing anti-NKp46 antibodies or antigen-binding fragments thereof.
  • an antigen-binding domain comprises any of the foregoing anti-NKp46 antibodies or antigen-binding fragments thereof.
  • an antibody drug conjugate comprising any of the aforementioned anti-NKp46 antibodies or antigen-binding fragments thereof, and the antibody drug conjugate is formed by interconnecting antibody-linker-toxin.
  • a chimeric antigen receptor is provided, the extracellular recognition unit of which comprises any of the aforementioned anti-NKp46 antibodies or antigen-binding fragments thereof.
  • nucleic acids encoding any of the foregoing anti-NKp46 antibodies or antigen-binding fragments thereof are provided.
  • recombinant vectors of the nucleic acids described in the present disclosure are provided.
  • a host cell that comprises a recombinant vector of the present disclosure or a nucleic acid of the present disclosure integrated into the genome.
  • a method for providing the anti-NKp46 antibody or antigen-binding fragment thereof of the present disclosure comprises: culturing the host cell of the present disclosure under suitable conditions, and purifying the expression product from the cell.
  • use of the anti-NKp46 antibody or antigen-binding fragment thereof described in the present disclosure is provided in the manufacture of a medicament that specifically targets cells expressing NKp46, preferably, the cells are NK cells.
  • the tumor includes: leukemia , aggressive lymphoma, non-Hodgkin lymphoma, glioma, cervical cancer, head and neck cancer, rectal cancer, kidney cancer, liver cancer, lung cancer, pancreatic cancer, stomach cancer, etc.
  • an anti-NKp46 antibody or antigen-binding fragment thereof described in the present disclosure in the preparation of a diagnostic reagent for NKp46-expressing NK is provided.
  • a solution formulation of an antibody to NKp46 comprising an antibody to NKp46 or an antigen-binding fragment thereof and a buffer.
  • a method of the present disclosure for identifying the presence of NKp46-expressing cells in an individual comprising obtaining a biological sample from an individual comprising cells, combining the cells with the present disclosure An anti-NKp46 antibody or antigen-binding fragment thereof is contacted, and whether the antibody binds to the cell is assessed.
  • a pharmaceutical composition comprising an effective amount of any of the foregoing anti-NKp46 antibodies or antigen-binding fragments thereof, or an effective amount of a bispecific antibody, or an effective amount of a multispecific antibody , or comprising an effective amount of an antibody drug conjugate, or comprising an effective amount of a chimeric antigen receptor, or comprising an effective amount of nucleic acid, or comprising an effective amount of a recombinant vector, or comprising an effective amount of a host cell.
  • a pharmaceutical composition of the present disclosure is provided, further comprising a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions described in the present disclosure are provided, further comprising one or more additional additional therapeutic agents, preferably the one or more additional additional therapeutic agents include: chemotherapeutic agents, cells Toxic agents, radiotherapeutics, cancer vaccines, tumor reducing agents, targeted anticancer agents, antiangiogenic agents, biological response modifiers, cytokines, hormones, antimetastatic agents and immunotherapeutics.
  • additional additional therapeutic agents include: chemotherapeutic agents, cells Toxic agents, radiotherapeutics, cancer vaccines, tumor reducing agents, targeted anticancer agents, antiangiogenic agents, biological response modifiers, cytokines, hormones, antimetastatic agents and immunotherapeutics.
  • a kit or kit that includes a container, and a pharmaceutical composition of the present disclosure located in the container.
  • Figure 1 shows the anti-NKp46 positive control antibody detected by flow cytometry to verify the expression of human NKp46 on CHOK1 cells; the solid line in the figure shows the expression of human NKp46 on the CHOK1 parental cells without any plasmids. The dotted line shows the overexpression of human NKp46 protein after CHOK1 parental cells were transfected with human NKp46 plasmid.
  • Figure 2 shows the anti-NKp46 positive control antibody detected by flow cytometry to verify the expression of cynomolgus monkey NKp46 on CHOK1 cells; the solid line in the figure shows the expression of cynomolgus monkey NKp46 on CHOK1 parental cells without any plasmid transfection situation, the dotted line in the figure shows the overexpression of cynomolgus monkey NKp46 protein after CHOK1 parental cells were transfected with cynomolgus monkey NKp46 plasmid.
  • Figures 3-6 show the binding of anti-NKp46 chimeric antibodies of the present disclosure to NKp46-expressing cells (human NKp46-CHOK1).
  • Figures 7-10 show the binding of anti-NKp46 chimeric antibodies of the present disclosure to cells expressing NKp46 (cynomolgus NKp46-CHOK1).
  • Figures 11-12 show the results of in vitro NK cell activation by anti-NKp46 chimeric antibodies of the present disclosure.
  • FIG. 13-16 show the binding of anti-NKp46 humanized antibodies of the present disclosure to NKp46 expressing cells (human NKp46-CHOK1).
  • FIG. 17-20 show the binding of anti-NKp46 humanized antibodies of the present disclosure to NKp46 expressing cells (cynomolgus NKp46-CHOK1).
  • Figures 21-24 show the results of in vitro NK cell activation by anti-NKp46 humanized antibodies of the present disclosure.
  • the term “about” is meant to include ⁇ 20% of the specified value, or in some cases ⁇ 10%, or in some cases ⁇ 5%, or within ⁇ 1% in some cases, or ⁇ 0.1% in some cases.
  • VHH variable domain of heavy chain of heavy-chain antibody
  • the VHH can have additional disulfide bonds between dromedary CDR1 and CDR3, and between llama CDR2 and CDR3 (Harmsen and De Haard 2007 Appl Microbiol Biotechnol., 77, 13-22; Muyldermans 2001 J Biotechnol., 74, 277-302).
  • Expanded CDR3 loops can adopt a convex conformation, whereas conventional paratopes are restricted to concave or planar structures (Muyldermans 2001 J Biotechnol., 74, 277-302). These features allow VHHs to recognize unique epitopes that are less immunogenic for conventional antibodies (Lafaye et al. 2009 Mol Immuno., 46, 695-704; Wernery 2001 J Vet Med B Infect Dis Vet Public Health., 48, 561-568) . Although VHHs are defined as monovalent antibodies, which by default exclude any avidity effects, the biological activity measured as IC50 in vitro can be similar to conventional bivalent antibody molecules (Thys et al. 2010 Antiviral Res., 87:257-264) .
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies contained in the population are identical (except for possible natural mutations that may be present in minor amounts). Monoclonal antibodies are highly specific for a single antigenic site. Furthermore, unlike conventional polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes) on an antigen, each monoclonal antibody is directed against only a single determinant or epitope on an antigen.
  • the present disclosure may relate to chimeric camelid/human antibodies, particularly wherein the VH and/or VL domains are entirely camelid sequences (eg, llama or alpaca), The remainder of the antibody is entirely chimeric with human sequences.
  • VH and/or VL domains are entirely camelid sequences (eg, llama or alpaca)
  • the remainder of the antibody is entirely chimeric with human sequences.
  • "humanized” or “germlined” camelid antibodies and camelid/human chimeric antibodies are also included, wherein the VH and/or VL domains are relative to those obtained by active immunization
  • the camelid VH and/or VL domains contain one or more amino acid substitutions in the framework regions.
  • the present disclosure includes native, recombinant VHH or VH.
  • Recombination involves the use of methods of genetic engineering (cloning, amplification) to produce the VHH or VH.
  • VHHs according to the present disclosure can be in the form of monomers or in the form of homomultimers, such as homodimers or homotrimers.
  • Antibodies of the present disclosure include alpaca-derived antibodies, chimeric antibodies, humanized antibodies, preferably humanized antibodies.
  • a "chimeric antibody” is an antibody obtained by fusing the variable region of an alpaca-derived antibody with the constant region (or Fc region) of a human antibody, which can reduce the immune response induced by the alpaca-derived antibody .
  • To establish a chimeric antibody first establish a hybridoma or antibody library that secretes alpaca-derived specific monoclonal antibodies, and then link the variable region gene of the alpaca antibody with the human constant region gene (or Fc region gene) to form a chimeric gene. Insert into expression vector and finally express chimeric antibody molecule in eukaryotic system or prokaryotic system.
  • Humanized antibody refers to an antibody produced by grafting the CDR sequences of alpaca into the framework of human antibody variable regions, ie, different types of human germline antibody framework sequences. It can overcome the heterologous reaction induced by chimeric antibodies due to carrying a large amount of alpaca protein components.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the germline DNA sequences of the human heavy and light chain variable region genes are available in the "VBase" human germline sequence database (https://www2.mrc-lmb.cam.ac.uk/vbase/), and Found in Kabat, E.A.
  • the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain the activity.
  • VHH refers to variable antigen binding domains of heavy chain antibodies from the family Camelidae (camelids, dromedaries, llamas, alpacas, etc.) (see Nguyen et al. 2000 EMBO J., 19, 921-930; Muyldermans 2001 J Biotechnol., 74, 277-302 and reviewed Vanlandschoot et al. 2011 Antiviral Res. 92, 389-407).
  • Nanobodies can be defined as amino acid sequences having the following (universal) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • FR1-FR4 refer to framework regions (Frame) 1-4, respectively, and wherein CDR1-CDR3 refer to complementarity determining regions 1-3, respectively.
  • IgA immunoglobulin A
  • IgD immunoglobulin D
  • IgE immunoglobulin G
  • IgG immunoglobulin M
  • the corresponding heavy chain constant domains are called ⁇ , ⁇ , ⁇ , ⁇ and ⁇ , respectively
  • IgG and IgA can be further divided into different
  • the subclasses, such as IgG can be divided into IgG1, IgG2, IgG3, IgG4, and IgA can be divided into IgA1 and IgA2.
  • the light chains of antibodies from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains.
  • the constant region comprises three domains called CH1, CH2 and CH3 (IgM and IgE have a fourth domain CH4).
  • the CH1 and CH2 domains are separated by a flexible hinge region, which is a variable length proline and cysteine rich segment.
  • Each class of antibodies further comprises interchain and intrachain disulfide bonds formed by paired cysteine residues.
  • Fc is used herein to define the C-terminal region of an immunoglobulin heavy chain, which region comprises at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions. Unless otherwise stated, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also known as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • sequence identity or “sequence similarity” or “sequence homology” mean that the sequences are aligned (and where necessary introduced gaps) for maximum percent sequence identity without any The percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference polypeptide sequence after conservative substitutions are considered part of sequence identity. Sequence alignments to determine percent amino acid sequence identity can be performed using various methods in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to obtain maximal alignment over the full length of the sequences being compared.
  • antibody fragment encompasses at least a portion of an intact antibody.
  • a “fragment” of an antibody molecule includes an "antigen-binding fragment” of an antibody, and the term “antigen-binding fragment” refers to an immunoglobulin or antibody that specifically binds or reacts with a selected antigen or epitope thereof Polypeptide fragments, or fusion protein products further derived from such fragments, such as single-chain antibodies, extracellular binding domains in chimeric antigen receptors, etc.
  • Exemplary antibody fragments or antigen-binding fragments thereof include, but are not limited to, variable light chain fragments (VL), variable heavy chain fragments (VH), Fab fragments, F(ab') 2 fragments, Fd fragments, Fv fragments, Single-domain antibodies, linear antibodies, single-chain antibodies (scFv), and bispecific or multispecific antibodies formed from antibody fragments, etc.
  • VL variable light chain fragments
  • VH variable heavy chain fragments
  • Fab fragments F(ab') 2 fragments
  • Fd fragments Fv fragments
  • Single-domain antibodies linear antibodies
  • single-chain antibodies scFv
  • bispecific or multispecific antibodies formed from antibody fragments etc.
  • multispecific antibody refers to an antibody that is functionally linked (eg, chemically conjugated, genetically fused, non-covalently bound, or otherwise) to one or more other binding molecules, thereby forming a New antibody constructs for site and/or target binding.
  • bispecific antibodies are more commonly used, which specifically refer to antibody constructs specific for two different antigens.
  • bispecific or multispecific antibodies comprise at least two antigen binding domains.
  • an antigen refers to a substance that is recognized and specifically bound by an antibody or antibody-binding fragment.
  • an antigen can include any immunogenic fragment or determinant of a selected target, including mono-epitopes, poly-epitopes, mono-structures domain, multi-domain, or complete extracellular domain (ECD) or protein.
  • ECD extracellular domain
  • Peptides, proteins, glycoproteins, polysaccharides, and lipids, parts thereof, and combinations thereof can constitute antigens.
  • Non-limiting exemplary antigens include tumor antigens or pathogen antigens, and the like.
  • Antigen can also refer to a molecule that elicits an immune response.
  • the antigen can be an isolated full-length protein, a cell surface protein (eg, immunized with a cell expressing at least a portion of the antigen on its surface), or a soluble protein (eg, immunized with only the ECD portion of the protein), or a protein Constructs (eg, Fc antigens).
  • the antigen can be produced in genetically modified cells. Any of the foregoing antigens may be used alone or in combination with one or more immunogenicity enhancing adjuvants known in the art.
  • the DNA encoding the antigen can be genomic or non-genomic (eg, cDNA) and can encode at least a portion of the ECD sufficient to elicit an immunogenic response.
  • Cells in which the antigen is expressed can be transformed using any vector including, but not limited to, adenoviral vectors, lentiviral vectors, plasmids, and non-viral vectors such as cationic lipids.
  • epitopes refers to the site on an antigen to which an immunoglobulin or antibody specifically binds.
  • Epitopes can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents.
  • Epitopes typically include at least 3-15 amino acids in a unique spatial conformation.
  • Methods for determining the epitope to which a given antibody binds are well known in the art and include immunoblotting and immunoprecipitation assays, among others. Methods for determining the spatial conformation of epitopes include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, among others.
  • polypeptide polypeptide
  • peptide protein
  • polymer may be linear, cyclic or branched, it may contain modified amino acids, especially conservatively modified amino acids, and it may be interrupted by non-amino acids.
  • amino acid polymers such as those that have been processed by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, prenylation, elimination Amino acid polymers modified by spination, selenylation, transfer-RNA mediated amino addition such as arginylation, ubiquitination, or any other manipulation such as conjugation to labeling components.
  • amino acid refers to natural and/or unnatural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptidomimetics.
  • a polypeptide or amino acid sequence "derived from" a specified protein refers to the source of the polypeptide.
  • the term also includes polypeptides expressed from the specified nucleic acid sequence.
  • amino acid modification includes amino acid substitutions, insertions and/or deletions in a polypeptide sequence.
  • amino acid substitution or “substitution” herein means replacing an amino acid at a particular position in the parent polypeptide sequence with another amino acid.
  • substitution S32A refers to the replacement of serine at position 32 with alanine.
  • sequence identity or homology of the humanized antibody variable regions to the human receptor variable regions can be determined as discussed herein, and when so measured will preferably share at least 60% or 65% of the sequence identity, more preferably at least 70%, 75%, 80%, 85% or 90% sequence identity, even more preferably at least 93%, 95%, 98% or 99% sequence identity.
  • residue positions that are not identical differ by conservative amino acid substitutions.
  • a "conservative substitution” is an amino acid substitution in which one amino acid residue is replaced by another amino acid residue on a side chain (R group) of similar chemical properties (eg, charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein. Families of amino acid residues with similar side chains have been defined in the art.
  • amino acids with basic side chains eg, lysine, arginine, histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains eg, glycine, asparagine, serine, threonine, tyrosine, cysteine, tryptophan
  • non-polar side chains eg, alanine, valine, leucine, isoleucine
  • amino acid proline
  • beta branched side chains eg, threonine, valine, isoleucine
  • aromatic side chains eg, tyrosine, phenylalanine, tryptophan, histidine
  • amino acid residues in the CDR regions or in the framework regions of the antibodies of the disclosure can be replaced with amino acid residues of other similar side chains.
  • percent sequence identity or degree of similarity can be adjusted upwards to correct for the conservative nature of the substitution.
  • affinity refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen).
  • KD refers to the dissociation constant for a particular antibody-antigen interaction. Binding affinity can be determined using various techniques known in the art, such as surface plasmon resonance, biolayer interferometry, dual polarization interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultrafast Centrifugation and flow cytometry, etc.
  • competitive binding or “competing antibody” generally refers to an antibody that binds to the same epitope as an antibody of the present disclosure, the binding of which results in the inhibition or blocking of binding of the antibody of the present disclosure to the epitope, in a competition assay The degree of competitive inhibition can be obtained.
  • composition refers to a formulation that is in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain additional ingredients that would be unacceptably toxic to the subject to whom the formulation is administered .
  • pharmaceutically acceptable carrier refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic agent is administered.
  • an effective amount refers to the dose of a pharmaceutical formulation of an antibody or fragment of the present disclosure which, after administration to the patient in single or multiple doses, produces the desired effect in the treated patient.
  • An effective amount can be readily determined by the attending physician, who is skilled in the art, by taking into account a variety of factors such as ethnic differences; weight, age, and health; the specific disease involved; the severity of the disease; the individual patient's response; The particular antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapy.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages.
  • the progeny may not be identical in nucleic acid content to the parental cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell.
  • transfection refers to the introduction of exogenous nucleic acid into a eukaryotic cell. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, Liposome fusion, lipofection, protoplast fusion, retroviral infection and biolistics.
  • stable transfection or “stable transfection” refers to the introduction and integration of exogenous nucleic acid, DNA or RNA into the genome of a transfected cell.
  • stable transfectant refers to a cell that stably integrates foreign DNA into its genomic DNA.
  • nucleic acid molecule encoding refers to the sequence of deoxyribonucleotides along a deoxyribonucleic acid chain. The sequence of these deoxyribonucleotides determines the sequence of amino acids along the polypeptide (protein) chain. Thus, a nucleic acid sequence encodes an amino acid sequence.
  • the antibodies or antigen-binding fragments thereof described in the present disclosure are genetically engineered to add one or more human FR regions to non-human CDR regions.
  • Human FR germline sequences are available from the ImMunoGeneTics (IMGT) website (http://imgt.cines.fr), or from The Immunoglobulin FactsBook, (2001) ISBN: 012441351.
  • the engineered antibodies or antigen-binding fragments thereof of the present disclosure can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian-like expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones were expanded in serum-free medium in bioreactors for antibody production.
  • the antibody-secreted culture medium can be purified and collected by conventional techniques.
  • Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange.
  • subject refers to any animal, such as a mammal or a marsupial.
  • Subjects of the present disclosure include, but are not limited to, humans, non-human primates (eg, cynomolgus or rhesus or other types of rhesus monkeys), mice, pigs, horses, donkeys, cattle, sheep, rats, and any species of poultry.
  • tumor refers to a disease characterized by the pathological proliferation of cells or tissues, and its subsequent migration or invasion of other tissues or organs. Tumor growth is usually uncontrolled and progressive, and does not induce or inhibit normal cell proliferation. Tumors can affect a variety of cells, tissues or organs, including but not limited to those selected from the group consisting of bladder, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, Nervous tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, urethra, ureter, urethra, uterus, vaginal organs, or tissue or corresponding cells.
  • Tumors include cancers such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells).
  • Tumors described in the present disclosure may include, but are not limited to, leukemias (eg, acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, Acute monocytic leukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphoma (Hodgkin's disease, non-Hodgkin's disease), primary macroglobulinemia, severe Chain disease, solid tumors such as sarcomas and cancers (eg, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endothelial
  • the "tumor” includes but is not limited to: pancreatic cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, Kidney cancer, leukemia, multiple myeloma, ovarian cancer, cervical cancer and glioma.
  • the terms “disease” or “condition” or “disorder” and the like refer to any alteration or disorder that impairs or interferes with the normal function of a cell, tissue or organ.
  • the “disease” includes, but is not limited to, tumor, pathogen infection, autoimmune disease, T cell dysfunctional disease, or deficiency of immune tolerance (eg, transplant rejection).
  • treatment refers to clinical intervention in an attempt to alter an individual or to manipulate a cell-induced disease process, either prophylactically or in a clinical pathological process.
  • Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing the direct or indirect pathological consequences of any disease, preventing metastasis, slowing the rate of disease progression, improving or relieving the condition, relieving or improving the prognosis, etc.
  • kits or kits includes an effective amount of one or more pharmaceutical compositions of the present disclosure in unit dosage form.
  • kits or kits may contain sterile containers; such containers may be in the form of boxes, ampoules, bottles, vials, tubes, bags, blister packs, or other suitable container forms known in the art .
  • Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding drugs.
  • the kit also includes instructions for administering the pharmaceutical composition of the present disclosure to a subject. Methods of treating diseases using the pharmaceutical compositions of the present disclosure are generally included in the instructions.
  • the nucleotide sequence encoding the amino acid of human NKp46 shown in SEQ ID NO: 1 was cloned into the pCMV3 (SinoBiological, Cat. No. CV011) vector to obtain a vector for the construction of human NKp46 cell line.
  • the obtained vector was transfected into CHOK1 cells (ATCC, Cat. No. CCL-61) to obtain a CHOK1 cell line expressing human NKp46 (abbreviation: human NKp46-CHOK1 cell line).
  • the nucleotide sequence encoding the cynomolgus monkey NKp46 amino acid shown in SEQ ID NO: 2 was cloned into the pCMV3 vector to obtain a vector for the construction of the cynomolgus monkey NKp46 cell line.
  • the obtained vector was transfected into CHOK1 cells to obtain a CHOK1 cell line expressing cynomolgus NKp46 (abbreviation: cynomolgus NKp46-CHOK1 cell line).
  • a positive control antibody (Santa Cruz Biotechnology, Cat. No. sc-59343) was used to detect the expression of human NKp46 in the human NKp46-CHOK1 cell line obtained above by FACS assay, and a negative control anti-HEL human IgG1 LALA was set up in the experiment (Baiying Bio, Cat. No. B109802).
  • cynomolgus monkey NKp46 in the cynomolgus monkey NKp46-CHOK1 cell line obtained above was detected by using a positive control antibody (Santa Cruz Biotechnology, product number sc-59343), and a negative control anti- HEL human IgG1 LALA (Baiying Bio, Cat. No. B109802).
  • NKp46 The expression of human NKp46 in the human NKp46-CHOK1 cell line is shown in Figure 1. The results show that human NKp46 is well overexpressed in CHOK1 cells and can be used for subsequent experiments to verify the relationship between NKp46 mAb and human NKp46 at the cellular level. Combined situation.
  • Alpacas/camels were immunized with the human NKp46 antigen shown in SEQ ID NO:3 and the cynomolgus monkey NKp46 antigen spacer shown in SEQ ID NO:4, respectively.
  • the dates of immunization were day 0, day 14, day 28, and day 42, a total of 4 times.
  • On the 28th day, the 42nd day and the 56th day blood samples were drawn to separate the serum, and the immune response in the serum was detected by flow cytometry. The immunization was terminated when the serum titer was greater than 1:10000.
  • lymphocyte separation solution (Solarbio) to separate the camel/alpaca PBMC according to the operation;
  • SEQ ID NO: 5 upstream primer: GTCCTGGCTGCTCTTCTACAAGG;
  • SEQ ID NO: 6 downstream primer: GGTACGTGCTGTTGAACTGTTCC.
  • SEQ ID NO: 8 upstream primer
  • SEQ ID NO: 12 (downstream primer):
  • the camel/alpaca VHH fragment obtained by the above amplification was recovered, digested with Sfi I, connected to the pADL-23c (Biovector) phagemid carrier, and electrotransformed TG1 E. coli competent cells to build alpaca/camel VHH heavy chain antibody immune library.
  • the library capacity of the NKp46 camel immune library was 5.2E7
  • the library capacity of the alpaca immune library was 7.8E7.
  • the above-mentioned library was amplified and added to M13K07 helper phage to assemble into phage, and then put into 1x10 12 pfu camel and alpaca immune libraries, respectively.
  • Pigmented human NKp46 protein (8 ⁇ g/mL) was incubated at room temperature for 1h, and 0.05% PBST was used to wash away unbound phage, and 100mM triethylamine was used to elute the phage that specifically binds to NKp46, and the infection was logarithmic after serial dilution.
  • Escherichia coli SS320 grown at the stage of growth was cultured overnight at 37°C on ampicillin plates; single clones were picked for IPTG-induced expression, and the supernatant was used for ELISA detection.
  • ELISA plates were coated with 2 ⁇ g/mL human NKp46 or cynomolgus monkey NKp46 antigen overnight at 4°C, washed 3 times with 0.05% PBST, blocked with 5% skim milk for 1 h at room temperature, washed 3 times with 0.05% PBST, and then added to each well of induced epitopes.
  • NKp46-PC1 SEQ ID NO: 14
  • NKp46-PC2 SEQ ID NO: 15
  • NKp46-PC3 SEQ ID NO: 16
  • NKp46-PC4 SEQ ID NO: 17
  • NKp46-PC6 SEQ ID NO: 18
  • NKp46-PC7 SEQ ID NO: 19
  • NKp46-PC8 SEQ ID NO: 20
  • NKp46-PC9 SEQ ID NO: 21
  • NKp46-PC11 SEQ ID NO: 22
  • NKp46-PC12 SEQ ID NO: 23
  • NKp46-PC13 SEQ ID NO: 24
  • NKp46-PC16 SEQ ID NO: 25
  • NKp46-PA2 SEQ ID NO: 26
  • NKp46-PA3 SEQ ID NO: 27
  • NKp46-PA11 SEQ ID NO: 28 NKp46-PA15 SEQ ID NO: 29 NKp46-PA19 SEQ ID NO: 30 NKp46-PA22 SEQ ID NO: 31 NKp46-PA24 SEQ ID NO: 32 NKp46-PA26 SEQ ID NO: 33 NKp46-PA27 SEQ ID NO: 34 NKp46-PA28 SEQ ID NO: 35 NKp46-PA29 SEQ ID NO: 36 NKp46-PA31 SEQ ID NO: 37 NKp46-PA34 SEQ ID NO: 38 NKp46-PA36 SEQ ID NO: 39 NKp46-PA40 SEQ ID NO: 40 NKp46-PA45 SEQ ID NO: 41 NKp46-PA63 SEQ ID NO: 42 NKp46-PA69 SEQ ID NO: 43
  • the CDRs and FRs of the variable region of the antibody are divided by the Kabat numbering rule, and the composition of the three CDR sequences of each antibody is shown in Table 3 below.
  • the numbers in parentheses in Table 3 represent sequence numbers, for example (44) represents SEQ ID NO: 44.
  • the target gene fragment generated by splicing the sequenced monoclonal antibody variable region of the present disclosure and the human IgG1 constant region whose amino acid sequence is shown in SEQ ID NO: 134 was cloned into the pTT5 expression vector (Nova lifetech) to prepare the transfection level expression plasmid.
  • Expi293F TM cells (Thermo Fisher Scientific) were grown in serum-free medium, cells were seeded in shake flasks (Corning), and cultured on a shaker at 37°C, 8% CO 2 . Adjust the cell density, mix the pTT5 recombinant expression vector containing the target gene fragment and PEI transfection reagent according to the appropriate ratio, and add it to the cell culture shake flask. After 6 days of cell culture, collect the expression supernatant, remove cell debris by high-speed centrifugation, and use Affinity purification was performed on a Protein A column. The column was rinsed with PBS until the A280 reading dropped to baseline.
  • the target protein was eluted with an acidic eluent of pH 3.0-pH 3.5, and neutralized with 1M Tris-HCl, pH 8.0-9.0. After the eluted sample is appropriately concentrated, the medium is changed to PBS for dispensing. The final purified chimeric antibody was subjected to SDS-PAGE and HPLC purity analysis and A280 concentration determination.
  • the stable human NKp46-CHOK1 and cynomolgus monkey NKp46-CHOK1 cell lines prepared in Example 2 were cultured, the cells were digested with 0.25% trypsin (containing EDTA) for about 5 minutes, and the reaction was terminated by adding complete medium. The supernatant was discarded by centrifugation at 1500 rpm for 5 minutes, and counted after resuspending in PBS containing 1% BSA.
  • the cell density was adjusted to 1E6/mL, 100 ⁇ L/well, inoculated into a corning-3799 96-well culture plate and cultured overnight at 37°C in an 8% CO 2 environment, after which the 96-well culture plate was centrifuged at 1500 rpm The supernatant was discarded for 5 minutes and placed at 4°C for later use.
  • PBMC peripheral blood cells
  • NK cells were isolated and counted using EasySep TM Human NK Cell Isolation Kit (StemCell).
  • Cells were cultured in ⁇ -MEM medium supplemented with 15% heat-inactivated FBS, 0.2 mM inositol, 0.1 mM ⁇ -mercaptoethanol, 0.02 mM folic acid, 200 U/mL IL-2. Adjust the NK cell density to 1E6/mL and culture in a 37°C 5% CO2 incubator for 6-8 days.
  • Antibodies were diluted in PBS up to a maximum concentration of 20 nM and diluted 3-fold to obtain seven gradient concentrations of 20 nM, 6.67 nM, 2.23 nM, 0.74 nM, 0.247 nM, 0.083 nM and 0.027 nM, respectively.
  • the diluted antibody was coated in corning-3599 96-well culture plate, 100 ⁇ L/well, and coated overnight at 4°C. The 96-well plate was washed twice with 150 ⁇ L/well PBS.
  • Activated NK cells were adjusted to a density of 1E6/mL using complete medium, then plated into washed 96-well plates, 100 ⁇ L/well, and cultured in a 37°C 5% CO 2 incubator for 4 hours. The cells were transferred from the corning-3599 culture plate to the corning-3799 culture plate, centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded for use.
  • CD3-APC Dilute CD3-APC separately according to the instructions CD56-BV421 CD107a-PE
  • the spare cells obtained above were resuspended, and the above-diluted CD3-APC, CD56-BV421, and CD107a-PE antibodies were added at a concentration of 50 ⁇ L/well, and incubated at 4°C for 0.5 hour. Centrifuge at 1500 rpm for 5 minutes, discard the supernatant, wash once with 160 ⁇ L PBS containing 1% BSA, discard the supernatant, wash once with 100 ⁇ L PBS containing 1% BSA, and filter the cells with 300-mesh gauze. The proportion of CD107a positive cells in NK was then analyzed on a flow cytometer.
  • FCS file exported from the flow cytometer, use Flowjo software to analyze the proportion of CD107a positive cells in NK (CD3 - CD56 + ) cells, and import it into Graphpad to obtain the half effective concentration of NK activation (hereinafter referred to as EC 50 ) and the highest average fluorescence Intensity (Top MFI), the results are shown in Table 5 and Figures 11-12.
  • Anti-NKp46 chimeric antibodies exhibited different in vitro NK cell activation abilities.
  • anti-NKp46 chimeric antibodies numbered NKp46-PC11, NKp46-PA27, NKp46-PA31 and NKp46-PA45 were selected for humanization design.
  • NKp46-PC11 The germline gene sequences with higher homology to clone number NKp46-PC11 were selected as VHH transplantation framework templates by sequence alignment: IGHV3-23*04 (sequence identity 64.3%) and IGHJ4_01 (sequence identity 73.3%).
  • IGHV3-23*04 sequence identity 64.3%
  • IGHJ4_01 sequence identity 73.3%
  • SEQ ID NO: 135 The germline gene sequences with higher homology to clone number NKp46-PC11 were selected as VHH transplantation framework templates by sequence alignment: IGHV3-23*04 (sequence identity 64.3%) and IGHJ4_01 (sequence identity 73.3%).
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in the sequence italic is the FR sequence, the underline is the CDR sequence, and the graft represents the humanized variable obtained after the CDR of the chimeric antibody is implanted into the human germline FR region Region VHH sequence.
  • H1-PC11 (sequence shown in SEQ ID NO: 136), H2-PC11 (sequence shown in SEQ ID NO: 137), H3-PC11 (sequence shown in SEQ ID NO: 138) ), H4-PC11 (sequence shown in SEQ ID NO: 139) and H5-PC11 (sequence shown in SEQ ID NO: 140), 5 humanized antibodies.
  • NKp46-PA27 The germline gene sequences with higher homology to clone number NKp46-PA27 were selected as VHH transplantation framework templates by sequence alignment: IGHV3-30*15 (sequence identity 74.5%) and IGHJ4_01 (sequence identity 80%).
  • sequence alignment IGHV3-30*15 (sequence identity 74.5%) and IGHJ4_01 (sequence identity 80%).
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in the sequence italic is the FR sequence, the underline is the CDR sequence, and the graft represents the humanized variable obtained after the CDR of the chimeric antibody is implanted into the human germline FR region Region VHH sequence.
  • H1-PA27 (sequence shown in SEQ ID NO: 141, original grafted sequence)
  • H2-PA27 (sequence shown in SEQ ID NO: 142)
  • H3-PA27 (sequence shown in SEQ ID NO: 142) : shown in SEQ ID NO: 143
  • H4-PA27 (sequence shown in SEQ ID NO: 144), 4 humanized antibodies.
  • NKp46-PA31 The germline gene sequences with higher homology to clone number NKp46-PA31 were selected as VHH transplantation framework templates by sequence alignment: IGHV3-23*04 (sequence identity 74.5%) and IGHJ4_01 (sequence identity 73.3%).
  • a humanized antibody hPA31 is obtained, and the humanized variable region hPA31 VHH-CDR graft sequence is shown in SEQ ID NO: 145:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in the sequence italic is the FR sequence, the underline is the CDR sequence, and the graft represents the humanized variable obtained after the CDR of the chimeric antibody is implanted into the human germline FR region Region VHH sequence.
  • H1-PA31 sequence as shown in SEQ ID NO: 145, original graft sequence
  • H2-PA31 sequence as shown in SEQ ID NO: 146
  • H3-PA31 sequence as shown in SEQ ID NO: 146) : 147
  • H4-PA31 sequence shown in SEQ ID NO: 148
  • 4 humanized antibodies H1-PA31 (sequence as shown in SEQ ID NO: 145, original graft sequence)
  • H2-PA31 sequence as shown in SEQ ID NO: 146
  • H3-PA31 sequence as shown in SEQ ID NO: 146) : 147
  • H4-PA31 sequence shown in SEQ ID NO: 148
  • NKp46-PA45 The germline gene sequences with higher homology to clone number NKp46-PA45 were selected as VHH transplantation framework templates by sequence alignment: IGHV3-23*04 (sequence identity 79.6%) and IGHJ4_01 (sequence identity 73.3%).
  • IGHV3-23*04 sequence identity 79.6%
  • IGHJ4_01 sequence identity 73.3%
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in the sequence italic is the FR sequence, the underline is the CDR sequence, and the graft represents the humanized variable obtained after the CDR of the chimeric antibody is implanted into the human germline FR region Region VHH sequence.
  • H1-PA45 sequence shown in SEQ ID NO: 149, original graft sequence
  • H2-PA45 sequence shown in SEQ ID NO: 150
  • H3-PA45 sequence shown in SEQ ID NO: 150
  • H4-PA45 sequence shown in SEQ ID NO: 152
  • 4 humanized antibodies H1-PA45 (sequence shown in SEQ ID NO: 149, original graft sequence)
  • H2-PA45 sequence shown in SEQ ID NO: 150
  • H3-PA45 sequence shown in SEQ ID NO: 150
  • H4-PA45 sequence shown in SEQ ID NO: 152
  • 4 humanized antibodies 4 humanized antibodies.
  • the target gene fragment generated by splicing the variable region of the humanized antibody with the human IgG1 constant region was subcloned into the pTT5 expression vector (Nova lifetech), and transfected with ExpiFectamine TM 293.
  • the reagents were transiently transfected into Expi293F TM cells in logarithmic growth phase, and the culture supernatant was collected and subjected to affinity purification.
  • the final purified humanized antibody was subjected to SDS-PAGE and HPLC purity analysis and A280 concentration determination.
  • Example 10 In vitro cell binding verification of humanized antibody that specifically binds to NKp46
  • the human NKp46-CHOK1 and cynomolgus monkey NKp46-CHOK1 stable cell lines obtained in Example 2 were cultured, and the cells were digested with 0.25% trypsin EDTA for about 5 minutes, and the complete medium was terminated. After centrifugation at 1500 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in PBS containing 1% BSA and counted. The cell density was adjusted to 1E6/mL, and plated in corning-3799 96-well culture plate, 100 ⁇ L/well. The supernatant was discarded by centrifugation at 1500 rpm for 5 minutes and kept at 4°C for later use.
  • Antibodies were prepared in PBS containing 1% BSA at a starting concentration of 100 nM and diluted 10-fold to obtain 7 gradients: 10 -4 nM, 10 -3 nM, 10 -2 nM, 10 -1 nM, 10 0 nM, 101 nM and 102 nM. Resuspend the cells with the prepared antibody, 100 ⁇ L/well. Incubate the resuspended cell culture plate in a refrigerator at 4°C for 1 hour. Centrifuge at 1500 rpm for 5 minutes, discard the supernatant, wash once with 160 ⁇ L of PBS containing 1% BSA, and discard the supernatant for use.
  • the secondary antibody (goat anti human IgG Fc PE) was prepared in PBS containing 1% BSA, diluted 1:200. Resuspend the cells with the prepared secondary antibody, 100 ⁇ L/well, and incubate in a refrigerator at 4°C for 0.5 hours. Centrifuge at 1500 rpm for 5 minutes, discard the supernatant, wash with 160 ⁇ L of PBS containing 1% BSA, discard the supernatant, resuspend the cells in 100 ⁇ L of PBS containing 1% BSA, and filter the cells with 300-mesh gauze. The mean fluorescence intensity of antibody binding to cells was then analyzed on a flow cytometer.
  • FCS file from the flow cytometer, use Flowjo software to analyze the PE channel mean fluorescence intensity (hereinafter referred to as MFI), and import the analyzed mean fluorescence intensity into Graphpad to analyze the half binding concentration of antibody to cells (hereinafter referred to as EC 50 ) and the highest Mean fluorescence intensity (Top MFI), the results are shown in Table 6 and Figures 13-20.
  • MFI mean fluorescence intensity
  • EC 50 half binding concentration of antibody to cells
  • Top MFI Mean fluorescence intensity
  • PBMC peripheral blood cells
  • NK cells were isolated and counted using EasySep TM Human NK Cell Isolation Kit.
  • Cells were cultured in ⁇ -MEM medium supplemented with 15% heat-inactivated FBS, 0.2 mM inositol, 0.1 mM ⁇ -mercaptoethanol, 0.02 mM folic acid, 200 U/mL IL-2. Adjust the NK cell density to 1E6/mL and culture in a 37°C 5% CO2 incubator for 6-8 days.
  • Antibodies were diluted in PBS up to a concentration of 20 nM to obtain seven gradient concentrations of 20 nM, 6.67 nM, 2.23 nM, 0.74 nM, 0.247 nM, 0.083 nM and 0.027 nM.
  • the diluted antibody was coated in corning-3599 96-well culture plate, 100 ⁇ L/well, and coated overnight at 4°C.
  • the 96-well plate was washed twice with 150 ⁇ L/well PBS.
  • Activated NK cells were adjusted to a density of 1E6/mL using complete medium, then plated into washed 96-well plates, 100 ⁇ L/well, and cultured in a 37°C 5% CO 2 incubator for 4 hours.
  • the cells were transferred from the corning-3599 culture plate to the corning-3799 culture plate, centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded for use.
  • CD3-APC Dilute the antibodies (CD3-APC, CD56-BV421, CD107a-PE) according to the instructions, resuspend the cells, 50 ⁇ L/well, and incubate at 4°C for 0.5 hours. Centrifuge at 1500 rpm for 5 minutes, discard the supernatant, wash with 160 ⁇ L of PBS containing 1% BSA, discard the supernatant, suspend the cells in 100 ⁇ L of PBS containing 1% BSA, and filter the cells with 300-mesh gauze. The proportion of CD107a positive cells in NK was then analyzed on a flow cytometer.
  • FCS file exported from the flow cytometer, use Flowjo software to analyze the proportion of CD107a positive cells in NK (CD3 - CD56 + ) cells, and import it into Graphpad to obtain the half effective concentration of NK activation (hereinafter referred to as EC 50 ) and the highest average fluorescence Intensity (Top MFI), the results are shown in Table 7 and Figures 21-24.
  • EC 50 half effective concentration of NK activation
  • Top MFI highest average fluorescence Intensity
  • the affinity and kinetic properties of the humanized antibody that specifically binds to NKp46 with human NKp46 (Kenbio, NKP-HM146) and cynomolgus monkey NKp46 (Kenbio, NKP-CM146) were analyzed using a Biacore 8K instrument.
  • the Biacore instrument CM5 chip was first activated with ethyl-dimethylaminopropyl-carbodiimide EDC and hydroxysuccinimide NHS, then anti-human Fc mouse monoclonal antibody was immobilized, and then blocked with ethanolamine.
  • NKp46 humanized antibody was diluted to 0.8 ⁇ g/mL with HBS-EP+ buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20) at 10 ⁇ L/mL The flow rate of min was captured for 60s.
  • Human NKp46 was two-fold serially diluted to serial concentrations (400 nM-3.125 nM), bound for 90 s and dissociated for 500 s at a flow rate of 50 ⁇ L/min.
  • NKp46 humanized antibody was diluted to 0.8 ⁇ g/mL with HBS-EP+ buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20). Capture for 60 s at a flow rate of 10 ⁇ L/min. Cynomolgus monkey NKp46 was two-fold serially diluted to serial concentrations (400nM-3.125nM), bound for 90s and dissociated for 500s at a flow rate of 50 ⁇ L/min.
  • the anti-NKp46 humanized antibody basically maintained the affinity with the human NKp46 antigen protein.
  • NKp46-PC11 7.02E+04 3.40E-04 4.84E-09 72.4 H1-PC11 5.08E+04 3.07E-04 6.05E-09 93.9 H2-PC11 6.13E+04 1.26E-04 2.05E-09 55.1 H3-PC11 6.78E+04 7.75E-05 1.14E-09 50 NKp46-PA27 1.12E+05 3.62E-03 3.25E-08 94.7 H2-PA27 9.77E+04 2.16E-03 2.21E-08 119.3 H4-PA27 1.12E+05 2.66E-03 2.37E-08 117.1 NKp46-PA31 1.68E+05 1.00E-03 5.97E-09 77.3 H2-PA31 8.78E+04 9.59E-04 1.09E-08 45.4 H4-PA31 1.75E+05 1.03E-03 5.89E-09 46.6 NKp46-PA
  • the purity of the samples was monitored by SEC-HPLC to investigate the periodic stability under certain concentration conditions.
  • Exemplary conditions such as controlling the sample concentration at 10 mg/mL, 10 mM acetate, 9% trehalose (pH 5.5) buffer and controlling the concentration at 1 mg/mL PBS (pH 7.4) buffer, compared to 40 Stored at °C for 0, 7, and 14 days of stability.
  • Waters Xbridge Protein BEH SEC 3.5 ⁇ m 7.8*300mm column was used for detection, the mobile phase was PBS buffer, adjusted to pH 6.8 with H 3 PO 4 ; the flow rate was 0.5 mL/min.
  • Table 10 The experimental results are shown in Table 10.
  • the molecular integrity of the samples was monitored by CE-SDS, and the periodic stability under certain concentration conditions was investigated.
  • the sample concentration was controlled at 10 mg/mL, 10 mM acetate, 9% trehalose (pH 5.5) buffer, and the molecular integrity at 40°C for 0, 7, and 14 days was compared under reducing and non-reducing conditions.
  • Detection using PA 800Plus (Beckman Sciex), electrophoresis time 40 minutes.
  • the experimental results are shown in Table 11.
  • the charge heterogeneity of the sample molecules was monitored by cIEF.
  • Antibodies have many post-translational modifications, including deamidation, isomerization, end changes, glycosylation, oxidation, cleavage, polymerization, and the like. These modifications may cause changes in the surface charge of the antibody, resulting in charge heterogeneity.
  • cIEF is based on the charge of the antibody, which is separated, and then the antibody charge heterogeneity is analyzed.
  • the sample concentration was controlled in 10 mg/mL, 10 mM acetate, 9% trehalose (pH 5.5) buffer, and the charge heterogeneity at 40°C for 0, 7, and 14 days was compared. Take 20ug sample, add it to Separation Mix buffer, and use Maurice.C (ProteinSimple) to focus for 7 minutes to detect the charge distribution of the antibody sample.
  • the experimental results are shown in Table 12.
  • Antibody samples were detected for post-translational modifications by LC-MS technology.
  • Deamidation modification is a common chemical modification in antibodies that may affect the later stability, especially the highly deamidated modification of some amino acids in the CDR region is generally selected to be avoided as much as possible or the mutation is reduced.
  • Exemplary conditions such as controlling the concentration of the antibody to be tested at 10 mg/mL, 10 mM acetate, 9% trehalose (pH 5.5) buffer and controlling the concentration at 1 mg/mL PBS (pH 7.4) buffer, 40 Stored in a °C incubator; samples were taken at 0, 7, and 14 days for enzymatic hydrolysis experiments.
  • N represents the detection of modified asparagine
  • M represents the detection of modified methionine
  • the number represents the position where the light chain or the N-terminal of the heavy chain starts counting.
  • Percentage represents the proportion of deamidation detected by LC-MS in the total peptide signal at the site.

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Abstract

提供NKp46受体的抗体或其抗原结合片段、包含所述抗体或其抗原结合片段的衍生物、药物组合物及其在治疗癌症方面的相关应用。

Description

针对NKp46的抗体及其应用 技术领域
本公开属于免疫学领域，更具体地，本公开涉及针对NKp46受体的抗体或其抗原结合片段、包含所述抗体或其抗原结合片段的衍生物、药物组合物及其在治疗癌症方面的相关应用。
背景技术
NK细胞属于先天性免疫系统中的效应淋巴细胞，是机体抵抗病毒感染和防止肿瘤发生的第一道防线。它与获得性免疫系统中的细胞毒性T细胞，扮演着相近的角色，但它们之间不同的是：通常情况下，细胞毒性T细胞需要检测到病毒细胞表面的MHC，才会引起细胞因子的释放，进而导致靶细胞裂解或凋亡；而NK细胞可以在没有抗体或MHC的情况下，识别这些细胞并进行快速的免疫响应。对于那些失去自身标记的MHC-I型的细胞，NK细胞不经过激活就可以进行杀伤，而这些细胞通常是有害但不能被其他免疫细胞发现并消灭的。
NK细胞通过表面活化性受体和抑制性受体的平衡调控NK细胞的活性。NK细胞表面的活化性受体分为人类白细胞抗原I(human leukocyte antigen-I，HLA-I)类分子相关受体和非相关受体，前者包括杀伤细胞免疫球蛋白样受体二区域(killer cell immunogloblin-like receptor，2 domains short，KIR2DS)，杀伤细胞免疫球蛋白样受体三区域(killer cell immunogloblin-like receptor，3 domains short，KIR3DS)及NK细胞受体2C(natural killer cell group 2C，CD94/NKG2C)；后者主要有NK细胞受体2区域(natural killer cell group 2D，NKG2D)和自然细胞毒性受体(natural cytotoxicity receptors,NCR；包括NKp46，NKp30，NKp44)及DNAX辅助分子1(DNAX accessory molecule-1，DNAM-1)；相应的抑制性受体为KIR2DL，KIR3DL及CD94/NKG2A等。
Sivori等于1997年发现了NKp46受体，是最早发现的自然细胞毒性受体，也是最主要的自然细胞毒受体，表达于所有NK细胞(包括成熟、未成熟、静止和活化NK细胞)表面，在自然杀伤作用中起关键作用。因其特异表达于NK细胞表面，分子量大小为46kD，故命名为NKp46。NKp46基因位于19号染色体，为I型跨膜糖蛋白，NKp46胞外区含有2个Ig样结构域，即D1和D2，D2(靠近细胞膜区域)是肿瘤及病毒感染细胞配体结合的区域。跨膜区有一个带正电荷的精氨酸和一个缺乏基于免疫受体酪氨酸活化序列的细胞质尾部，可以与CD3ζ和FcγR穿膜区天冬氨酸残基建立盐桥，后者通过受体结合使得酪氨酸磷酸化故而介导NKp46的信号转导。目前为止鉴定的NKp46的配体只有流感病毒血凝素和副流感病毒血凝素，而细胞性配体还不知晓。
NK细胞杀伤靶细胞时，毒性颗粒将达到浆膜面并与细胞膜融合，引起颗粒内容物释放，最终导致靶细胞的死亡。随着脱颗粒的发生，CD107a分子被转运到细胞膜表面，并且CD107a分子的表达上调与穿孔素的分泌一致。故，CD107a分子阳性表达的NK细胞可代表具有杀伤活性的NK细胞。
NKp46在抗肿瘤中的作用：活化的NK细胞具有较强的杀伤肿瘤细胞作用，而NKp46受体是传递活化刺激所必需的。已经知道NKp46的表达减少涉及宫颈癌及其前驱病损的免疫逃逸，NKp46/NCR1表达缺失，增加淋巴瘤的生长。Yasser等证实NKp46对所有MM细胞株的杀伤都是必须的。此外，NKp46在中枢神经系统中小胶质细胞的清除也有着重要的作用，Lunemann等证实，大脑内浸润的NK细胞通过NKp46，NKG2D的介导识别，使活化的NK细胞与小胶质细胞形成突触连接，后者的穿孔素极化至细胞界面而发挥杀伤作用。
发明概述
本公开提供了一种抗NKp46的抗体或其抗原结合片段，所述抗体或其抗原结合片段特异性地结合人类和食蟹猴NKp46。
在一些实施方案中，提供一种抗NKp46的抗体或其抗原结合片段，所述抗体或其抗原结合片段能够特异性结合NKp46，其包含3个选自SEQ ID NO:44-133序列所示的CDR。
在一些实施方案中，提供一种抗NKp46的抗体或其抗原结合片段，所述抗体或其抗原结合片段能够特异性结合NKp46，其包含选自SEQ ID NO:44，47，50，53，56，59，62，65，68，71，74，77，80，83，86，89，92，95，98，101，104，107，110，113，116，119，122，125，128，131所示的HCDR1；以及，选自SEQ ID NO:45，48，51，54，57，60，63，66，69，72，75，78，81，84，87，90，93，96，99，102，105，108，111，114，117，120，123，126，129，132所示的HCDR2；以及，选自SEQ ID NO:46，49，52，55，58，61，64，67，70，73，76，79，82，85，88，91，94，97，100，103，106，109，112，115，118，121，124，127，130，133所示的HCDR3。
在一些实施方案中，提供一种抗NKp46的抗体或其抗原结合片段，所述抗体或其抗原结合片段能够特异性结 合NKp46，包含重链可变区，所述重链可变区包含选自下组的HCDR1、HCDR2和HCDR3：SEQ ID NO:44、SEQ ID NO:45和SEQ ID NO:46；或SEQ ID NO:47、SEQ ID NO:48和SEQ ID NO:49；或SEQ ID NO:50、SEQ ID NO:51和SEQ ID NO:52；或SEQ ID NO:53、SEQ ID NO:54和SEQ ID NO:55；或SEQ ID NO:56、SEQ ID NO:57和SEQ ID NO:58；或SEQ ID NO:59、SEQ ID NO:60和SEQ ID NO:61；或SEQ ID NO:62、SEQ ID NO:63和SEQ ID NO:64；或SEQ ID NO:65、SEQ ID NO:66和SEQ ID NO:67；或SEQ ID NO:68、SEQ ID NO:69和SEQ ID NO:70；或SEQ ID NO:71、SEQ ID NO:72和SEQ ID NO:73；或SEQ ID NO:74、SEQ ID NO:75和SEQ ID NO:76；或SEQ ID NO:77、SEQ ID NO:78和SEQ ID NO:79；或SEQ ID NO:80、SEQ ID NO:81和SEQ ID NO:82；或SEQ ID NO:83、SEQ ID NO:84和SEQ ID NO:85；或SEQ ID NO:86、SEQ ID NO:87和SEQ ID NO:88；或SEQ ID NO:89、SEQ ID NO:90和SEQ ID NO:91；或SEQ ID NO:92、SEQ ID NO:93和SEQ ID NO:94；或SEQ ID NO:95、SEQ ID NO:96和SEQ ID NO:97；或SEQ ID NO:98、SEQ ID NO:99和SEQ ID NO:100；或SEQ ID NO:101、SEQ ID NO:102和SEQ ID NO:103；或SEQ ID NO:104、SEQ ID NO:105和SEQ ID NO:106；或SEQ ID NO:107、SEQ ID NO:108和SEQ ID NO:109；或SEQ ID NO:110、SEQ ID NO:111和SEQ ID NO:112；或SEQ ID NO:113、SEQ ID NO:114和SEQ ID NO:115；或SEQ ID NO:116、SEQ ID NO:117和SEQ ID NO:118；或SEQ ID NO:119、SEQ ID NO:120和SEQ ID NO:121；或SEQ ID NO:122、SEQ ID NO:123和SEQ ID NO:124；或SEQ ID NO:125、SEQ ID NO:126和SEQ ID NO:127；或SEQ ID NO:128、SEQ ID NO:129和SEQ ID NO:130；或SEQ ID NO:131、SEQ ID NO:132和SEQ ID NO:133。
在一些实施方案中，提供一种抗NKp46的抗体或其抗原结合片段，所述抗体或其抗原结合片段能够特异性结合NKp46，其包含HCDR1、HCDR2和HCDR3，所述HCDR1、HCDR2和HCDR3来自于SEQ ID NO:14-43，136-152所示的重链可变区。
在一些实施方案中，提供一种抗NKp46的抗体或其抗原结合片段，所述抗体或其抗原结合片段能够特异性结合NKp46，包含重链可变区，所述重链可变区具有与SEQ ID NO:14-43，136-152所示序列至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性。
在一些实施方案中，提供所述的抗NKp46的抗体或其抗原结合片段，其中所述单克隆抗体是重组抗体，优选的，为羊驼源抗体、嵌合抗体或人源化抗体。
在一些实施方案中，提供根据本公开所述的抗NKp46的抗体或其抗原结合片段，其中所述单克隆抗体是纳米抗体，优选的，为人源化的骆驼科VHH。
在一些实施方案中，提供本公开所述的抗NKp46的抗体或其抗原结合片段，其还包括重链恒定区和/或轻链恒定区，优选的，所述重链恒定区包括Fc或变体Fc，Fc来源于鼠或人。
在一些实施方案中，提供本公开所述的抗NKp46的抗体或其抗原结合片段，其为IgG1、IgG2、IgG3或IgG4形式。
在一些实施方案中，提供本公开所述的一种偶联物，将前述任一抗NKp46的抗体或其抗原结合片段与捕获标记物或检测标记物偶联形成，所述的检测标记物包括放射性核素、发光物质、有色物质或酶。
在一些实施方案中，提供一种双特异性抗体，其中的一个抗原结合结构域包含前述任一抗NKp46的抗体或其抗原结合片段。
在一些实施方案中，提供一种多特异性抗体，其中的一个抗原结合结构域包含前述任一抗NKp46的抗体或其抗原结合片段。
在一些实施方案中，提供一种抗体药物缀合物，含有前述任一抗NKp46的抗体或其抗原结合片段，所述的抗体药物缀合物由抗体-接头-毒素相互连接形成。
在一些实施方案中，提供一种嵌合抗原受体，其胞外识别单元包含前述任一抗NKp46的抗体或其抗原结合片段。
在一些实施方案中，提供编码前述任一抗NKp46的抗体或其抗原结合片段的核酸。
在一些实施方案中，提供本公开所述的核酸的重组载体。
在一些实施方案中，提供一种宿主细胞，其包含本公开所述的重组载体或基因组中整合本公开所述的核酸。
在一些实施方案中，提供本公开所述抗NKp46的抗体或其抗原结合片段的方法，包括：在适合的条件下培养 本公开所述的宿主细胞，并从所述细胞中纯化获得表达产物。
在一些实施方案中，提供本公开所述的抗NKp46的抗体或其抗原结合片段在制备特异性靶向表达NKp46的细胞的药物中的用途，优选的，所述细胞是NK细胞。
在一些实施方案中，提供本公开所述的抗NKp46的抗体或其抗原结合片段在制备癌症、感染性疾病或者炎性或自身免疫性疾病药物中的用途，优选的，所述肿瘤包括：白血病，侵袭性淋巴瘤，非霍奇金淋巴瘤，脑胶质瘤，宫颈癌，头颈癌，直肠癌，肾癌，肝癌，肺癌，胰腺癌，胃癌等。
在一些实施方案中，提供本公开所述的抗NKp46的抗体或其抗原结合片段在制备表达NKp46的NK的诊断试剂中的用途。
在一些实施方案中，提供一种含有NKp46的抗体的溶液制剂，其包含抗NKp46的抗体或其抗原结合片段和缓冲液。
在一些实施方案中，提供本公开所述的一种用于鉴定个体中NKp46表达细胞的存在的方法，所述方法包括从包含细胞的个体获得生物样品，使所述细胞与本公开所述的抗NKp46的抗体或其抗原结合片段接触，以及评估所述抗体是否与所述细胞结合。
在一些实施方案中，提供一种药物组合物，其包含有效量的前述任一抗NKp46的抗体或其抗原结合片段、或包含有效量的双特异性抗体、或包含有效量的多特异性抗体、或包含有效量的抗体药物缀合物、或包含有效量的嵌合抗原受体、或包含有效量的核酸、或包含有效量的重组载体、或包含有效量的宿主细胞。
在一些实施方案中，提供本公开所述的药物组合物，还包含药学上可接受的载体。
在一些实施方案中，提供本公开所述的药物组合物，还包含一种或多种额外的其他治疗剂，优选的所述一种或多种额外的其他治疗剂包括：化学治疗剂、细胞毒性剂、放射性治疗剂、癌症疫苗、减瘤剂、靶向性抗癌剂、抗血管生成剂、生物反应修饰剂、细胞因子、激素、抗转移剂和免疫治疗剂。
在一些实施方案中，提供一种药盒或试剂盒，其包括容器，以及位于容器中的本公开所述的药物组合物。
附图说明
附图更进一步说明了本说明书所公开的新特性。参照这些附图将能更好地理解本说明书中所公开的特性和优点，但应当理解，这些附图仅用于说明本文所公开原理的具体的实施方案，而非意欲对所附权利要求的范围加以限制。
图1显示了流式细胞术检测的抗NKp46阳性对照抗体验证CHOK1细胞上人NKp46的表达情况；图中实线所示为未转任何质粒的CHOK1母本细胞上人NKp46的表达情况，图中虚线所示为CHOK1母本细胞转染人NKp46质粒后，人NKp46蛋白过表达情况。
图2显示了流式细胞术检测的抗NKp46阳性对照抗体验证CHOK1细胞上食蟹猴NKp46的表达情况；图中实线所示为未转任何质粒的CHOK1母本细胞上食蟹猴NKp46的表达情况，图中虚线所示为CHOK1母本细胞转染食蟹猴NKp46质粒后，食蟹猴NKp46蛋白过表达情况。
图3-6显示了本公开的抗NKp46嵌合抗体与表达NKp46细胞(人NKp46-CHOK1)的结合情况。
图7-10显示了本公开的抗NKp46嵌合抗体与表达NKp46细胞(食蟹猴NKp46-CHOK1)的结合情况。
图11-12显示了本公开的抗NKp46嵌合抗体的体外NK细胞激活结果。
图13-16显示了本公开的抗NKp46人源化抗体与表达NKp46细胞(人NKp46-CHOK1)的结合情况。
图17-20显示了本公开的抗NKp46人源化抗体与表达NKp46细胞(食蟹猴NKp46-CHOK1)的结合情况。
图21-24显示了本公开的抗NKp46人源化抗体的体外NK细胞激活结果。
具体实施方式
术语
本说明书中提及的所有公布、专利和专利申请都以引用的方式并入本文，所述引用的程度就如同已特定地和个别地指示将各个别公布、专利或专利申请以引用的方式并入一般。
在下文详细描述本公开前，应理解本公开不限于本文中描述的特定方法学、方案和试剂，因为这些可以变化。还应理解本文中使用的术语仅为了描述具体实施方案，而并不意图限制本公开的范围。除非另外定义，本文中使用的所有技术和科学术语与本公开所属领域中普通技术人员通常的理解具有相同的含义。
本文所公开的某些实施方案包含了数值范围，并且本公开的某些方面可采用范围的方式描述。除非另有说明，应当理解数值范围或者以范围描述的方式仅是出于简洁、便利的目的，并不应当认为是对本公开的范围的严格限定。因此，采用范围方式的描述应当被认为具体地公开了所有可能的子范围以及在该范围内的所有可能的具体数值点，正如这些子范围和数值点在本文中已经明确写出。例如，从1至6的范围的描述应当被认为具体公开了从1至3、1至4、1至5、2至4、2至6、3至6等的子范围，以及在这些范围内的具体的数值点，例如1、2、3、4、5、6。不论所述数值的宽窄，上述原则均同等适用。当采用范围描述时，该范围包括范围的端点。
当涉及可测量值比如量、暂时持续时间等时，术语“约”是指包括指定值的±20％、或在某些情况下±10％、或在某些情况下±5％、或在某些情况下±1％、或在某些情况下±0.1％的变化。
本文所用氨基酸三字母代码和单字母代码如J.Biol.Chem，243，p3558(1968)中所述。
常规的免疫球蛋白是四聚体，由两条重链和两条轻链组成，组合分子量约150kDa。在骆驼科(Camelidae)成员中，相当比例的血清抗体是同源二聚体IgG，分子量约80kD(Hamers-Casterman等人.1993Nature,363,446-448)。这些重链免疫球蛋白(Ig)包含三个结构域，其可变区被称为VHH(variable domain of heavy chain of heavy-chain antibody)。重组VHH(约12至14kD)构成完整的抗原结合结构域并显示出广阔的抗原结合谱。扩大它们的高变区，并表现出独特的特性，如三至四个(与常规抗体VL相互作用的)疏水框架残基被更多亲水性氨基酸取代。为了稳定扩大的CDR，除了常规的二硫键以外，在单峰骆驼CDR1和CDR3之间，在美洲驼的CDR2和CDR3之间，VHH可具有额外的二硫键(Harmsen和De Haard 2007 Appl Microbiol Biotechnol.,77,13-22；Muyldermans 2001 J Biotechnol.,74,277-302)。扩大的CDR3环可以采取凸面构象，而常规的互补位被限制在凹的或者平面结构(Muyldermans 2001 J Biotechnol.,74,277-302)。这些特征允许VHH识别对于常规抗体而言免疫原性较差的独特表位(Lafaye等人.2009 Mol Immuno.,46,695-704；Wernery 2001 J Vet Med B Infect Dis Vet Public Health.,48,561-568)。尽管VHH被定义为单价抗体，默认排除任何亲合力效果，被测量表示为体外IC 50的生物活性可类似于常规的二价抗体分子(Thys等人.2010 Antiviral Res.,87:257-264)。
术语“单克隆抗体”是指由一群基本同源的抗体获得的抗体，即包含在该群体中的各个抗体是相同的(除了可能以少量存在的可能的天然突变以外)。单克隆抗体针对单个抗原位点具有高度特异性。此外，与常规的多克隆抗体制剂(其通常包括针对抗原上不同决定簇(表位)的不同抗体)不同的是，每个单克隆抗体只针对抗原上的单个决定簇或表位。
在某些实施方式中，本公开可涉及嵌合的骆驼科动物/人抗体，特别是其中VH和/或VL结构域完全是骆驼科动物序列(例如大羊驼或阿尔帕卡羊驼)，而抗体的其余部分完全是人序列的嵌合抗体。在本公开的一些优选实施方式中，还包括“人源化”或“种系化”的骆驼科抗体以及骆驼科/人嵌合抗体，其中VH和/或VL结构域相对于通过主动免疫获得的骆驼科VH和/或VL结构域来说在框架区包含一个或多个氨基酸替换。用人种系VH或VL结构域中的对应残基来取代起始骆驼科VH或VL结构域中的不匹配氨基酸残基，这样的“人源化”过程提高了与人种系VH或VL结构域的序列一致性百分比。
本公开包括天然、重组的VHH或VH。
“重组”涉及采用遗传工程的方法(克隆、扩增)来生产所述VHH或VH。
根据本公开的VHH能够是单体的形式或者同源多聚体的形式，如同源二聚体或同源三聚体。
本公开的抗体包括羊驼源抗体、嵌合抗体、人源化抗体，优选人源化抗体。
“嵌合抗体(chimeric antibody)”，是将羊驼源性抗体的可变区与人抗体的恒定区(或Fc区)融合而成的抗体，可以减轻羊驼源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌羊驼源性特异性单抗的杂交瘤或抗体文库，然后将羊驼抗体可变区基因与人恒定区基因(或Fc区基因)连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。
“人源化抗体(humanized antibody)”，是指将羊驼的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量羊驼蛋白成分，从而诱导的异源性反应。此类框架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(https://www2.mrc-lmb.cam.ac.uk/vbase/)中获得，以及在Kabat，E.A.等人，1991 Sequences of Proteins of Immunological Interest第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。
“VHH”涉及来自骆驼科(骆驼、单峰骆驼、美洲驼、羊驼等)重链抗体的可变抗原结合结构域(参见Nguyen等人.2000 EMBO J.，19，921-930；Muyldermans 2001 J Biotechnol.，74，277-302以及综述Vanlandschoot等人.2011 Antiviral Res.92，389-407)。
通常纳米抗体可以定义为具有下述(通用)结构的氨基酸序列：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。其中，FR1-FR4分别是指框架区(Frame)1-4，并且其中CDR1-CDR3分别是指互补决定区1-3。
本领域已知五个主要类别的抗体：IgA，IgD，IgE，IgG和IgM，对应的重链恒定结构域分别被称为α，δ，ε，γ和μ，IgG和IgA可以进一步分为不同的亚类，例如IgG可分为IgG1，IgG2，IgG3，IgG4，IgA可分为IgA1和IgA2。来自任何脊椎动物物种的抗体的轻链基于其恒定结构域的氨基酸序列可以被分配到两种明显相异的类型之一，称为κ和λ。
在IgG、IgA和IgD抗体的情形中，该恒定区包含称为CH1、CH2和CH3的三个结构域(IgM和IgE具有第四结构域CH4)。在IgG、IgA和IgD类别中，CH1和CH2结构域被柔性铰链区分离，该铰链区是可变长度的富含脯氨酸和半胱氨酸的区段。每类抗体进一步包含由配对半胱氨酸残基形成的链间和链内二硫键。
术语“Fc”在本文中用于定义免疫球蛋白重链的C端区域，所述区域包含至少一部分的恒定区。该术语包括天然序列Fc区和变体Fc区。除非另外说明，Fc区或恒定区中的氨基酸残基的编号是根据EU编号系统，其也被称为EU索引，如在Kabat等，Sequences of Proteins of Immunological Interest，5thEd.Public Health Service，National Institutes of Health，Bethesda，MD，1991中所述。
需说明的是，本公开的单克隆抗体可变区的CDR和FR的划分是根据Kabat定义确定的。而其他命名和编号系统，例如Chothia、IMGT或AHo等，也是本领域技术人员已知的。因此，以本公开的单抗序列为基础，包含任何命名系统衍生的一种或多种CDR的人源化抗体均明确地保持在本公开的范围内。
术语“序列同一性”或“序列相似性”或“序列同源性”，是指在将所述序列进行比对(并在必要时导入空位)以获取最大百分比序列同一性，且不将任何保守置换视为序列同一性的部分之后，候选序列中的氨基酸残基与参比多肽序列中的相同氨基酸残基的百分比。可使用本领域各种方法进行序列比对以便测定百分比氨基酸序列同一性，例如，使用公众可得到的计算机软件如BLAST、BLAST-2、ALIGN或MEGALIGN(DNASTAR)软件。本领域技术人员可以决定测量比对的适宜参数，包括对所比较的序列全长获得最大比对所需的任何算法。
术语“抗体片段”包含完整抗体的至少一部分。如在此所使用，抗体分子的“片段”包括抗体的“抗原结合片段”，并且术语“抗原结合片段”是指免疫球蛋白或抗体中与所选抗原或其抗原表位特异性结合或反应的多肽片段，或由此片段进一步衍生的融合蛋白产物，例如单链抗体，嵌合抗原受体中的胞外结合区等。示例性的抗体片段或其抗原结合片段包括但不限于：可变轻链片段(VL)、可变重链片段(VH)、Fab片段、F(ab’) 2片段、Fd片段、Fv片段、单结构域抗体、线性抗体、单链抗体(scFv)及由抗体片段形成的双特异性抗体或多特异性抗体等。
术语“多特异性抗体”是指，将抗体通过功能连接(例如化学偶联、基因融合、非共价结合或其他方法)至一个或多个其他结合分子上，从而形成与两个以上不同位点和/或靶点结合的新的抗体构建体。其中，使用较多的是“双特异性抗体”，其特指针对两种不同抗原具有特异性的抗体构建体。通常，双特异性抗体或多特异性抗体至少包括2个抗原结合结构域。
术语“抗原”是指被抗体或抗体结合片段识别并特异性结合的物质，广义上，抗原可以包括所选靶标的任何免疫原性片段或决定簇，包括单表位、多表位、单结构域、多结构域、或完整的胞外结构域(ECD)或蛋白质。肽、蛋白质、糖蛋白、多糖和脂质，其部分及其组合均可构成抗原。非限制性示例性抗原包括肿瘤抗原或病原体抗原等。“抗原”也可以指引发免疫反应的分子。任何形式的抗原或含有该抗原的细胞或制剂都可以用于生成对抗原决定簇具有特异性的抗体。抗原可以是分离的全长蛋白质、细胞表面蛋白(例如，用在其表面上表达至少一部分抗原的细胞进行免疫的)、或可溶性蛋白质(例如，仅用该蛋白质的ECD部分进行免疫的)或蛋白质构建体(例如，Fc抗原)。该抗原可以在基因修饰的细胞中产生。前述任何抗原可以单独或与本领域已知的一种或多种免疫原性增强佐剂组合使用。编码该抗原的DNA可以是基因组的或非基因组的(例如，cDNA)，并且可以编码足以引起免疫原性应答的至少一部分ECD。可以使用任何载体来转化其中表达抗原的细胞，所述载体包括但不限于腺病毒载体、慢病毒载体、质粒以及非病毒载体如阳离子脂质。
术语“表位”是指抗原上与免疫球蛋白或抗体特异性结合的位点。表位可以由相邻的氨基酸、或通过蛋白质的三级折叠而并列的不相邻的氨基酸形成。由相邻的氨基酸形成的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象包括至少3-15个氨基酸。由给定的抗体确定其 结合的表位的方法是本领域熟知的，包括免疫印迹和免疫沉淀检测分析等。确定表位的空间构象的方法包括本领域中的技术和本文所述的技术，例如X射线晶体分析法和二维核磁共振等。
术语“多肽”、“肽”和“蛋白质”在本文中可互换使用以指任何长度的氨基酸的聚合物。聚合物可以是直链、环状或支链的，它可以包含修饰的氨基酸，特别是保守修饰的氨基酸，并且它可以被非氨基酸中断。该术语还包括改性的氨基酸聚合物例如已经通过硫酸化、糖基化、脂化、乙酰化、磷酸化、碘化、甲基化、氧化、蛋白水解加工、异戊二烯化、外消旋化、硒酰化、转移-RNA介导的氨基加成如精氨酸化、泛在化、或任何其他操作如与标记组分缀合等改性的氨基酸聚合物。如本文所用，术语“氨基酸”是指天然和/或非天然或合成氨基酸，包括甘氨酸以及D或L光学异构体，以及氨基酸类似物和肽模拟物。“衍生自”指定的蛋白质的多肽或氨基酸序列是指多肽的来源。该术语还包括由指定的核酸序列表达的多肽。
术语“氨基酸修饰”(或“修饰的氨基酸”)包括在多肽序列中的氨基酸取代、插入和/或缺失。本文中的“氨基酸取代”或“取代”意指用另一种氨基酸替换亲本多肽序列中特定位置上的氨基酸。例如，取代S32A指第32位的丝氨酸被丙氨酸替换。
人源化抗体可变区与人类受体可变区的序列同一性或同源性可以如在此所论述的进行测定，并且当这样测量时，将优选地共享至少60％或65％的序列同一性，更优选至少70％、75％、80％、85％或90％的序列同一性，甚至更优选至少93％、95％、98％或99％的序列同一性。优选地，不相同的残基位置因保守性氨基酸置换而不同。“保守取代”是一个氨基酸残基被具有类似化学特性(例如，电荷或疏水性)的侧链(R基团)的另一个氨基酸残基替换的氨基酸取代。一般而言，保守氨基酸取代不会实质上改变蛋白质的功能特性。本领域已经定义了具有相似侧链的氨基酸残基家族。这些家族包括含碱性侧链的氨基酸(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电的极性侧链(例如，甘氨酸、天冬酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因而，可以用其他相似侧链的氨基酸残基替换本公开抗体的CDR区中或框架区中的一个或多个氨基酸残基。在两个或更多个氨基酸序列因保守取代而彼此不同的情形中，序列同一性百分比或相似性程度可以向上调整以校正该取代的保守性质。
术语“亲和力”或“结合亲和力”指分子(例如抗体)的单一结合位点与其结合配偶体(例如抗原)之间全部非共价相互作用总和的强度。术语“K D”是指特定的抗体-抗原相互作用的解离常数。可以使用本领域已知的各种技术来确定结合亲和力，例如表面等离子体共振、生物层干涉法、双极化干涉法、静态光散射、动态光散射、等温滴定量热法、ELISA、分析超速离心和流式细胞术等。
术语“竞争结合”或“竞争性抗体”通常是指，与本公开的抗体结合相同表位的抗体，其结合导致本公开的抗体与抗原表位的结合被抑制或阻断，在竞争测定中可以得到竞争性抑制的程度。
术语“药物组合物”指这样的制剂，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述制剂的受试者具有不可接受的毒性的另外的成分。
术语“药用载体”或“药学上可接受的载体”指与治疗剂一起施用的稀释剂、佐剂(例如弗氏佐剂(完全和不完全的))、赋形剂或媒介物。
术语“有效量”指本公开的抗体或片段的药物制剂的剂量，其以单一或多次剂量施用患者后，在治疗的患者中产生预期效果。有效量可以由作为本领域技术人员的主治医师通过考虑以下多种因素来容易地确定：诸如人种差异；体重、年龄和健康状况；涉及的具体疾病；疾病的严重程度；个体患者的应答；施用的具体抗体；施用模式；施用制剂的生物利用率特征；选择的给药方案；和任何伴随疗法的使用。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可交换地使用且是指其中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“转化的细胞”，其包括初级转化的细胞和来源于其的后代，而不考虑传代的数目。后代在核酸含量上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括与在最初转化的细胞中筛选或选择的具有相同功能或生物学活性的突变体后代。
本文所用的术语“转染”是指将外源核酸引入真核细胞。转染可以通过本领域已知的各种手段来实现，包括磷酸钙-DNA共沉淀、DEAE-葡聚糖介导的转染、聚凝胺介导的转染、电穿孔、显微注射、脂质体融合、脂质转染、原生质体融合、逆转录病毒感染和生物弹道技术(biolistics)。
术语“稳定转染”或“稳转”是指将外源核酸、DNA或RNA引入和整合到转染细胞的基因组中。术语“稳定转染体”(stable transfectant)是指将外来DNA稳定地整合到基因组DNA中的细胞。
术语“核酸分子编码”、“编码DNA序列”和“编码DNA”是指沿着脱氧核糖核酸链的脱氧核糖核苷酸的顺序。这些脱氧核糖核苷酸的顺序决定了沿着多肽(蛋白质)链的氨基酸的顺序。因此，核酸序列编码氨基酸序列。
生产和纯化抗体和抗原结合片段的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南，5-8章和15章。本公开所述的抗体或其抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人FR区。人FR种系序列可以从ImMunoGeneTics(IMGT)的网站(http://imgt.cines.fr)得到，或者从免疫球蛋白杂志(The Immunoglobulin FactsBook，(2001)ISBN：012441351)上获得。
本公开工程化的抗体或其抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。
本文所用的术语“个体”或“受试者”是指任何动物，例如哺乳动物或有袋动物。本公开的个体包括但不限于人类、非人类灵长类动物(例如食蟹猴或恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、绵羊、大鼠和任何种类的家禽。
本文所用的术语“肿瘤”指的是一种以细胞或组织的病理性增生为特征的疾病，及其随后的迁移或侵袭其他组织或器官。肿瘤生长通常是不受控制的和进行性的，不诱导或抑制正常细胞增殖。肿瘤可影响多种细胞、组织或器官，包括但不限于选自膀胱、骨、脑、乳腺、软骨、神经胶质细胞、食管、输卵管、胆囊、心脏、肠、肾、肝、肺、淋巴结、神经组织、卵巢、胰腺、前列腺、骨骼肌、皮肤、脊髓、脾、胃、睾丸、胸腺、甲状腺、气管、尿道、输尿管、尿道、子宫、阴道器官，或组织或相应的细胞。肿瘤包括癌症，如肉瘤，癌，或浆细胞瘤(浆细胞的恶性肿瘤)。本公开所述的肿瘤，可包括，但不限于白血病(如急性白血病、急性淋巴细胞白血病、急性髓细胞性白血病，急性粒细胞白血病，急性早幼粒细胞白血病、急性粒-单核细胞白血病、急性单核细胞白血病、慢性白血病、慢性粒细胞白血病、慢性淋巴细胞白血病、真性红细胞增多症)，淋巴瘤(霍奇金病、非霍奇金病)、原发性巨球蛋白血症，重链病，实体瘤如肉瘤和癌症(如纤维肉瘤、粘液肉瘤、脂肪肉瘤、软骨肉瘤、骨肉瘤、脊索瘤、内皮肉瘤、淋巴管肉瘤、血管肉瘤、淋巴管内皮肉瘤，间皮瘤，尤文氏瘤、平滑肌肉瘤、横纹肌肉瘤、结肠癌、胰腺癌、乳腺癌、卵巢癌、前列腺癌、鳞状细胞癌、基底细胞癌、腺癌、汗腺癌、皮脂腺癌、乳头状癌、乳头状腺癌、支气管癌、髓样癌、肾细胞癌、肝癌，尼罗河管癌，绒癌、精原细胞瘤、胚胎癌、肾母细胞瘤、宫颈癌、子宫癌、睾丸癌、肺癌、小细胞肺癌、膀胱癌、上皮癌、胶质瘤、星形细胞瘤、髓母细胞瘤，颅咽管瘤、室管膜瘤、松果体瘤、血管母细胞瘤，听神经瘤，少突胶质瘤、神经鞘瘤、脑膜瘤、黑色素瘤、神经母细胞瘤、视网膜母细胞瘤)、食管癌、胆囊癌、肾癌、多发性骨髓瘤。较佳地，所述的“肿瘤”包括但不限于：胰腺癌、肝癌、肺癌、胃癌、食管癌、头颈部鳞状细胞癌、前列腺癌、结肠癌、乳腺癌、淋巴瘤、胆囊癌、肾癌、白血病、多发性骨髓瘤、卵巢癌、宫颈癌和胶质瘤。
本文所用的术语“疾病”或“病症”或“紊乱”等是指任何损害或干扰细胞、组织或器官的正常功能的改变或失调。例如，所述的“疾病”包括但不限于：肿瘤、病原体感染、自身免疫性疾病、T细胞功能障碍性疾病、或免疫耐受能力缺陷(如移植排斥)。
本文所用的术语“治疗”是指在试图改变个人或处理细胞引起的疾病过程中的临床干预，既可以进行预防也可以在临床病理过程干预。治疗效果包括但不限于，防止疾病的发生或复发、减轻症状、减少任何疾病直接或间接的病理后果、防止转移、减慢疾病的进展速度、改善或缓解病情、缓解或改善预后等。
术语“药盒”或“试剂盒”包括有效量的一种或多种单位剂型的本公开的药物组合物。在一些实施方案中，药盒或“试剂盒”可含有无菌容器；这样的容器可以是盒、安瓿、瓶、小瓶、管、袋、泡罩包装或本领域已知的其它合适的容器形式。这种容器可以由塑料、玻璃、层压纸、金属箔或其他适合于保持药物的材料制成。此外，药盒还包括将本公开的药物组合物给予个体的说明书。说明书中通常包含使用本公开的药物组合物来治疗疾病的方法。
实施例
下面结合具体实施例，进一步阐述本公开。应理解，这些实施例仅用于说明本公开而不用于限制本公开的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如分子克隆实验指南(J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002)中所述的条件，或按照制造厂商所建议的条件。
实施例1 人NKp46及食蟹猴NKp46抗原信息
表1 序列信息
Figure PCTCN2022079236-appb-000001
实施例2 人NKp46及食蟹猴NKp46细胞系的制备
将编码SEQ ID NO:1所示的人NKp46氨基酸的核苷酸序列，克隆至pCMV3(SinoBiological，货号CV011)载体中，获得用于人NKp46细胞系构建的载体。将所获得的载体转染至CHOK1细胞(ATCC，货号CCL-61)中，获得表达人NKp46的CHOK1细胞系(简称：人NKp46-CHOK1细胞系)。
将编码SEQ ID NO:2所示的食蟹猴NKp46氨基酸的核苷酸序列，克隆至pCMV3载体中，获得用于食蟹猴NKp46细胞系构建的载体。将所获得的载体转染至CHOK1细胞中，获得表达食蟹猴NKp46的CHOK1细胞系(简称：食蟹猴NKp46-CHOK1细胞系)。
使用阳性对照抗体(Santa Cruz Biotechnology，货号sc-59343)，使用FACS测定方法，检测上述获得的人NKp46-CHOK1细胞系中人NKp46的表达情况，并在实验中设立阴性对照anti-HEL human IgG1 LALA(百英生物，货号B109802)。
使用阳性对照抗体(Santa Cruz Biotechnology，货号sc-59343)，使用FACS测定方法，检测上述获得的食蟹猴NKp46-CHOK1细胞系中食蟹猴NKp46的表达情况，并在实验中设立阴性对照anti-HEL human IgG1 LALA(百英生物，货号B109802)。
结果显示：
人NKp46-CHOK1细胞系中人NKp46的表达情况如图1所示，结果表明人NKp46在CHOK1细胞中有很好的过表达现象，可以用于后续实验验证NKp46单抗与人NKp46在细胞水平的结合情况。
食蟹猴NKp46-CHOK1细胞系中食蟹猴NKp46的表达情况如图2所示，结果表明食蟹猴NKp46在CHOK1细胞中有很好的过表达现象，可以用于后续实验验证NKp46单抗与食蟹猴NKp46在细胞水平的结合情况。
实施例3 羊驼/骆驼VHH重链抗体免疫文库的构建
分别用SEQ ID NO:3所示的人NKp46抗原和SEQ ID NO:4所示的食蟹猴NKp46抗原间隔对羊驼/骆驼进行免疫。免疫的日期分别为第0天，第14天，第28天，第42天，共免疫4次。分别在第28天，第42天，第56天抽取血样分离出血清，利用流式细胞术检测血清中免疫应答反应情况。检测血清效价大于1：10000时，结束免疫。对免疫的羊驼/骆驼重新抽取血样各100mL，进行如下操作：
1.使用淋巴细胞分离液(Solarbio公司)，按照操作分别分离骆驼/羊驼PBMC；
2.使用细胞总RNA提取试剂盒(OMEGA公司)，按照操作分别提取骆驼/羊驼总RNA；
3.使用PrimeScript TM II反转录试剂盒(Takara公司)，按照操作分别合成骆驼/羊驼cDNA；
4.以上述获得的cDNA做模板，使用下述SEQ ID NO:5-SEQ ID NO:13所示的特异性引物进行巢式PCR扩增；第一轮PCR
SEQ ID NO:5(上游引物)：GTCCTGGCTGCTCTTCTACAAGG；
SEQ ID NO:6(下游引物)：GGTACGTGCTGTTGAACTGTTCC。
第二轮PCR
SEQ ID NO:7(上游引物)：
Figure PCTCN2022079236-appb-000002
SEQ ID NO:8(上游引物)：
Figure PCTCN2022079236-appb-000003
SEQ ID NO:9(上游引物)：
Figure PCTCN2022079236-appb-000004
SEQ ID NO:10(下游引物)：
Figure PCTCN2022079236-appb-000005
SEQ ID NO:11(下游引物)：
Figure PCTCN2022079236-appb-000006
SEQ ID NO:12(下游引物)：
Figure PCTCN2022079236-appb-000007
SEQ ID NO:13(下游引物)：
Figure PCTCN2022079236-appb-000008
5.扩增获得骆驼/羊驼VHH基因片段。
回收上述扩增获得的骆驼/羊驼VHH片段，通过Sfi I酶切，连接到pADL-23c(Biovector)噬菌粒载体上，电转化TG1大肠杆菌感受态细胞建成羊驼/骆驼VHH重链抗体免疫文库。其中，NKp46骆驼免疫文库库容为5.2E7，羊驼免疫文库库容为7.8E7。
实施例4 特异性结合NKp46的阳性克隆的筛选
为了获得可与人NKp46和食蟹猴NKp46交叉结合的阳性抗体，将上述文库扩增加入M13K07 helper phage组装成噬菌体后，分别投入1x10 12pfu骆驼和羊驼免疫文库，噬菌体与结合在磁珠上生物素化的人NKp46蛋白(8μg/mL)室温孵育1h，0.05％PBST洗去未结合噬菌体后，用100mM三乙基胺将与NKp46特异性结合的噬菌体洗脱下来，梯度稀释后侵染对数期生长的大肠杆菌SS320，在氨苄平板上37℃过夜培养；挑取单克隆进行IPTG诱导表达，上清用于ELISA检测。ELISA板上分别包被2μg/mL人NKp46或食蟹猴NKp46抗原4℃过夜，0.05％PBST清洗3次后用5％脱脂牛奶室温封闭1h，0.05％PBST清洗3次后每孔加入诱导的上清30μL，阴性对照孔加入培养基，室温孵育1h，最后用anti-Myc HRP检测(IPTG诱导表达出的VHH带有his和c-Myc标签)；将ELISA测试得到的结合人和食蟹猴NKp46的OD450值大于1.0，且与结合培养基阴性对照的ELISA OD450比值均大于3的克隆进行测序，获得本公开的30个重链抗体可变区的氨基酸序列，结果如表2所示：
表2 30个重链抗体可变区的氨基酸序列
克隆号 重链可变区(VHH)
NKp46-PC1 SEQ ID NO:14
NKp46-PC2 SEQ ID NO:15
NKp46-PC3 SEQ ID NO:16
NKp46-PC4 SEQ ID NO:17
NKp46-PC6 SEQ ID NO:18
NKp46-PC7 SEQ ID NO:19
NKp46-PC8 SEQ ID NO:20
NKp46-PC9 SEQ ID NO:21
NKp46-PC11 SEQ ID NO:22
NKp46-PC12 SEQ ID NO:23
NKp46-PC13 SEQ ID NO:24
NKp46-PC16 SEQ ID NO:25
NKp46-PA2 SEQ ID NO:26
NKp46-PA3 SEQ ID NO:27
NKp46-PA11 SEQ ID NO:28
NKp46-PA15 SEQ ID NO:29
NKp46-PA19 SEQ ID NO:30
NKp46-PA22 SEQ ID NO:31
NKp46-PA24 SEQ ID NO:32
NKp46-PA26 SEQ ID NO:33
NKp46-PA27 SEQ ID NO:34
NKp46-PA28 SEQ ID NO:35
NKp46-PA29 SEQ ID NO:36
NKp46-PA31 SEQ ID NO:37
NKp46-PA34 SEQ ID NO:38
NKp46-PA36 SEQ ID NO:39
NKp46-PA40 SEQ ID NO:40
NKp46-PA45 SEQ ID NO:41
NKp46-PA63 SEQ ID NO:42
NKp46-PA69 SEQ ID NO:43
在上述氨基酸序列的基础上，利用Kabat编号规则划分抗体可变区的CDR和FR，每个抗体的3个CDR序列组成如下表3所示。表3中括号内数字表示序列号，例如(44)即代表SEQ ID NO:44。
表3 30个重链抗体的CDR序列
Figure PCTCN2022079236-appb-000009
Figure PCTCN2022079236-appb-000010
实施例5 抗NKp46嵌合抗体的构建及其在真核细胞中的瞬时转染表达
将测序完成的本公开的单抗可变区与氨基酸序列如SEQ ID NO:134所示的人IgG1恒定区拼接后生成的目的基因片段克隆到pTT5表达载体(Nova lifetech)中，制备转染级别的表达质粒。
在无血清培养基中培养Expi293F TM细胞(Thermo Fisher Scientific)，将细胞接种在摇瓶(Corning公司)中，并在37℃，8％CO 2的环境中置于摇床上培养。调整细胞密度，将含有目的基因片段的pTT5重组表达载体和PEI转染试剂按照合适的比例混合，并添加进细胞培养摇瓶中，细胞培养6天后收集表达上清，高速离心去除细胞碎片，用Protein A柱进行亲和纯化。用PBS冲洗柱子，至A280读数降至基线。用pH3.0-pH3.5的酸性洗脱液洗脱目的蛋白，用1M Tris-HCl，pH8.0-9.0中和。洗脱样品适当浓缩后，换液到PBS分装备用。对最终纯化的嵌合抗体进行SDS-PAGE及HPLC纯度分析和A280浓度测定。
实施例6 抗NKp46嵌合抗体与表达NKp46细胞的结合
培养实施例2中所制备得到的人NKp46-CHOK1和食蟹猴NKp46-CHOK1稳定细胞株，用0.25％胰酶(含EDTA)消化细胞约5分钟，加入完全培养基终止反应。1500rpm离心5分钟弃上清，使用含有1％BSA的PBS重悬后计 数。将细胞密度调整到1E6/mL，以100μL/孔，接种到corning-3799号96孔培养板中培养，在37℃，8％CO 2的环境中培养过夜，之后将96孔培养板以1500rpm离心5分钟弃上清，4℃放置备用。
选取上述实施例中获得的24个抗NKp46嵌合抗体，用含有1％BSA的PBS分别配置抗NKp46嵌合抗体溶液，起始浓度为100nM，10倍稀释，获得7个梯度，分别为：10 -4nM、10 -3nM、10 -2nM、10 -1nM、10 0nM、10 1nM和10 2nM。
用配置好的抗体重悬细胞培养板，100μL/孔。将重悬好的细胞培养板在4℃冰箱孵育1小时。1500rpm离心5分钟，弃上清，160μL含1％BSA的PBS洗涤一遍，弃上清备用。用含1％BSA的PBS配置二抗(goat anti human IgG Fc PE)，1：200稀释。用配置好的二抗重悬细胞，100μL/孔，4℃冰箱孵育0.5小时。1500rpm离心5分钟，弃上清，160μL含1％BSA的PBS洗涤一遍，弃上清，100μL含1％BSA的PBS重悬细胞，300目纱布过滤细胞，然后在流式细胞仪上分析抗体与细胞结合的平均荧光强度。从流式细胞仪导出FCS文件，用Flowjo软件分析PE通道平均荧光强度(以下简称MFI)，将分析得出的平均荧光强度导入Graphpad分析抗体与细胞的半数结合浓度(以下简称EC 50)和最高平均荧光强度(Top MFI)，结果如表4及附图3-10所示。抗NKp46嵌合抗体均与过表达人及食蟹猴NKp46细胞有较好的结合。
表4A 抗NKp46嵌合抗体与表达NKp46细胞的结合
Figure PCTCN2022079236-appb-000011
表4B 抗NKp46嵌合抗体与表达NKp46细胞的结合
Figure PCTCN2022079236-appb-000012
Figure PCTCN2022079236-appb-000013
实施例7 抗NKp46嵌合抗体的体外NK细胞激活实验
取人新鲜外周血细胞(PBMC)，使用EasySep TM Human NK Cell Isolation Kit(StemCell)分离NK细胞并计数。使用α-MEM培养基培养细胞，添加15％热灭活FBS，0.2mM肌醇，0.1mMβ-巯基乙醇，0.02mM叶酸，200U/mL IL-2。将NK细胞密度调整到1E6/mL，在37℃5％CO 2培养箱中培养6-8天。用PBS稀释抗体，最高浓度为20nM，3倍稀释，获得7个梯度浓度，分别为20nM、6.67nM、2.23nM、0.74nM、0.247nM、0.083nM和0.027nM。将稀释好的抗体包被在corning-3599号96孔培养板中，100μL/孔，4℃包被过夜。使用150μL/孔PBS将96孔板洗涤两次。使用完全培养基将激活后的NK细胞密度调整到1E6/mL，然后铺到洗涤过的96孔板中，100μL/孔，37℃5％CO 2培养箱中培养4小时。将细胞从corning-3599号培养板转移到corning-3799号培养板，1500rpm离心5分钟，弃上清，备用。
按照说明书要求分别稀释CD3-APC
Figure PCTCN2022079236-appb-000014
CD56-BV421
Figure PCTCN2022079236-appb-000015
CD107a-PE
Figure PCTCN2022079236-appb-000016
将上述获得的备用细胞重悬，以50μL/孔的浓度添加上述稀释后的CD3-APC，CD56-BV421，CD107a-PE抗体，4℃孵育0.5小时。1500rpm离心5分钟，弃上清，160μL含1％BSA的PBS洗涤一遍，弃上清，100μL含1％BSA的PBS洗涤一遍，300目纱布过滤细胞。然后在流式细胞仪上分析CD107a阳性的细胞在NK中的比例。从流式细胞仪导出FCS文件，用Flowjo软件分析CD107a阳性细胞在NK(CD3 -CD56 +)细胞中的比例，并导入Graphpad求出NK活化的半数有效浓度(以下简称EC 50)和最高平均荧光强度(Top MFI)，结果如表5及附图11-12所示。抗NKp46嵌合抗体表现出不同的体外NK细胞激活能力。
表5A 抗NKp46嵌合抗体的体外NK细胞激活
Figure PCTCN2022079236-appb-000017
表5B 抗NKp46嵌合抗体的体外NK细胞激活
Figure PCTCN2022079236-appb-000018
实施例8 抗NKp46抗体的人源化设计
选择上述编号为NKp46-PC11，NKp46-PA27，NKp46-PA31和NKp46-PA45的抗NKp46嵌合抗体进行人源化设计。
实施例8.1 NKp46-PC11的人源化
(1)NKp46-PC11人源化框架选择
通过序列比对选择与克隆号NKp46-PC11同源性较高的胚系基因序列作为VHH移植框架模板：IGHV3-23*04(序列一致性64.3％)和IGHJ4_01(序列一致性73.3％)。将NKp46-PC11 CDR区嫁接至选定的人抗体可变区框架后，得到人源化抗体hPC11，人源化可变区hPC11 VHH-CDR graft序列如SEQ ID NO:135所示：
Figure PCTCN2022079236-appb-000019
Figure PCTCN2022079236-appb-000020
顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列，graft代表嵌合抗体的CDR植入人种系FR区后获得的人源化可变区VHH序列。
对hPC11进行回复突变，获得H1-PC11(序列如SEQ ID NO:136所示)、H2-PC11(序列如SEQ ID NO:137所示)、H3-PC11(序列如SEQ ID NO:138所示)、H4-PC11(序列如SEQ ID NO:139所示)和H5-PC11(序列如SEQ ID NO:140所示)，5条人源化抗体。
实施例8.2 NKp46-PA27的人源化
(1)NKp46-PA27人源化框架选择
通过序列比对选择与克隆号NKp46-PA27同源性较高的胚系基因序列作为VHH移植框架模板：IGHV3-30*15(序列一致性74.5％)和IGHJ4_01(序列一致性80％)。将NKp46-PA27CDR区嫁接至选定的人抗体可变区框架后，得到人源化抗体hPA27，人源化可变区hPA27VHH-CDR graft序列SEQ ID NO:141所示：
Figure PCTCN2022079236-appb-000021
Figure PCTCN2022079236-appb-000022
顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列，graft代表嵌合抗体的CDR植入人种系FR区后获得的人源化可变区VHH序列。
对hPA27进行回复突变，获得H1-PA27(序列如SEQ ID NO:141所示，原grafted序列)、H2-PA27(序列如SEQ ID NO:142所示)、H3-PA27(序列如SEQ ID NO:143所示)、H4-PA27(序列如SEQ ID NO:144所示)，4条人源化抗体。
实施例8.3 NKp46-PA31的人源化
(1)NKp46-PA31人源化框架选择
通过序列比对选择与克隆号NKp46-PA31同源性较高的胚系基因序列作为VHH移植框架模板：IGHV3-23*04(序列一致性74.5％)和IGHJ4_01(序列一致性73.3％)。将NKp46-PA31 CDR区嫁接至选定的人抗体可变区框架后，得到人源化抗体hPA31，人源化可变区hPA31 VHH-CDR graft序列如SEQ ID NO:145所示：
Figure PCTCN2022079236-appb-000023
Figure PCTCN2022079236-appb-000024
顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列，graft代表嵌合抗体的CDR植入人种系FR区后获得的人源化可变区VHH序列。
对hPA27进行回复突变，获得H1-PA31(序列如SEQ ID NO:145所示，原graft序列)、H2-PA31(序列如SEQ ID NO:146所示)、H3-PA31(序列如SEQ ID NO:147所示)、H4-PA31(序列如SEQ ID NO:148所示)，4条人源化抗体。
实施例8.4 NKp46-PA45的人源化
(1)NKp46-PA45人源化框架选择
通过序列比对选择与克隆号NKp46-PA45同源性较高的胚系基因序列作为VHH移植框架模板：IGHV3-23*04(序列一致性79.6％)和IGHJ4_01(序列一致性73.3％)。将NKp46-PA45CDR区嫁接至选定的人抗体可变区框架后，得到人源化抗体hPA45，人源化可变区hPA45VHH-CDR graft序列SEQ ID NO:149所示：
Figure PCTCN2022079236-appb-000025
Figure PCTCN2022079236-appb-000026
顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列，graft代表嵌合抗体的CDR植入人种系FR区后获得的人源化可变区VHH序列。
对hPA45进行回复突变，获得H1-PA45(序列如SEQ ID NO:149所示，原graft序列)、H2-PA45(序列如SEQ ID NO:150所示)、H3-PA45(序列如SEQ ID NO:151所示)、H4-PA45(序列如SEQ ID NO:152所示)，4条人源化抗体。
实施例9 特异性结合NKp46的人源化抗体的制备
通过本领域技术人员已知的标准方法，将人源化抗体的可变区与人IgG1恒定区拼接后生成的目的基因片段亚克隆到pTT5表达载体(Nova lifetech)中，采用ExpiFectamine TM 293转染试剂瞬时转染对数生长期的Expi293F TM细胞，收集培养上清，并进行亲和纯化，对最终纯化的人源化抗体进行SDS-PAGE及HPLC纯度分析和A280浓度测定。
实施例10 特异性结合NKp46人源化抗体的体外细胞结合验证
培养实施例2得到的人NKp46-CHOK1和食蟹猴NKp46-CHOK1稳定细胞株，用0.25％胰酶EDTA消化细胞约5分钟，完全培养基终止。1500rpm离心5分钟弃上清，用含有1％BSA的PBS重悬后计数。将细胞密度调整到1E6/mL，铺到corning-3799号96孔培养板中，100μL/孔。1500rpm离心5分钟弃上清，4℃放置备用。用含有1％BSA的PBS配置抗体，起始浓度为100nM，10倍稀释，获得7个梯度，分别为：10 -4nM、10 -3nM、10 -2nM、10 -1nM、10 0nM、10 1nM和10 2nM。配置好的抗体重悬细胞，100μL/孔。将重悬好的细胞培养板在4℃冰箱孵育1小时。1500rpm离心5分钟，弃上清，160μL含有1％BSA的PBS洗涤一遍，弃上清备用。
用含有1％BSA的PBS配置二抗(goat anti human IgG Fc PE)，1：200稀释。用配置好的二抗重悬细胞，100μL/孔，4℃冰箱孵育0.5小时。1500rpm离心5分钟，弃上清，160μL含有1％BSA的PBS洗涤一遍，弃上清，100μL含有1％BSA的PBS重悬细胞，300目纱布过滤细胞。然后在流式细胞仪上分析抗体与细胞结合的平均荧光强度。从流式细胞仪导出FCS文件，用Flowjo软件分析PE通道平均荧光强度(以下简称MFI)，将分析得出的平均荧光强度导入Graphpad分析抗体与细胞的半数结合浓度(以下简称EC 50)和最高平均荧光强度(Top MFI)，结果如表6及附图13-20所示。抗NKp46人源化抗体和对应嵌合抗体相比，在过表达人及食蟹猴NKp46细胞上的结合能力相当。
表6 抗NKp46人源化抗体与表达NKp46细胞的结合
Figure PCTCN2022079236-appb-000027
Figure PCTCN2022079236-appb-000028
实施例11 抗NKp46人源化抗体的体外NK细胞激活实验
取人新鲜外周血细胞(PBMC)，使用EasySep TM Human NK Cell Isolation Kit分离NK细胞并计数。使用α-MEM培养基培养细胞，添加15％热灭活FBS，0.2mM肌醇，0.1mMβ-巯基乙醇，0.02mM叶酸，200U/mL IL-2。将NK细胞密度调整到1E6/mL，在37℃5％CO 2培养箱中培养6-8天。用PBS稀释抗体，最高浓度为20nM，获得7个梯度浓度，分别为20nM、6.67nM、2.23nM、0.74nM、0.247nM、0.083nM和0.027nM。将稀释好的抗体包被在corning-3599号96孔培养板中，100μL/孔，4℃包被过夜。使用150μL/孔PBS将96孔板洗涤两次。使用完全培养基将激活后的NK细胞密度调整到1E6/mL，然后铺到洗涤过的96孔板中，100μL/孔，37℃5％CO 2培养箱中培养4小时。将细胞从corning-3599培养板转移到corning-3799培养板，1500rpm离心5分钟，弃上清，备用。
按照说明书稀释抗体(CD3-APC，CD56-BV421，CD107a-PE)，重悬细胞，50μL/孔，4℃孵育0.5小时。1500rpm离心5分钟，弃上清，160μL含有1％BSA的PBS洗涤一遍，弃上清，100μL的含有1％BSA的PBS悬细胞，300目纱布过滤细胞。然后在流式细胞仪上分析CD107a阳性的细胞在NK中的比例。从流式细胞仪导出FCS文件，用Flowjo软件分析CD107a阳性细胞在NK(CD3 -CD56 +)细胞中的比例，并导入Graphpad求出NK活化的半数有效浓度(以下简称EC 50)和最高平均荧光强度(Top MFI)，结果如表7及附图21-24所示。抗NKp46人源化抗体和对应嵌合抗体相比，体外NK细胞的激活能力保持或略微减弱。
表7 抗NKp46人源化抗体的体外NK细胞激活
Figure PCTCN2022079236-appb-000029
实施例12 特异性结合NKp46人源化抗体的Biacore亲和力实验
使用Biacore 8K仪器分析特异性结合NKp46人源化抗体与人NKp46(恺佧生物，NKP-HM146)和食蟹猴NKp46(恺佧生物，NKP-CM146)的亲和力和动力学性质。将Biacore仪器CM5芯片先用乙基-二甲氨基丙基-碳二亚胺EDC和羟基丁二酰亚胺NHS活化，然后固定抗人Fc的鼠单抗，再用乙醇胺封闭。
为测定与人NKp46的亲和力与动力学性质，NKp46人源化抗体用HBS-EP+缓冲液(10mM HEPES，pH 7.4，150mM NaCl，3mM EDTA，0.05％P20)稀释至0.8μg/mL，以10μL/min的流速捕获60s。人NKp46两倍逐级稀释至系列浓度(400nM-3.125nM)，以50μL/min的流速结合90s，解离500s。
为测定与食蟹猴NKp46的亲和力与动力学性质，NKp46人源化抗体用HBS-EP+缓冲液(10mM HEPES，pH 7.4，150mM NaCl，3mM EDTA，0.05％P20)稀释至0.8μg/mL，以10μL/min的流速捕获60s。食蟹猴NKp46两倍逐级稀释至系列浓度(400nM-3.125nM)，以50μL/min的流速结合90s，解离500s。
每一轮实验结束后，使用3M MgCl 2溶液以30μL/min的流速冲洗30s，将捕获的抗体连同抗原一起去除，完成芯片的再生。原始数据使用Biacore Insight Evaluation Software(版本3.0.12.15655)软件进行分析，以(1:1)Langmuir模型进行拟合，结果如表8所示。抗NKp46人源化抗体和对应嵌合抗体相比，与人NKp46抗原蛋白的亲和力基本保持。
表8A 抗NKp46人源化抗体与人NKp46抗原蛋白的亲和力测定结果
克隆号 ka(1/Ms) kd(1/s) KD(M) Rmax(RU)
NKp46-PC11 7.02E+04 3.40E-04 4.84E-09 72.4
H1-PC11 5.08E+04 3.07E-04 6.05E-09 93.9
H2-PC11 6.13E+04 1.26E-04 2.05E-09 55.1
H3-PC11 6.78E+04 7.75E-05 1.14E-09 50
NKp46-PA27 1.12E+05 3.62E-03 3.25E-08 94.7
H2-PA27 9.77E+04 2.16E-03 2.21E-08 119.3
H4-PA27 1.12E+05 2.66E-03 2.37E-08 117.1
NKp46-PA31 1.68E+05 1.00E-03 5.97E-09 77.3
H2-PA31 8.78E+04 9.59E-04 1.09E-08 45.4
H4-PA31 1.75E+05 1.03E-03 5.89E-09 46.6
NKp46-PA45 8.59E+04 1.87E-03 2.18E-08 48.2
H2-PA45 6.38E+04 1.54E-03 2.41E-08 59.7
H4-PA45 9.30E+04 2.04E-03 2.19E-08 68.2
表8B 抗NKp46人源化抗体与猴NKp46抗原蛋白的亲和力测定结果
克隆号 ka(1/Ms) kd(1/s) KD(M) Rmax(RU)
H1-PC11 5.13E+04 5.89E-03 1.15E-07 102.1
H2-PA27 2.52E+05 5.90E-03 2.34E-08 171.7
H2-PA31 7.75E+04 5.96E-04 7.69E-09 44.8
H2-PA45 7.67E+04 5.63E-04 7.34E-09 39.5
实施例13 抗NKp46人源化抗体的物理稳定性测试
利用NanoDSF(差示荧光扫描技术)检测不同抗体在pH7.4PBS缓冲液中的热稳定性。样品浓度在1mg/mL左右，利用Prometheus NT.Plex仪器进行检测。检测前，将各个样品10000g离心10分钟。样品板每个孔加入40μL样品(仪器上样量为10μL，每个样品均有一个复孔)。扫描温度从30℃开始到95℃结束，扫描速率0.5℃/min。实验结果如表9所示。
表9 抗NKp46人源化抗体的NanoDSF检测结果
克隆号 Tm Onset Tm1 Tm2 Tm3 Tagg
NKp46-PC11 62.6℃ 68.5℃ 76.9℃ / 76.5℃
H1-PC11 61.2℃ 69.0℃ 79.4℃ / 77.3℃
H2-PC11 61.1℃ 68.4℃ 77.2℃ / 76.4℃
H3-PC11 62.5℃ 67.6℃ 75.6℃ / 76.8℃
H4-PC11 62.0℃ 65.8℃ 74.8℃ / 76.0℃
H5-PC11 62.0℃ 67.3℃ 75.8℃ / 77.0℃
NKp46-PA27 62.4℃ 67.3℃ 73.6℃ 80.3℃ 70.1℃
H2-PA27 58.9℃ 62.5℃ / / 62.1℃
H3-PA27 57.6℃ 60.9℃ 79.9℃ / 60.1℃
H4-PA27 58.9℃ 61.4℃ 80.7℃ / 61.2℃
NKp46-PA31 62.7℃ 64.2℃ 80.0℃ / 64.9℃
H2-PA31 60.7℃ 67.9℃ 80.3℃ / 69.0℃
H3-PA31 / 64.9℃ 80.4℃ / 65.8℃
H4-PA31 73.9℃ 80.1℃ / / 66.3℃
NKp46-PA45 65.3℃ 67.4℃ 80.4℃ / 67.9℃
H2-PA45 62.1℃ 69.5℃ 76.8℃ 81.3℃ 75.6℃
H3-PA45 63.6℃ 66.3℃ 73.1℃ 81.0℃ 72.1℃
H4-PA45 63.1℃ 67.0℃ 73.9℃ 81.3℃ 72.9℃
通过SEC-HPLC监测样品纯度考察一定浓度条件下周期性稳定性。示例性的条件比如将样品浓度控制在10mg/mL，10mM醋酸盐，9％海藻糖(pH 5.5)缓冲液中和将浓度控制在1mg/mL PBS(PH 7.4)缓冲液中，比较在40℃保存0、7、14天的稳定性情况。利用Waters Xbridge Protein BEH SEC 3.5μm 7.8*300mm柱子检测，流动相为PBS缓冲液，使用H 3PO 4调整至pH 6.8；流速为0.5mL/min。实验结果如表10所示。
表10 抗NKp46人源化抗体的周期稳定性结果(SEC-HPLC)
Figure PCTCN2022079236-appb-000030
通过CE-SDS监测样品分子完整性，考察一定浓度条件下周期性稳定性。将样品浓度控制在10mg/mL，10mM醋酸盐，9％海藻糖(pH 5.5)缓冲液中，在还原和非还原条件下比较在40℃保存0、7、14天的分子完整性情况。取200μg样本(100μg还原，100μg非还原)加入样本缓冲液至97μL，还原样本加入5μL 2-巯基乙醇，非还原样本加入5μL250mM碘乙酰胺，样本均在70℃加热10分钟。利用PA 800Plus(Beckman Sciex)检测，电泳时间40分钟。实验结果如表11所示。
表11 抗NKp46人源化抗体的周期稳定性结果(CE-SDS)
Figure PCTCN2022079236-appb-000031
实施例14 抗NKp46人源化抗体的化学稳定性测试
通过cIEF监测样品分子的电荷异质性。抗体具有很多翻译后修饰，包括脱酰氨作用、异构化、末端的改变、糖基化、氧化、断裂、聚合等。这些修饰可能会引起抗体表面电荷的改变，导致电荷异质性。cIEF是基于抗体所带电荷，对其进行分离，进而对抗体电荷异质性进行分析。将样品浓度控制在10mg/mL，10mM醋酸盐，9％海藻糖(pH 5.5)缓冲液中，比较在40℃保存0、7、14天的电荷异质性情况。取20ug样本，加入到Separation Mix缓冲液中，利用Maurice.C(ProteinSimple)聚焦7分钟检测抗体样本的电荷分布。实验结果如表12所示。
表12 抗NKp46人源化抗体的周期稳定性结果(cIEF)
  H1-PC11 H2-PA27 H2-PA31 H2-PA45
酸性峰变化 +11.5％ +11.4％ +7.7％ +9.7％
主峰变化 -16.3％ -12.5％ -20.5％ -15.3％
碱性峰变化 +4.7％ +1％ +12.8％ +5.6％
通过LC-MS技术检测抗体样本的翻译后修饰。脱酰胺修饰是抗体中可能影响后期稳定性的一种常见的化学修饰，尤其是CDR区域的部分氨基酸高度脱酰胺修饰一般选择尽量避免或者突变降低。示例性的条件比如将待测抗体浓度控制在10mg/mL，10mM醋酸盐，9％海藻糖(pH 5.5)缓冲液中和将浓度控制在1mg/mL PBS(pH 7.4)缓冲液中，40℃恒温箱存放；分别于0、7、14天取样，用于酶解实验。取30μg的抗体样品，加入6μL 8M盐酸胍和3μL 200mM DTT，60℃水浴20min，再加入46μL 20mM Histidine缓冲液(pH6.0)和2μL PNGase F酶(Promega，V5111)，37℃水浴16h。在Q-Exactive Plus仪器上，进行LC-MS检测脱酰胺修饰情况。实验结果如表13所示。
表13 抗NKp46人源化抗体的周期稳定性结果(化学修饰)
Figure PCTCN2022079236-appb-000032
备注：N代表检测到修饰的天冬酰胺，M代表检测到修饰的甲硫氨酸，数字代表所处轻链或者重链N端开始计数所处的位置。百分含量代表LC-MS检测到的脱酰胺修饰占该位点所处全部肽段信号的比例。
从上述实验数据可以看出，H1-PC11，H2-PA27，H2-PA31和H2-PA45等4个人源化抗体表现出较高的热变形温度，且在40℃高温条件下储存14天后，聚体及碎片比例变化较小，发生化学修饰的变化比例较低，这4个人源化抗体的物理稳定性及化学稳定性均较好。
上文所述的本公开的实施方案仅为示例性的，任何本领域技术人员都可以认识到或者可以确定无数的特定化合物、材料和操作的等价物，而不需要进行超出常规的试验。所有这些等价物都是在本公开范围之内的，并且被权利要求所包含。

Claims (17)

  1. 一种抗NKp46的抗体或其抗原结合片段，所述抗体或其抗原结合片段能够特异性结合NKp46，其包含3个选自SEQ ID NO:44-133序列所示的CDR。
  2. 一种抗NKp46的抗体或其抗原结合片段，所述抗体或其抗原结合片段能够特异性结合NKp46，其包含重链可变区，所述重链可变区包含选自SEQ ID NO:44，47，50，53，56，59，62，65，68，71，74，77，80，83，86，89，92，95，98，101，104，107，110，113，116，119，122，125，128，131所示的HCDR1；以及，选自SEQ ID NO:45，48，51，54，57，60，63，66，69，72，75，78，81，84，87，90，93，96，99，102，105，108，111，114，117，120，123，126，129，132所示的HCDR2；以及，选自SEQ ID NO:46，49，52，55，58，61，64，67，70，73，76，79，82，85，88，91，94，97，100，103，106，109，112，115，118，121，124，127，130，133所示的HCDR3；
    优选的，所述重链可变区包含选自下组的HCDR1、HCDR2和HCDR3：SEQ ID NO:44、SEQ ID NO:45和SEQ ID NO:46；或SEQ ID NO:47、SEQ ID NO:48和SEQ ID NO:49；或SEQ ID NO:50、SEQ ID NO:51和SEQ ID NO:52；或SEQ ID NO:53、SEQ ID NO:54和SEQ ID NO:55；或SEQ ID NO:56、SEQ ID NO:57和SEQ ID NO:58；或SEQ ID NO:59、SEQ ID NO:60和SEQ ID NO:61；或SEQ ID NO:62、SEQ ID NO:63和SEQ ID NO:64；或SEQ ID NO:65、SEQ ID NO:66和SEQ ID NO:67；或SEQ ID NO:68、SEQ ID NO:69和SEQ ID NO:70；或SEQ ID NO:71、SEQ ID NO:72和SEQ ID NO:73；或SEQ ID NO:74、SEQ ID NO:75和SEQ ID NO:76；或SEQ ID NO:77、SEQ ID NO:78和SEQ ID NO:79；或SEQ ID NO:80、SEQ ID NO:81和SEQ ID NO:82；或SEQ ID NO:83、SEQ ID NO:84和SEQ ID NO:85；或SEQ ID NO:86、SEQ ID NO:87和SEQ ID NO:88；或SEQ ID NO:89、SEQ ID NO:90和SEQ ID NO:91；或SEQ ID NO:92、SEQ ID NO:93和SEQ ID NO:94；或SEQ ID NO:95、SEQ ID NO:96和SEQ ID NO:97；或SEQ ID NO:98、SEQ ID NO:99和SEQ ID NO:100；或SEQ ID NO:101、SEQ ID NO:102和SEQ ID NO:103；或SEQ ID NO:104、SEQ ID NO:105和SEQ ID NO:106；或SEQ ID NO:107、SEQ ID NO:108和SEQ ID NO:109；或SEQ ID NO:110、SEQ ID NO:111和SEQ ID NO:112；或SEQ ID NO:113、SEQ ID NO:114和SEQ ID NO:115；或SEQ ID NO:116、SEQ ID NO:117和SEQ ID NO:118；或SEQ ID NO:119、SEQ ID NO:120和SEQ ID NO:121；或SEQ ID NO:122、SEQ ID NO:123和SEQ ID NO:124；或SEQ ID NO:125、SEQ ID NO:126和SEQ ID NO:127；或SEQ ID NO:128、SEQ ID NO:129和SEQ ID NO:130；或SEQ ID NO:131、SEQ ID NO:132和SEQ ID NO:133。
  3. 一种抗NKp46的抗体或其抗原结合片段，所述抗体或其抗原结合片段能够特异性结合NKp46，其包含HCDR1、HCDR2和HCDR3，所述HCDR1、HCDR2和HCDR3来自于SEQ ID NO:14-43，136-152所示的重链可变区；优选的，所述重链可变区具有与SEQ ID NO:14-43，136-152所示序列至少80％至100％的序列同一性。
  4. 根据权利要求1-3任一项所述的抗NKp46的抗体或其抗原结合片段，其中所述抗体是重组抗体，优选的，为羊驼源抗体、嵌合抗体或人源化抗体；更优选的，所述抗体为纳米抗体；进一步优选的，所述抗体为人源化的骆驼科VHH。
  5. 如前述权利要求任一项所述的抗NKp46的抗体或其抗原结合片段，其还包括重链恒定区和/或轻链恒定区，优选的，所述重链恒定区包括Fc或变体Fc，Fc来源于鼠或人；和/或，
    所述的抗NKp46的抗体或其抗原结合片段为IgG1、IgG2、IgG3或IgG4形式。
  6. 一种偶联物，将前述权利要求任一项所述的抗NKp46的抗体或其抗原结合片段与捕获标记物或检测标记物偶联形成，所述的检测标记物包括放射性核素、发光物质、有色物质或酶。
  7. 一种双特异性抗体或多特异性抗体，其中的一个抗原结合结构域包含前述任一权利要求所述的抗NKp46的抗体或其抗原结合片段。
  8. 一种抗体药物缀合物，含有前述任一权利要求所述的抗NKp46的抗体或其抗原结合片段，所述的抗体药物缀合物由抗体-接头-毒素相互连接形成。
  9. 一种嵌合抗原受体，其胞外识别单元包含前述任一权利要求所述的抗NKp46的抗体或其抗原结合片段。
  10. 编码前述任一权利要求所述的抗NKp46的抗体或其抗原结合片段的核酸；或包含所述核酸的重组载体；或包含所述重组载体或基因组中整合有所述核酸的宿主细胞。
  11. 制备前述任一权利要求所述的抗NKp46的抗体或其抗原结合片段的方法，包括：在适合的条件下培 养权利要求10的宿主细胞，并从所述细胞中纯化获得表达产物。
  12. 前述任一权利要求所述的抗NKp46的抗体或其抗原结合片段在制备特异性靶向表达NKp46的细胞的药物中的用途；前述任一权利要求所述的抗NKp46的抗体或其抗原结合片段在治疗、预防或检测特异性表达NKp46疾病中的用途；优选的，所述细胞是NK细胞。
  13. 前述任一权利要求所述的抗NKp46的抗体或其抗原结合片段在制备癌症、感染性疾病或者炎性或自身免疫性疾病药物中的用途；前述任一权利要求所述的抗NKp46的抗体或其抗原结合片段在治疗癌症、感染性疾病或者炎性或自身免疫性疾病中的用途；优选的，所述癌症包括：白血病，侵袭性淋巴瘤，非霍奇金淋巴瘤，脑胶质瘤，宫颈癌，头颈癌，直肠癌，肾癌，肝癌，肺癌，胰腺癌，胃癌等。
  14. 前述任一权利要求所述的抗NKp46的抗体或其抗原结合片段在制备表达NKp46的诊断试剂中的用途。
  15. 一种含有抗NKp46的抗体的溶液制剂，其包含前述任一权利要求所述的抗NKp46的抗体或其抗原结合片段和缓冲液。
  16. 一种用于鉴定个体中NKp46表达细胞的存在的方法，所述方法包括从包含细胞的个体获得生物样品，使所述细胞与如权利要求1-5所述的抗NKp46的抗体或其抗原结合片段接触，以及评估所述抗体是否与所述细胞结合。
  17. 一种药物组合物，其包含有效量的前述权利要求1-5任一项所述的抗NKp46的抗体或其抗原结合片段、或有效量的包含权利要求6所述的偶联物，或包含有效量的权利要求7所述的双特异性抗体或多特异性抗体、或包含有效量的权利要求8所述的抗体药物缀合物、或包含有效量的权利要求9所述的嵌合抗原受体、或包含有效量的权利要求10所述的核酸、重组载体、或宿主细胞；
    优选的，还包含药学上可接受的载体；
    更优选的，还包含一种或多种额外的其他治疗剂，优选的，所述一种或多种额外的其他治疗剂包括：化学治疗剂、细胞毒性剂、放射性治疗剂、癌症疫苗、减瘤剂、靶向性抗癌剂、抗血管生成剂、生物反应修饰剂、细胞因子、激素、抗转移剂和免疫治疗剂。
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