WO2022124864A1 - Anti-tigit antibody and use thereof - Google Patents

Anti-tigit antibody and use thereof Download PDF

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WO2022124864A1
WO2022124864A1 PCT/KR2021/018795 KR2021018795W WO2022124864A1 WO 2022124864 A1 WO2022124864 A1 WO 2022124864A1 KR 2021018795 W KR2021018795 W KR 2021018795W WO 2022124864 A1 WO2022124864 A1 WO 2022124864A1
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seq
antibody
variable region
antigen
chain variable
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French (fr)
Korean (ko)
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권병세
황선희
김혜정
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주식회사 유틸렉스
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • 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
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention provides an anti-TIGIT (T cell immunoglobulin and ITIM domain) antibody or antigen-binding fragment thereof, a nucleic acid encoding the same, a recombinant expression vector containing the nucleic acid, a host cell transfected with the recombinant expression vector, the antibody or antigen thereof
  • a method for producing a binding fragment, a dual or multispecific antibody comprising the antibody or antigen-binding fragment thereof, an scFv of the antibody and one or more scFv of an antibody binding to an immune cell activating antigen, immune cell engaging (immune) cell engage) a bispecific or multispecific antibody, an antibody-drug conjugate (ADC) in which the antibody or antigen-binding fragment thereof is bound to a drug, and the scFv of the anti-TIGIT antibody as an antigen-binding site of the extracellular domain
  • a chimeric antigen receptor (CAR) an immune cell into which the chimeric antigen receptor is introduced, a
  • TIGIT T cell immune receptor with Ig and ITIM domains
  • TIGIT is an immunomodulatory receptor expressed primarily on activated T cells and NK cells.
  • TIGIT is VSIG9; VSTM3; and WUCAM.
  • the structure of TIGIT contains one extracellular immunoglobulin domain, a type 1 transmembrane region and two ITIM motifs.
  • TIGIT forms part of a co-stimulatory network consisting of positive (CD226) and negative (TIGIT) immunomodulatory receptors on T cells and ligands (CD155 and CD112) expressed on APCs ( Boles KS, et al., 2009 Eur J Immunol, 39:695-703) .
  • TIGIT initiates inhibitory signaling in immune cells when bound to its ligands CD155 and CD112.
  • the binding affinity of TIGIT for CD155 (Kd: ⁇ 1 nM) is much higher than that of CD112, and whether the TIGIT:CD112 interaction is functionally involved in mediating inhibitory signaling remains to be determined.
  • the costimulatory receptor CD226 (DNAM-1) binds the same ligand with low affinity (Kd: -100 nM), but transmits a positive signal ( Bottino C, et al., 2003 J Exp Med 198:557-67 ).
  • the “TIGIT-like” receptor CD96 (Tactile) also plays a similar inhibitory role in the same pathway ( Chan CJ, et al., 2014 Nat. Immunol 15:431-8 ).
  • TIGIT signaling In the case of cancer and viral infections, activation of TIGIT signaling promotes immune cell dysfunction, resulting in cancer growth or increased viral infection. Inhibition of TIGIT-mediated inhibitory signaling by therapeutic agents can restore functional activity of immune cells, including T cells, NK cells and dendritic cells (DCs), thereby enhancing immunity against cancer or chronic viral infection.
  • TIGIT-mediated inhibitory signaling can restore functional activity of immune cells, including T cells, NK cells and dendritic cells (DCs), thereby enhancing immunity against cancer or chronic viral infection.
  • the present inventors completed the present invention by confirming that, as a result of diligent efforts to develop a novel anti-TIGIT antibody, excellent properties and efficacy of the antibody, and it can be used for the treatment of a desired cancer or infectious disease, did.
  • An object of the present invention is to provide a novel antibody or antigen-binding fragment thereof against TIGIT (T cell immunoglobulin and ITIM domain).
  • Another object of the present invention is to provide a nucleic acid encoding the antibody or antigen-binding fragment thereof.
  • Another object of the present invention is to provide a recombinant expression vector containing the nucleic acid or a host cell transfected with the recombinant expression vector.
  • Another object of the present invention is to provide a method for producing an antibody or antigen-binding fragment thereof that specifically binds to TIGIT.
  • Another object of the present invention is to provide a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof.
  • Another object of the present invention is to provide an immune cell engaging bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof.
  • Another object of the present invention is to provide an antibody-drug conjugate (ADC) in which the antibody or antigen-binding fragment thereof is bound to a drug.
  • ADC antibody-drug conjugate
  • Another object of the present invention is a chimeric antigen receptor (CAR) comprising the scFv of the anti-TIGIT antibody as an antigen-binding site of an extracellular domain, an immune cell into which the chimeric antigen receptor is introduced, and combination therapy comprising the immune cell
  • CAR chimeric antigen receptor
  • Another object of the present invention is to provide a composition for combination therapy comprising the antibody or antigen-binding fragment thereof or the immune cell engaging bispecific or multispecific antibody.
  • Another object of the present invention is an antibody or antigen-binding fragment thereof, a bi- or multispecific antibody comprising the antibody or antigen-binding fragment thereof, an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof, the antibody or antigen thereof
  • a chimeric antigen receptor comprising a binding fragment
  • a composition for treating cancer comprising the chimeric antigen receptor, or a method for preventing or treating cancer.
  • Another object of the present invention is an antibody or antigen-binding fragment thereof, a bi- or multispecific antibody comprising the antibody or antigen-binding fragment thereof, an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof, the antibody or antigen thereof
  • a chimeric antigen receptor comprising a binding fragment
  • a composition for treating an infectious disease comprising the chimeric antigen receptor, or a method for preventing or treating an infectious disease.
  • Another object of the present invention is the antibody or antigen-binding fragment thereof; a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof; an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof; a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof; Or an immune cell comprising the chimeric antigen receptor; to provide a method for preventing or treating cancer, comprising administering to an individual in need thereof.
  • Another object of the present invention is the antibody or antigen-binding fragment thereof; a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof; an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof; a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof; Or an immune cell comprising the chimeric antigen receptor; to provide a method for preventing or treating an infectious disease, comprising administering to an individual in need thereof.
  • the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to TIGIT (T cell immunoglobulin and ITIM domain) comprising:
  • heavy chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID Nos: 1, 9, 17, 25 and 33;
  • a heavy chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 10, 18, 26, 34 and 45;
  • a heavy chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11, 19, 27 and 35, and
  • a light chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 12, 20, 28, 36, 41, 46 and 79;
  • a light chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37, 42 and 47;
  • a light chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID Nos: 6, 14, 22, 30, 38 and 43.
  • the present invention also provides a heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 83 to 89; And it provides an antibody or antigen-binding fragment thereof that specifically binds to TIGIT (T cell immunoglobulin and ITIM domain), including; and a light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 90 and 91 .
  • the present invention provides a nucleic acid encoding the antibody or antigen-binding fragment thereof.
  • the present invention also provides a recombinant expression vector comprising the nucleic acid.
  • the present invention also provides a host cell transfected with the recombinant expression vector.
  • the present invention also comprises the steps of culturing a host cell to produce an antibody; And it provides a method for producing an antibody or antigen-binding fragment thereof that specifically binds to TIGIT, comprising the step of isolating and purifying the produced antibody.
  • the present invention also provides a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof.
  • the present invention also provides an immune cell engage bispecific or multispecific antibody comprising at least one scFv of the antibody and an scFv of the antibody that binds to an immune cell activating antigen.
  • the present invention also provides an antibody-drug conjugate (ADC) wherein the antibody or antigen-binding fragment thereof is bound to a drug.
  • ADC antibody-drug conjugate
  • the present invention also provides a chimeric antigen receptor (CAR) comprising an extracellular domain comprising an antigen-binding site, a transmembrane domain and an intracellular signaling domain, wherein the antigen-binding site of the extracellular domain is an scFv of the antibody.
  • CAR chimeric antigen receptor
  • the present invention also provides an immune cell comprising the chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the present invention also provides a composition for combination therapy comprising the immune cells.
  • the present invention also provides a composition for combination therapy comprising the antibody or antigen-binding fragment thereof and at least one selected from the group consisting of:
  • immune cells comprising an antigen receptor (CAR) comprising an scFv fragment for an antibody against an immune checkpoint inhibitor as an extracellular domain; and
  • CAR antigen receptor
  • the present invention also provides a composition for combination therapy comprising the bispecific or multispecific antibody and at least one selected from the group consisting of:
  • immune cells comprising an antigen receptor (CAR) comprising an scFv fragment for an antibody against an immune checkpoint inhibitor as an extracellular domain; and
  • CAR antigen receptor
  • the present invention also relates to the antibody or antigen-binding fragment thereof, a bi- or multispecific antibody comprising the antibody or antigen-binding fragment thereof, an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof, the antibody or antigen thereof It provides a composition for treating cancer, comprising a chimeric antigen receptor containing a binding fragment or immune cells into which the chimeric antigen receptor is introduced.
  • the present invention also relates to the antibody or antigen-binding fragment thereof, a bi- or multispecific antibody comprising the antibody or antigen-binding fragment thereof, an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof, the antibody or antigen thereof It provides a composition for treating infectious diseases, comprising a chimeric antigen receptor containing a binding fragment or immune cells into which the chimeric antigen receptor is introduced.
  • the present invention also provides the antibody or antigen-binding fragment thereof; a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof; an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof; a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof; Or an immune cell comprising the chimeric antigen receptor; provides a method for preventing or treating cancer, comprising administering to an individual in need thereof.
  • the present invention also provides the antibody or antigen-binding fragment thereof; a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof; an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof; a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof; Or an immune cell comprising the chimeric antigen receptor; provides a method for preventing or treating an infectious disease, comprising administering to an individual in need thereof
  • the anti-TIGIT antibody or antigen-binding fragment thereof according to the present invention exhibits excellent binding ability to TIGIT, and can be usefully used for the prevention or treatment of a desired tumor, cancer, or infectious disease.
  • 1 is a diagram showing the results of analyzing the antigen-binding ability of a candidate clone through flow cytometry.
  • FIG. 2 is a diagram showing the results of confirming the TIGIT and CD155 binding inhibitory ability of the candidate antibody through the TIGIT/CD155 Blockade Bioassay.
  • FIG. 3 is a diagram showing the results of comparing the production of chimeric antibodies through cloning.
  • FIG. 4 is a diagram showing the results of analyzing the purity and binding affinity of the chimeric candidate antibody through HPLC and SPR.
  • FIG. 5 is a diagram showing the results of confirming the binding ability of the chimeric candidate antibody to CD4 T cells and CD8 T cells through flow cytometry.
  • FIG. 6 is a diagram showing the results of confirming the TIGIT and CD155 binding inhibitory ability of the chimeric candidate antibody through the TIGIT/CD155 Blocking Assay.
  • FIG. 7 is a diagram showing the results of analysis of whether the humanized candidate antibody was purified and quantitative binding ability through SDS-PAGE and ELISA.
  • FIG. 8 is a diagram showing the results of analyzing the purity and binding affinity of the humanized candidate antibody through HPLC and SPR.
  • FIG. 9 is a view showing the results of confirming the binding ability of the humanized candidate antibody to artificial effector T cells through flow cytometry.
  • FIG. 10 is a diagram showing the results of confirming the TIGIT and CD155 binding inhibitory ability of the humanized candidate antibody through the TIGIT/CD155 Blocking Assay.
  • FIG. 11 is a diagram showing the results of measuring the tumor size of a tumor mouse model according to administration of a humanized candidate antibody.
  • FIG. 13 is a diagram showing the results of analyzing the ratio of CD8 T cells from the blood of a tumor mouse model sacrificed after administration of a humanized candidate antibody.
  • FIG. 14 is a diagram showing the results of measuring liver toxicity using blood from a tumor mouse model sacrificed after administration of a humanized candidate antibody.
  • 15 is a diagram showing the results of analyzing the characteristics of a gene engineered candidate antibody with improved affinity through SDS-PAGE and SEC-HPLC.
  • 16 is a diagram showing the results of analyzing the characteristics of a gene engineered candidate antibody with improved affinity through SPR.
  • 17A is a diagram showing the results of confirming the TIGIT and CD155 binding inhibition ability of a gene engineered candidate antibody with improved affinity through the TIGIT/CD155 Blocking Assay.
  • Figure 17b is a graph prepared based on the TIGIT and CD155 binding inhibitory ability confirmation results.
  • FIG. 18 is a diagram showing six CDRs defined by analyzing the sequence of the humanized candidate antibody 62F (hu62F).
  • 19 is a diagram showing the sequences of 22 primers designed for CDR library construction.
  • 20 is a diagram showing a cleavage map of the prepared plasmid pYD5.
  • 21 is a schematic diagram and experimental conditions of PCR (1 st PCR) for the CDR region.
  • 22 is a diagram showing PCR results for a CDR region.
  • 23 is a diagram showing a schematic diagram and reaction conditions of 2nd PCR.
  • 24 is a diagram showing 2nd PCR results.
  • 25 is a diagram illustrating an experimental process of yeast transformation.
  • 26 is a diagram showing the results of staining the scFv expressed on the yeast cell surface through FACS.
  • FIG. 27 is a diagram showing the results of analyzing the antigen-binding capacity of individual clones for affinity enhancement through FACS.
  • FIG. 28 is a diagram showing a cleavage map of the pOptivec (AAA) vector and pcDNA3.3 vector.
  • 29 is a diagram showing the results of analyzing the characteristics of the antigen-binding ability enhancing candidate antibody through SDS-PAGE.
  • FIG. 30 is a diagram showing the results of analyzing the IgG antibody produced through size exclusion chromatography.
  • Figure 31 is a diagram showing the results of analyzing the antigen-antibody binding force of the humanized antibodies Hu62F and T02.10 using Biacore.
  • the present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to TIGIT (T cell immunoglobulin and ITIM domain) comprising: a group consisting of SEQ ID NOs: 1, 9, 17, 25 and 33
  • a heavy chain CDR1 comprising at least one amino acid sequence selected from, a heavy chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 10, 18, 26, 34 and 45, SEQ ID NOs: 3, 11, 19, 27 and a heavy chain CDR3 comprising at least one amino acid sequence selected from the group consisting of 35
  • a light chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 12, 20, 28, 36, 41, 46 and 79
  • the sequence a light chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37, 42 and 47 and at least one amino acid selected from the group consisting of SEQ ID NOs: 6, 14, 22, 30, 38
  • antibody refers to an anti-TIGIT antibody that specifically binds to TIGIT.
  • the scope of the present invention includes not only complete antibody forms that specifically bind TIGIT, but also antigen-binding fragments of the antibody molecule.
  • a complete antibody has a structure having two full-length light chains and two full-length heavy chains, each light chain connected to the heavy chain by a disulfide bond.
  • the term “heavy chain” refers to a full-length heavy chain comprising a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and three constant region domains CH1, CH2 and CH3. and fragments thereof.
  • the term “light chain” refers to a full-length light chain comprising a variable region domain VL and a constant region CL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and fragments thereof. all means
  • the whole antibody includes subtypes of IgA, IgD, IgE, IgM and IgG, in particular, IgG includes IgG1, IgG2, IgG3 and IgG4.
  • the heavy chain constant region has gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ) and epsilon ( ⁇ ) types and subclasses gamma 1 ( ⁇ ), gamma 2 ( ⁇ ), gamma 3 ( ⁇ ). ), gamma 4 ( ⁇ ), alpha 1 ( ⁇ 1) and alpha 2 ( ⁇ 2).
  • the constant region of the light chain has a kappa ( ⁇ ) and a lambda ( ⁇ ) type.
  • Antigen-binding fragment or antibody fragment of an antibody refers to a fragment having an antigen-binding function, and includes Fab, F(ab'), F(ab')2 and Fv.
  • Fab has a structure having variable regions of light and heavy chains, constant regions of light chain and first constant region (CH1) of heavy chain, and has one antigen-binding site.
  • Fab' differs from Fab in that it has a hinge-region comprising one or more cysteine residues at the C-terminus of the heavy chain CH1 domain.
  • F(ab')2 is formed when a cysteine residue in the hinge region of Fab' forms a disulfide bond.
  • Fv corresponds to the smallest antibody fragment having only a heavy chain variable region and a light chain variable region.
  • double-chain Fv two-chain Fv
  • the heavy chain variable region and the light chain variable region are connected by a non-covalent bond
  • single-chain Fv scFv
  • scFv single-chain Fv
  • antibody fragments can be prepared by using proteolytic enzymes (for example, by restriction digestion of the intact antibody with papain to obtain Fab, and by digestion with pepsin to obtain F(ab')2), or by genetic recombination technology. can be produced using proteolytic enzymes (for example, by restriction digestion of the intact antibody with papain to obtain Fab, and by digestion with pepsin to obtain F(ab')2), or by genetic recombination technology. can be produced using proteolytic enzymes (for example, by restriction digestion of the intact antibody with papain to obtain Fab, and by digestion with pepsin to obtain F(ab')2), or by genetic recombination technology. can be produced using proteolytic enzymes (for example, by restriction digestion of the intact antibody with papain to obtain Fab, and by digestion with pepsin to obtain F(ab')2), or by genetic recombination technology. can be produced using proteolytic enzymes (for example, by restriction digestion of the intact antibody with papain to obtain
  • Fv fragment is an antibody fragment that contains a complete antibody recognition and binding site. This region is a dimer in which one heavy chain variable domain and one light chain variable domain are bound.
  • a “Fab” fragment comprises the variable and constant domains of a light chain and the variable and first constant domains (CH1) of a heavy chain.
  • F(ab')2 antibody fragments generally comprise a pair of Fab' fragments covalently linked by cysteines in the hinge region present at the C-terminus of the Fab' fragment.
  • a “single chain Fv (scFv)” antibody fragment is a construct consisting of a single polypeptide chain comprising the VH and VL domains of an antibody. It may further comprise a polypeptide linker between the VH domain and the VL domain that enables the scFv to form the desired structure for antigen binding.
  • the antibody of the invention is a monoclonal antibody, multispecific antibody, human antibody, humanized antibody, chimeric antibody, scFv, Fab fragment, F(ab')2 fragment, disulfide-bonded Fvs (sdFv) and anti-idiotypic (anti-Id) antibodies or epitope-binding fragments of the antibodies, and the like.
  • the heavy chain constant region may be selected from any one isotype of gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ), or epsilon ( ⁇ ).
  • the constant region is gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3) or gamma 4 (IgG4).
  • the light chain constant region may be kappa or lambda type.
  • Said monoclonal antibody refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in trace amounts.
  • Monoclonal antibodies are highly specific, being directed against a single antigenic site.
  • conventional (polyclonal) antibodies which typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody is directed against a single determinant on the antigen.
  • Epitope refers to a protein determinant to which an antibody can specifically bind. Epitopes are usually composed of a group of chemically active surface molecules, such as amino acids or sugar side chains, and generally have specific three-dimensional structural characteristics as well as specific charge properties. Conformational and non-steric epitopes are distinguished in that binding to the former is lost but not to the latter in the presence of a denaturing solvent.
  • non-human antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are of a non-human species (donor antibody) that retain the desired specificity, affinity and ability for residues from the hypervariable region of the recipient, eg, mouse, rat, rabbit or non-human primate. It is a human immunoglobulin (recipient antibody) replaced with residues from the hypervariable region of
  • human antibody is a molecule derived from human immunoglobulin, and means that the entire amino acid sequence constituting the antibody, including the complementarity determining region and structural region, is composed of human immunoglobulin.
  • a portion of the heavy and/or light chain is identical to or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remaining chain(s) are from another species or from another antibody class or subclass.
  • Included are "chimeric" antibodies (immunoglobulins) that are identical to or homologous to the corresponding sequence in antibodies belonging to the subclass, as well as fragments of such antibodies that exhibit the desired biological activity.
  • variable region of an antibody refers to the light and heavy chain portions of an antibody molecule comprising the amino acid sequences of complementarity determining regions (CDRs; ie, CDR1, CDR2, and CDR3) and framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • VH refers to the variable domain of a heavy chain
  • VL refers to the variable domain of the light chain.
  • CDR Complementary determining region
  • the anti-TIGIT antibody or antigen-binding fragment thereof according to the present invention may comprise, for example:
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 2 and the heavy chain CDR3 of SEQ ID NO: 3, and the light chain CDR1 of SEQ ID NO: 4, the light chain CDR2 of SEQ ID NO: 5 and the light chain of SEQ ID NO: 6 a light chain variable region comprising CDR3;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 9, the heavy chain CDR2 of SEQ ID NO: 10 and the heavy chain CDR3 of SEQ ID NO: 11, and the light chain CDR1 of SEQ ID NO: 12, the light chain CDR2 of SEQ ID NO: 13 and the light chain of SEQ ID NO: 14 a light chain variable region comprising CDR3;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 17, the heavy chain CDR2 of SEQ ID NO: 18 and the heavy chain CDR3 of SEQ ID NO: 19, and the light chain CDR1 of SEQ ID NO: 20, the light chain CDR2 of SEQ ID NO: 21 and the light chain of SEQ ID NO: 22 a light chain variable region comprising CDR3;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 25, the heavy chain CDR2 of SEQ ID NO: 26 and the heavy chain CDR3 of SEQ ID NO: 27, and the light chain CDR1 of SEQ ID NO: 28, the light chain CDR2 of SEQ ID NO: 29 and the light chain of SEQ ID NO: 30 a light chain variable region comprising CDR3;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 33, the heavy chain CDR2 of SEQ ID NO: 34 and the heavy chain CDR3 of SEQ ID NO: 35, and the light chain CDR1 of SEQ ID NO: 36, the light chain CDR2 of SEQ ID NO: 37 and the light chain of SEQ ID NO: 38 a light chain variable region comprising CDR3;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 33, the heavy chain CDR2 of SEQ ID NO: 34 and the heavy chain CDR3 of SEQ ID NO: 35, and the light chain CDR1 of SEQ ID NO: 41, the light chain CDR2 of SEQ ID NO: 42 and the light chain of SEQ ID NO: 43 a light chain variable region comprising CDR3;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 33, the heavy chain CDR2 of SEQ ID NO: 45 and the heavy chain CDR3 of SEQ ID NO: 35, and the light chain CDR1 of SEQ ID NO: 46, the light chain CDR2 of SEQ ID NO: 47 and the light chain of SEQ ID NO: 38 a light chain variable region comprising CDR3; or
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 17, the heavy chain CDR2 of SEQ ID NO: 18 and the heavy chain CDR3 of SEQ ID NO: 19, and the light chain CDR1 of SEQ ID NO: 79, the light chain CDR2 of SEQ ID NO: 21 and the light chain of SEQ ID NO: 22 A light chain variable region comprising CDR3.
  • a “framework region (FR)” is a variable domain residue other than CDR residues. Each variable domain typically has four FRs: FR1, FR2, FR3 and FR4.
  • the binding affinity of the anti-TIGIT antibody to TIGIT is in the range of 10 -5 M to 10 -12 M.
  • the binding affinity of an anti-TIGIT antibody to TIGIT is 10 -6 M to 10 -12 M, 10 -7 M to 10 -12 M, 10 -8 M to 10 -12 M, 10 -9 M to 10 -12 M, 10 -5 M to 10 -11 M, 10 -6 M to 10 -11 M, 10 -7 M to 10 -11 M, 10 -8 M to 10 -11 M, 10 -9 M to 10 -11 M, 10 -10 M to 10 -11 M, 10 -5 M to 10 -10 M, 10 -6 M to 10 -10 M, 10 -7 M to 10 -10 M, 10 -8 M to 10 -10 M, 10 -9 M to 10 -10 M, 10 -5 M to 10 -9 M, 10 -6 M to 10 -9 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -9 M, 10 -5 M to 10 -8 M, 10 -6 M to 10
  • the antibody or antigen-binding fragment thereof binding to TIGIT is a heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 7, 15, 23, 31, 39, 48, 50, 52, 54, 56 and 82 may include
  • the TIGIT-binding antibody or antigen-binding fragment thereof is a group consisting of SEQ ID NOs: 8, 16, 24, 32, 40, 44, 49, 51, 53, 55, 57, 80, 81, 101, 102 and 103 It may include a light chain variable region comprising one or more amino acid sequences selected from.
  • it may include:
  • the present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to TIGIT (T cell immunoglobulin and ITIM domain) comprising:
  • a heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 83 to 89;
  • a light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 90 and 91.
  • it may include:
  • the antibody or antigen-binding fragment thereof of the present invention may include single chain Fvs (scFv), single chain antibody, Fab, F(ab'), disulfide-bonded Fvs (sdFv).
  • scFv single chain Fvs
  • Fab single chain antibody
  • F(ab') single chain antibody
  • sdFv disulfide-bonded Fvs
  • An scFv is an antibody fragment, which is a construct consisting of a single polypeptide chain comprising the VH and VL domains of an antibody.
  • the scFv may have a heavy chain variable region and a light chain variable region linked through a linker, preferably a polypeptide linker between the VH domain and the VL domain.
  • the linker may be a peptide linker and may have a length of about 10-25 aa.
  • hydrophilic amino acids such as glycine and/or serine may be included, but are not limited thereto.
  • the linker may include, for example, (GS) n , (GGS) n , (GSGGS) n or (G n S) m (n and m are each 1 to 10), but the linker is For example, it may be (G n S) m (n and m are each 1 to 10).
  • the linker may include GGGGS.
  • Phage display is a technique for displaying a variant polypeptide as a fusion protein with at least a portion of an envelope protein on the surface of a phage, eg, a filamentous phage particle.
  • the usefulness of phage display resides in the fact that, by targeting a large library of randomized protein variants, it is possible to quickly and efficiently sort sequences that bind to a target antigen with high affinity. Displaying peptide and protein libraries on phage has been used to screen millions of polypeptides for polypeptides with specific binding properties.
  • Phage display technology has provided a powerful tool for generating and screening novel proteins that bind specific ligands (eg antigens). Phage display technology can be used to generate large libraries of protein variants and rapidly sort sequences that bind target antigens with high affinity.
  • a nucleic acid encoding a variant polypeptide is fused with a nucleic acid sequence encoding a viral envelope protein, such as a gene III protein or a gene VIII protein.
  • a monovalent phage display system has been developed in which a nucleic acid sequence encoding a protein or polypeptide is fused with a nucleic acid sequence encoding a portion of a gene III protein. In a monovalent phage display system, the gene fusion is expressed at low levels and the wild-type gene III protein is also expressed to maintain particle infectivity.
  • Proving the expression of peptides on the surface of filamentous phage and functional antibody fragments in the periplasm of E. coli is important for developing antibody phage display libraries.
  • Libraries of antibodies or antigen-binding polypeptides have been prepared in a number of ways, for example, by altering a single gene by inserting a random DNA sequence or by cloning a related gene family.
  • the library can be screened for expression of an antibody or antigen-binding protein accompanied by a desired characteristic.
  • Phage display technology has several advantages over conventional hybridoma and recombinant methods for producing antibodies with desired characteristics. This technology allows the generation of large antibody libraries with various sequences in a short time without using animals. Preparation of hybridomas or humanized antibodies may require a production period of several months. In addition, since no immunization is required, the phage antibody library can generate antibodies against antigens that are toxic or of low antigenicity. Phage antibody libraries can also be used to generate and identify novel therapeutic antibodies.
  • lymphoid tissues can be used to prepare na ⁇ ve or non-immune antigen-binding libraries.
  • a technology capable of identifying and isolating high-affinity antibodies from phage display libraries is important for isolating novel therapeutic antibodies.
  • Isolation of high affinity antibodies from a library may depend on the size of the library, production efficiency in bacterial cells, and diversity of the library.
  • the size of the library is reduced by improper folding of the antibody or antigen-binding protein and inefficient production due to the presence of stop codons.
  • Expression in bacterial cells can be inhibited if the antibody or antigen binding domain does not fold properly.
  • Expression can be improved by alternately mutating residues on the surface of the variable/constant interface or at selected CDR residues.
  • the sequence of the framework region is one element to provide for proper folding when generating antibody phage libraries in bacterial cells.
  • CDR3 regions have been found to often participate in antigen binding. Since the CDR3 regions on the heavy chain vary considerably in size, sequence and structural conformation, they can be used to prepare a variety of libraries.
  • diversity can be generated by randomizing the CDR regions of the variable heavy and light chains using all 20 amino acids at each position.
  • the use of all 20 amino acids can result in highly diverse variant antibody sequences and increase the chances of identifying novel antibodies.
  • the antibody or antibody fragment of the present invention may include not only the sequence of the anti-TIGIT antibody of the present invention described herein, but also a biological equivalent thereof to the extent that it can specifically recognize TIGIT.
  • additional changes may be made to the amino acid sequence of an antibody to further improve its binding affinity and/or other biological properties.
  • modifications include, for example, deletions, insertions and/or substitutions of amino acid sequence residues of the antibody.
  • amino acid variations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like.
  • arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine can be said to be biologically functional equivalents.
  • the antibody or nucleic acid molecule encoding the same of the present invention is interpreted to include a sequence showing substantial identity to the sequence set forth in SEQ ID NO:.
  • the substantial identity is at least 90% when the sequence of the present invention and any other sequences are aligned as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. refers to a sequence exhibiting homology, most preferably at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, at least 99% homology.
  • Alignment methods for sequence comparison are known in the art.
  • the NCBI Basic Local Alignment Search Tool (BLAST) can be accessed from NBCI, etc.
  • BLAST can be accessed at www.ncbi.nlm.nih.gov/BLAST/.
  • a method for comparing sequence homology using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.
  • the antibody or antigen-binding fragment thereof of the present invention is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% compared to the specified sequence or all of the sequences described in the specification. , 99% or more homology.
  • homology can be determined by sequence comparison and/or alignment by methods known in the art. For example, a sequence comparison algorithm (ie, BLAST or BLAST 2.0), manual alignment, or visual inspection can be used to determine the percent sequence homology of a nucleic acid or protein of the invention.
  • the present invention relates to a nucleic acid encoding the antibody or antigen-binding fragment thereof.
  • An antibody or antigen-binding fragment thereof can be recombinantly produced by isolating a nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention.
  • Nucleic acid has a meaning comprehensively encompassing DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acids, include natural nucleotides as well as analogues in which sugar or base regions are modified. .
  • the sequences of the nucleic acids encoding the heavy and light chain variable regions of the present invention may be modified. Such modifications include additions, deletions or non-conservative substitutions or conservative substitutions of nucleotides.
  • the nucleic acid encoding the TIGIT-binding antibody or antigen-binding fragment thereof may include a nucleic acid encoding a heavy chain variable region or a light chain variable region selected from the group consisting of SEQ ID NOs: 58 to 78. have.
  • it may include:
  • nucleic acid encoding a heavy chain variable region of SEQ ID NO: 58 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 59;
  • nucleic acid encoding a heavy chain variable region of SEQ ID NO: 60 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 61;
  • nucleic acid encoding a heavy chain variable region of SEQ ID NO: 62 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 63;
  • nucleic acid encoding a heavy chain variable region of SEQ ID NO: 64 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 65;
  • nucleic acid encoding a heavy chain variable region of SEQ ID NO: 66 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 67;
  • nucleic acid encoding a heavy chain variable region of SEQ ID NO: 66 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 68;
  • nucleic acid encoding a heavy chain variable region of SEQ ID NO: 69 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 70;
  • nucleic acid encoding a heavy chain variable region of SEQ ID NO: 71 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 72;
  • nucleic acid encoding a heavy chain variable region of SEQ ID NO: 73 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 74;
  • nucleic acid encoding a heavy chain variable region of SEQ ID NO: 75 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 76;
  • the nucleic acid encoding the antibody or antigen-binding fragment thereof binding to TIGIT comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs: 64, 92 to 98; Or it may include a nucleic acid encoding a light chain variable region selected from the group consisting of SEQ ID NOs: 65, 99 and 100.
  • it may include:
  • nucleic acid encoding a heavy chain variable region of SEQ ID NO: 92 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 65;
  • nucleic acid encoding a heavy chain variable region of SEQ ID NO: 93 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 65;
  • nucleic acid encoding a heavy chain variable region of SEQ ID NO: 94 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 65;
  • nucleic acid encoding a heavy chain variable region of SEQ ID NO: 95 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 65;
  • nucleic acid encoding a heavy chain variable region of SEQ ID NO: 64 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 99;
  • nucleic acid encoding a heavy chain variable region of SEQ ID NO: 64 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 100;
  • nucleic acid encoding a heavy chain variable region of SEQ ID NO: 96 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 65;
  • nucleic acid encoding a heavy chain variable region of SEQ ID NO: 97 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 65;
  • nucleic acid encoding a heavy chain variable region of SEQ ID NO: 98 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 65;
  • DNA encoding the antibody is easily isolated or synthesized using conventional molecular biological techniques (eg, by using an oligonucleotide probe capable of specifically binding to DNA encoding the antibody and the heavy and light chains)
  • the nucleic acid is isolated and inserted into a replicable vector for further cloning (amplification of DNA) or further expression.
  • the present invention relates to a recombinant expression vector comprising the nucleic acid from another aspect.
  • the term "vector” is a means for expressing a target gene in a host cell, and a viral vector such as a plasmid vector, a cosmid vector, a bacteriophage vector, an adenoviral vector, a retroviral vector, and an adeno-associated viral vector. etc.
  • Components of a vector generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more antibiotic resistance marker genes, an enhancer element, a promoter, a transcription termination sequence. Nucleic acids encoding antibodies are operatively linked, such as promoters and transcription termination sequences.
  • operably linked refers to a functional association between a nucleic acid expression control sequence (eg, an array of promoter, signal sequence or transcriptional regulator binding sites) and another nucleic acid sequence, such that the control sequence is the other nucleic acid sequence to regulate transcription and/or translation of
  • a nucleic acid expression control sequence eg, an array of promoter, signal sequence or transcriptional regulator binding sites
  • a strong promoter capable of propagating transcription eg, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL ⁇ promoter, pR ⁇ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter
  • a ribosome binding site for initiation of translation e.g, lac promoter, lacUV5 promoter, lpp promoter, pL ⁇ promoter, pR ⁇ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter
  • a promoter derived from the genome of a mammalian cell eg, metallotionine promoter, ⁇ -actin promoter, human hegglobin promoter, and human muscle creatine promoter
  • mammalian Promoters derived from viruses eg, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, Moloney virus promoter Epstein Barr virus (EBV) promoter and Loose Sacoma virus (RSV) promoter
  • viruses eg, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, Moloney virus promoter Epstein Barr virus (
  • the vector may be fused with other sequences to facilitate purification of the antibody expressed therefrom.
  • the sequence to be fused includes, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA).
  • the vector contains an antibiotic resistance gene commonly used in the art as a selection marker, and for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline. There is a resistance gene.
  • the present invention relates to a host cell transfected with the recombinant expression vector.
  • the host cell used to produce the antibody of the present invention may be, but is not limited to, a prokaryotic, yeast or higher eukaryotic cell.
  • Escherichia coli Escherichia coli
  • Bacillus subtilus Bacillus subtilus
  • Bacillus thuringiensis such as Bacillus genus strains, Streptomyces , Pseudomonas ( Pseudomonas ) (for example, Pseudomonas putida ), Proteus mirabilis ) and Staphylococcus ) (eg, Staphylocus carnosus ), such as prokaryotic host cells can be used.
  • animal cells are of greatest interest, and examples of useful host cell lines include COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO/-DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2/0, NS-0 , but may be U20S or HT1080, but is not limited thereto.
  • the present invention comprises the steps of culturing the host cell to generate an antibody; And it relates to a method for producing an antibody or antigen-binding fragment thereof that specifically binds to TIGIT, comprising the step of isolating and purifying the produced antibody.
  • the host cells may be cultured in various media. Among commercially available media, it can be used as a culture medium without limitation. All other essential supplements known to those skilled in the art may be included in appropriate concentrations. Culture conditions, such as temperature, pH, etc., are already used with the host cells selected for expression and will be apparent to those skilled in the art.
  • impurities may be removed by, for example, centrifugation or ultrafiltration, and the resultant product may be purified using, for example, affinity chromatography. Additional other purification techniques may be used, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, and the like.
  • the present invention relates to a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof.
  • Bispecific antibody refers to an antibody having binding ability or antagonistic ability to one or more targets, and a form in which an antibody having binding ability or antagonistic ability to two different targets is bound, or an antibody having binding ability to one target and a different target It refers to an antibody to which a substance having antagonistic ability is bound.
  • a multispecific antibody refers to an antibody having binding specificities for at least three different antigens.
  • a multi-specific antibody is a tri-specific antibody or more, for example, a tri-specific antibody, a tetra-specific antibody, or a target that targets more. may include antibodies.
  • Antibodies belonging to bispecific or multispecific antibodies may be classified into scFv-based antibodies, Fab-based antibodies, and IgG-based antibodies.
  • a bispecific or multispecific antibody since two or more signals can be simultaneously inhibited or amplified, it can be more effective than when one signal is inhibited/amplified, and each signal must be treated with a respective signal inhibitor. Compared with the case, low dose administration is possible, and it is possible to suppress/amplify two or more signals in the same time and space.
  • bispecific or multispecific antibodies Methods for making bispecific or multispecific antibodies are well known. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two or more immunoglobulin heavy/light chain pairs under conditions in which the two or more heavy chains have different specificities.
  • a diabody can be made by preparing a hybrid scFv in a heterodimeric form by combining the VL and VH of different scFvs, and linking different scFvs with each other
  • tendem ScFv can be prepared
  • heterodimeric miniantibodies can be prepared by expressing CH1 and CL of Fab at the ends of each scFv.
  • Fab's directed against a specific antigen can be combined with each other using a disulfide bond or a mediator to form a heterodimeric Fab, and the heavy or light chain of a specific Fab can be It can be prepared to have two antigen valencies by expressing scFvs for different antigens at the ends, or to have four antigen valencies in homodimeric form by providing a hinge region between Fab and scFv.
  • a dual-target bibody with three antigen binding values, and different scFvs to the light and heavy chain ends of the Fab are fused to the antigen. It can be obtained by chemically conjugating three different Fabs, a triple-targeted bibody having three valencies.
  • bispecific or multispecific antibodies based on IgG hybrid hybridomas, also known as quadromas, were prepared by re-crossing mouse and rat hybridomas by Trion Pharma. Thus, a method for producing a bispecific antibody is known.
  • a bispecific antibody can be prepared in the so-called 'Holes and Knob' form, which is produced in a heterodimeric form by modifying some amino acids of the CH3 homodimeric domain of Fc for different heavy chains while sharing the light chain portion.
  • two different scFvs can be fused to the constant domains instead of the light and heavy chain variable domains of IgG to produce homodimeric (scFv)4-IgG.
  • ImClone Inc. is based on IMC-1C11, a chimeric monoclonal antibody against human VEGFR-2, and mouse platelet-derived growth factor receptor- ⁇ at the amino terminus of the light chain of this antibody. ), a bispecific antibody was produced and reported by fusion of only a single variable domain.
  • bispecific or multispecific antibodies which are bispecific, trivalent or tetravalent or higher.
  • WO2001/077342 WO2009/080251, WO2009/080252, WO2009/080253, WO2009/080254, WO2010/112193, WO2010/115589
  • antibodies described in WO2010/136172, WO2010/145792, WO2010/145793 and WO2011/117330 which are bivalent, trivalent or tetravalent or higher.
  • a bivalent, trivalent or more tetravalent antibody indicates that two or more binding domains, three or more binding domains or four or more binding domains, respectively, are present in the antibody molecule.
  • the bi- or multispecific antibody according to the present invention comprises the anti-TIGIT antibody or antigen-binding fragment, specifically in the form of a complete IgG antibody or fragment thereof, for example, single-chain Fv, V H domain and/or V L It may be included in the form of a domain, Fab or (Fab) 2 .
  • the antibody or antigen-binding fragment thereof that binds to a different target than the antibody targeting the TIGIT may include one or more selected from the group consisting of PDGFRa and NRP1.
  • the antibody or antigen-binding fragment thereof may specifically include a complete IgG antibody or fragment thereof, for example, in the form of a single chain Fv, V H domain and/or V L domain, Fab or (Fab) 2 .
  • bi- or multispecific antibody Through the bi- or multispecific antibody according to the present invention, it is possible to secure additional binding specificity induced or mediated by other targets in addition to TIGIT.
  • the bispecific antibody according to the present invention comprises TIGIT and FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT) , MARCO, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c
  • One selected from the group consisting of -Met, EGFR, HER2, KDR, PDGFRa and NRP1 may be simultaneously targeted.
  • the multispecific antibody according to the present invention is TIGIT and FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT) , MARCO, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c Two or more selected from the group consisting of -Met, EGFR, HER2, KDR, PDGFRa and NRP1 may be simultaneously targeted.
  • the present invention relates to an immune cell engage bispecific or multispecific antibody comprising an scFv of an antibody and one or more scFvs of an antibody that bind to an immune cell activating antigen.
  • a cytolytic synapse is temporarily induced between a cytotoxic T cell and a cancer target cell to release a toxic substance.
  • the immune cell activating antigen may be selected from, for example, an antibody or antigen-binding fragment thereof binding thereto may serve as an immune cell engager:
  • T cell activating antigens include CD3, TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2 or CD226;
  • NK cell activating antigens are NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (e.g., CD16a, CD16b), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E or CD160;
  • CD16 e.g., CD16a, CD16b
  • CRTAM CD27, PSGL1, CD96, CD100 (SEMA4D)
  • NKp80 CD244
  • SLAMF4 or 2B4 SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2
  • B cell activating antigens include OX40, CD40 or CD70;
  • Macrophage activating antigens include CD2 agonists, CD40, CD70, Toll-like Receptor (TCR) agonists, CD47, STING or OX40L; or
  • the dendritic cell activating antigen is a CD2 agonist, OX40, OX40L, 41BB agonist, TCR agonist, CD47 agonist or STING agonist.
  • the immune cell engager is specifically described in US Patent Application Publication No. 2017/0368169, and may be incorporated herein by reference.
  • the immune cell-engaging bispecific or multispecific antibody comprises a tandem scFv and is capable of binding to the following antigens and surface antigens on cancer cells.
  • the surface antigen on said cancer cell is TIGIT which the antibody according to the invention targets:
  • CD3 TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ or CD226;
  • CD16 e.g., CD16a, CD16b
  • CRTAM CD27, PSGL1, CD96, CD100 (SEMA4D)
  • NKp80 CD244
  • SLAMF4 or 2B4 SLAMF7, KIR2DS2, SLAMF7, KIR2DS2 , KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1,
  • CD2 agonist CD40, CD70, TCR (Toll-like Receptor) agonist, CD47, STING or OX40L; or
  • CD2 agonist OX40, OX40L, 41BB agonist, TCR agonist, CD47 agonist or STING agonist.
  • Said immune cell engaging bispecific or multispecific antibody can be, for example, VL(TIGIT)-VH(TIGIT)-VH(CD3 or CD16A)-VL(CD3 or CD16A), VH(TIGIT)-VL(TIGIT) )-VH(CD3 or CD16A)-VL(CD3 or CD16A), VH(CD3 or CD16A)-VL(CD3 or CD16A)-VH(TIGIT )-VL(TIGIT ) or VH(CD3 or CD16A)-VL(CD3)
  • CD16A)-VL(TIGIT )-VH(TIGIT ) may include a structure.
  • the scFv includes, for example, a heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 17 to 24 and a light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 25 to 27 and the heavy chain variable region and the light chain variable region may be connected by a linker.
  • the linker may be a peptide linker and may have a length of about 10-25 aa.
  • hydrophilic amino acids such as glycine and/or serine may be included.
  • the linker may include, for example, (GS)n, (GGS)n, (GSGGS)n or (GnS)m (n and m are each 1 to 10), but the linker is, for example, (G n S) m (n and m are each 1 to 10).
  • the linker may include GGGGS.
  • Examples of the immune cell engaging bispecific or multispecific antibody include blinatumomab (Amgen) that binds to CD3 and CD19; solitomab (Amgen) that binds to CD3 and EpCAM; MEDI 565 (MedImmune, Amgen) that binds to CD3 and CEA; and BAY2010112 (Bayer, Amgen) that binds to CD3 and PSMA.
  • Exemplary DARTs include MGD006 (Macrogenics) that binds CD3 and CD123; and MGD007 (Macrogenics), which binds to CD3 and gpA33.
  • Exemplary TandAbs include AFM11 (Affimed Therapeutics) that binds to CD3 and CD19; and AFM13 (Affimed Therapeutics) that binds to CD30 and CD16A.
  • ADCs Antibody-Drug Conjugates
  • the present invention relates to an antibody-drug conjugate (ADC) in which the antibody or antigen-binding fragment thereof is bound to a drug.
  • ADC antibody-drug conjugate
  • the anticancer drug In the antibody-drug conjugate, the anticancer drug must be stably bound to the antibody until the anticancer drug is delivered to the target cancer cell.
  • the drug delivered to the target must be released from the antibody to induce the death of the target cell.
  • the drug when the drug is stably bound to the antibody and released from the target cell, it must have sufficient cytotoxicity to induce the death of the target cell.
  • the antibody or antigen-binding fragment thereof may be bound to a drug through a linker.
  • the linker is a site linking the anti-TIGIT antibody and the drug, and allows the drug to be released from the antibody in a form that is cleavable under intracellular conditions, that is, in the intracellular environment, and reflects the long half-life of the antibody, allowing the antibody to circulate throughout the body It should be stable, and binding of the linker to the drug should not affect the stability and pharmacokinetics of the antibody.
  • the linker may include, for example, a cleavable linker or a non-cleavable linker.
  • a cleavable linker such as a peptide linker
  • it can be cleaved by an intracellular peptidase or protease enzyme such as a lysosomal or endosomal protease
  • a non-cleavable linker e.g. a thioether linker, where the antibody is cleaved by intracellular hydrolysis.
  • the drug may be released after non-selective degradation.
  • the cleavable linker may include a peptide linker.
  • the peptide linker has a length of at least two or more amino acids.
  • dipeptides of Val-Cit, Val-Ala or Val-Cit or Phe-Leu or Gly-Phe-Leu-Gly may be included. Examples of linkers are specifically described in International Patent Application Publication No. WO2004/010957, which may be incorporated herein by reference.
  • the antibody-drug conjugate is encapsulated into the cancer cell through the endo-lysosomal pathway after the antibody region of the ADC binds to the antigen of the target cancer cell to form the ADC-antigen complex.
  • the intracellular release of the cytotoxic drug is regulated by the internal environment of the endosome/lysosome.
  • the cleavable linker or non-cleavable linker is an acid labile linker, disulfide linker, peptide linker, beta-glucuronide linker, thioether group or maleimido It may contain a caproyl group.
  • the cleavable linker is pH sensitive and may be susceptible to hydrolysis at certain pH values. In general, it is indicated that pH sensitive linkers can be hydrolyzed under acidic conditions.
  • acid labile linkers capable of being hydrolyzed in the lysosome, such as hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketal or the like.
  • the linker may be cleaved under reducing conditions, for example, a disulfide linker may correspond to this.
  • SATA N-succinimidyl-S-acetylthioacetate
  • SPDP N-succinimidyl-3-(2-pyridyldithio)propionate
  • SPDB N-succinimidyl-3-(2-pyridyldithio)butyrate
  • SMPT N-succinimidyl-oxycarbonyl -alpha-methyl-alpha-(2-pyridyl-dithio)toluene
  • This disulfide linker can be cleaved by disulfide exchange with thiols of intracellular glutathione.
  • the drug and/or drug-linker may be randomly conjugated via a lysine of the antibody, or may be conjugated via a cysteine exposed when the disulfide bond chain of the antibody is reduced.
  • the linker-drug may be bound through a genetically engineered tag, for example, a cysteine present in a peptide or protein.
  • the genetically engineered tag for example, a peptide or protein, may include an amino acid motif that can be recognized by, for example, isoprenoid transferase.
  • the peptide or protein has a deletion at the carboxy terminus of the peptide or protein, or has an addition through a covalent bond of a spacer unit to the carboxy (C) terminus of the peptide or protein.
  • the peptide or protein may be directly covalently bonded to an amino acid motif or may be covalently bonded to a spacer unit to be linked to the amino acid motif.
  • the amino acid spacer unit is composed of 1 to 20 amino acids, and among them, a glycine unit is preferable.
  • the isoprenoid transferase may be, for example, farnesyl transferase (FTase, farnesyl protein transferase) or geranylgeranyl transferase (GGTase, geranylgeranyl transferase), and FTase and GGTase I are CAAX motif and GGTase II can recognize XXCC, XCXC or CXX motif, where C is cysteine, A is an aliphatic amino acid, and X is an amino acid that determines the substrate specificity of an isoprenoid transferase. have.
  • FTase farnesyl transferase
  • GGTase I are CAAX motif
  • GGTase II can recognize XXCC, XCXC or CXX motif, where C is cysteine, A is an aliphatic amino acid, and X is an amino acid that determines the substrate specificity of an isoprenoid transferas
  • the linker may include a beta-glucuronide linker that is recognized and hydrolyzed by beta-glucuronidase ( ⁇ ), which is present in many lysosomes or overexpressed in some tumor cells.
  • ⁇ beta-glucuronidase
  • Peptide Unlike the linker, it has the advantage of increasing the solubility of the antibody-drug complex when combined with a drug with high hydrophobicity due to its high hydrophilicity.
  • beta-glucuronide linker disclosed in International Patent Application Publication No. WO2015/182984, for example, a beta-glucuronide linker including a self-immolative group may be used.
  • a beta-glucuronide linker including a self-immolative group may be used.
  • the linker may be, for example, a non-cleavable linker, and the drug is released through only one step of antibody hydrolysis in the cell, for example, to produce an amino acid-linker-drug complex.
  • This type of linker may be a thioether group or a maleimidocaproyl group, and may maintain stability in blood.
  • the linker-drug may be bound through a cysteine exposed when the disulfide bond chain of the antibody is reduced, or the linker-drug may be bound by random linkage or by introducing an antibody terminal binding peptide having the sequence GGGGGGGCVIM.
  • the drug may be bound to an antibody as an agent exhibiting a pharmacological effect, and specifically may be a chemotherapeutic agent, a toxin, micro RNA (miRNA), siRNA, shRNA, or a radioactive isotope.
  • the chemotherapeutic agent may be, for example, a cytotoxic agent or an immunosuppressive agent. Specifically, it may include a microtubulin inhibitor, a mitosis inhibitor, a topoisomerase inhibitor, or a chemotherapeutic agent capable of functioning as a DNA intercalator.
  • it may include an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an anthelmintic agent, or a combination thereof.
  • Such drugs include, for example, maytansinoids, auristatins (including MMAE, MMAF), aminopterin, actinomycin, bleomycin, thalisomycin, camptothecin, N8-acetyl spermidine, 1-(2 Chloroethyl)-1,2-dimethyl sulfonyl hydrazide, esperamicin, etoposide, 6-mercaptopurine, dolastatin, trichothecene, calicheamicin, taxol, taxane, paclitaxel , docetaxel, methotrexate, vincristine, vinblastine, doxorubicin, melphalan, mitomycin A, mitomycin C, chlorambucil, duocarmycin, L-asparaginase (L-asparaginase), mercaptopurine (mercaptopurine), thioguanine, hydroxyurea, cytarabine,
  • the drug is an amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, capable of reacting to form a covalent bond with an electrophilic group on the linker and linker reagent; and one or more nucleophilic groups selected from the group consisting of an arylhydrazide group.
  • ADC was prepared in which the antibody or antigen-binding fragment thereof according to the present invention was linked to a drug, for example, auristatin (MMAE) through the MC-vc-PAB linker. It was confirmed that these ADCs exhibit the desired cytotoxicity.
  • a drug for example, auristatin (MMAE)
  • MMAE auristatin
  • the present invention is a chimeric antigen receptor (CAR) comprising an extracellular domain comprising an antigen-binding site, a transmembrane domain and an intracellular signaling domain, wherein the antigen-binding site of the extracellular domain is the scFv of the antibody It relates to a chimeric antigen receptor, characterized in that.
  • CAR chimeric antigen receptor
  • Chimeric antigen receptors are synthetic constructs designed to induce an immune response against a target antigen and cells expressing the antigen.
  • the CAR comprises an extracellular domain, a transmembrane domain and an intracellular signaling domain.
  • a gene encoding a receptor recognizing a cancer cell surface antigen specifically expressed on the surface of cancer cells into immune cells, cancer cells can be killed.
  • immune cells containing a receptor that binds to an antigen specifically expressed in cancer cells it is possible to induce an immune response by targeting only cancer cells.
  • the CAR includes the scFv of the anti-TIGIT antibody according to the present invention as an antigen recognition site of an extracellular domain.
  • a second-generation CAR combining a co-stimulatory domain (CD28 or CD137/4-1BB) and CD3 ⁇ was prepared to improve responsiveness to immune cells. Compared with the first-generation CAR, the number of CAR-containing immune cells remaining in the body significantly increased.
  • the second generation CAR used one auxiliary stimulation domain, whereas the third generation CAR used two or more auxiliary stimulation domains.
  • a co-stimulatory domain can be combined with 4-1BB, CD28 or OX40, etc. to achieve expansion and persistence of immune cells including CAR in vivo.
  • the second-generation CAR is specifically described in U.S. Patent Nos. 7,741,465, 7,446,190 or 9,212,229, and the third-generation CAR is specifically described in U.S. Patent No. 8,822,647, which is incorporated herein by reference.
  • cytokines such as IL-12 or IL-15
  • additional genes encoding cytokines such as IL-12 or IL-15 are included to allow the expression of additional CAR-based immune proteins of cytokines
  • the fifth-generation CAR includes interleukins to enhance immune cells. It further comprises a receptor chain such as IL-2R ⁇ .
  • the 4th generation CAR is specifically described in US Patent No. 10,316,102, and the 5th generation CAR is specifically described in US Patent No. 10,336,810, which is incorporated herein by reference.
  • the antigen binding site of the extracellular domain is an scFv of an antibody.
  • the VH and VL domains may be linked via a linker.
  • the heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 17 to 24 may be linked to a light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 25 to 27 through a linker.
  • the linker may be a peptide linker and may have a length of about 10-25 aa.
  • hydrophilic amino acids such as glycine and/or serine may be included.
  • the linker may include, for example, (GS) n , (GGS) n , (GSGGS) n or (G n S) m (n and m are each 1 to 10), but the linker is, for example (G n S) m (n and m are each 1 to 10).
  • the linker may include GGGGS.
  • the trans membrane domain may be derived from a natural or synthetic source. When the source is natural, the domain may be derived from any membrane bound protein or trans membrane protein.
  • the transmembrane domain is the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS alpha, beta or zeta chains.
  • hydrophobic residues such as leucine and valine may be included, or peptides including phenylalanine, tryptophan, and valine may be included at each end.
  • a short oligo- or polypeptide linker of 2 to 10 amino acids in length can form a bond between the trans membrane domain and the cytoplasmic signaling domain of the CAR.
  • a glycine-serine peptide may be used as a linker.
  • the signal transduction domain can induce activation of the normal effector function of the immune cell in which the CAR is located. For example, it can induce cytolytic activation or helper activation through the secretion of cytokines.
  • the signaling domain may comprise a truncated fragment of an intracellular signaling domain sufficient to transduce an effector function signal.
  • the cytoplasm of the T cell receptor (TCR) and the co-receptor, which act in concert to initiate signal transduction after antigen receptor engagement with the signal transduction domain, may be included.
  • Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex in a stimulatory or inhibitory manner.
  • Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.
  • ITAMs containing primary cytoplasmic signaling sequences may include TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
  • the cytoplasmic domain of the CAR may include a CD3 zeta chain portion and a costimulatory signaling region.
  • Costimulatory signaling region refers to the portion of the CAR comprising the intracellular domain of a costimulatory molecule.
  • CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, Lymphocyte Function-Related Antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7- H3, and a ligand that specifically binds to CD83, and the like may be included.
  • Cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR may be linked via a peptide linker comprising 2 to 10 amino acids, eg, glycine-serine.
  • the present invention relates to an immune cell into which the chimeric antigen receptor (CAR) is introduced.
  • CAR chimeric antigen receptor
  • the immune cells are capable of inducing a desired cancer therapeutic effect by inducing immunity, for example, T cells, NK cells, cytokine-induced killer cells (CIK), activated cytotoxic T lymphocytes (Cytotoxic).
  • T Lymphocyte, CTL cytokine-induced killer cells
  • macrophages tumor tissue infiltrating T cells
  • TIL tumor tissue infiltrating Lymphocytes
  • the immune cells may be additionally introduced with a chimeric antigen receptor (CAR) in which an scFv for an antibody against an immune checkpoint inhibitor is included as an antigen-binding site of an extracellular domain.
  • CAR chimeric antigen receptor
  • the antibody against the checkpoint inhibitor is, for example, the antibody is FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), MARCO , CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met , EGFR, HER2, KDR, PDGFRa, and may be an antibody that targets one or more selected from the group consisting of NRP1.
  • the present invention provides an antibody or antigen-binding fragment thereof; a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof; an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof; a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof; Or immune cells comprising the chimeric antigen receptor; relates to a composition for preventing or treating cancer comprising.
  • the present invention includes, for example, (a) an antibody or antigen-binding fragment thereof against TIGIT according to the present invention, a bi- or multispecific antibody comprising the antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof a pharmaceutically effective amount of an antibody-drug conjugate, a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof, or an immune cell comprising the chimeric antigen receptor; And (b) may be a pharmaceutical composition for the prevention or treatment of cancer comprising a pharmaceutically acceptable carrier.
  • the present invention also provides an antibody or antigen-binding fragment thereof against TIGIT according to the present invention, a bi- or multispecific antibody comprising the antibody or antigen-binding fragment thereof, and an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof , It may be a method of preventing or treating cancer comprising administering to an individual in need thereof, a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof, or an immune cell comprising the chimeric antigen receptor.
  • the subject is a subject expected to develop cancer; affected individuals; Or it may be an individual who has been cured, but is not limited thereto.
  • the present invention in another aspect the antibody or antigen-binding fragment thereof; a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof; an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof; a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof; Or immune cells comprising the chimeric antigen receptor; relates to a composition for preventing or treating infectious diseases, including.
  • the present invention includes, for example, (a) an antibody or antigen-binding fragment thereof against TIGIT according to the present invention, a bi- or multispecific antibody comprising the antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof a pharmaceutically effective amount of an antibody-drug conjugate, a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof, or an immune cell comprising the chimeric antigen receptor; And (b) it may be a pharmaceutical composition for preventing or treating an infectious disease comprising a pharmaceutically acceptable carrier.
  • the present invention also provides an antibody or antigen-binding fragment thereof against TIGIT according to the present invention, a bi- or multispecific antibody comprising the antibody or antigen-binding fragment thereof, and an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof , It may be a method for preventing or treating an infectious disease comprising administering to an individual in need thereof, a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof, or an immune cell comprising the chimeric antigen receptor.
  • the subject is an individual expected to develop an infectious disease; affected individuals; Or it may be an individual who has been cured, but is not limited thereto.
  • Prevention means any action that suppresses or delays the progression of clinical symptoms of a disease by administration of the composition according to the present invention, and “treatment” refers to suppression of the development of clinical symptoms for a disease, alleviation of clinical symptoms for a disease, or means removal.
  • the infectious disease may mean a viral infection.
  • viruses include, for example, adeno-associated virus, Aichi virus, Australian bat lyssavirus, BK polyomavirus, Banna virus, Barmah forest virus, Bunyamwera Virus, Bunyavirus La Crosse, Bunyavirus snowshoe hare, Cercopithecine herpesvirus, Chandipura virus, Chikungunya virus, Cosavirus A (Cosavirus A), cowpox virus, Coxsackievirus, Crimean-Congo hemorrhagic fever virus, Dengue virus, Dhori virus, Dugbe virus, Duvenhage Virus, Eastern encephalitis virus, Ebola virus, echovirus, encephalomyocarditis virus, Epstein-Barr virus, European bat lyssavirus, GB virus Hepatitis C/G virus, Hantan virus, Hendra virus, Hepatitis A virus, Hepatitis B virus, hepatitis C virus,
  • the cancer is, for example, Hodgkin's lymphoma, non-Hodgkin's lymphoma (eg, B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, marginal area B-cell lymphoma, Burkitt's lymphoma) , lymphoid cell lymphoma, hairy cell leukemia), acute myeloid leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, multiple myeloma or acute lymphocytic leukemia.
  • non-Hodgkin's lymphoma eg, B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, marginal area B-cell lymphoma, Burkitt's lymphoma
  • Said cancer is, for example, ovarian cancer, rectal cancer, gastric cancer, testicular cancer, anal region cancer, uterine cancer, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, small intestine cancer, esophageal cancer, melanoma, Kaposi's sarcoma, Endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, brain stem glioma, pituitary adenocarcinoma, epidermal cancer, carcinoma of the cervical squamous cell carcinoma, fallopian tube Carcinoma, endometrial carcinoma, vaginal carcinoma, soft tissue sarcoma, urethral cancer, vulvar carcinoma, penile cancer, bladder cancer, kidney or ureter cancer, renal pelvic carcinoma, spinal tumor, neoplasm of central nervous system (CNS), primary CNS
  • the cancer may be, for example, glioblastoma, lung cancer, bladder cancer, oral cancer, head and neck squamous cell cancer, gallbladder cancer or cervical cancer.
  • the antibody or antigen-binding fragment thereof is a barrier formed by a tight junction in the capillary endothelial cell membrane of the brain that exists between the brain and spine and the surrounding circulatory system, the blood-brain barrier (blood-brain barrier). barrier) needs to be passed. It can be used in conjunction with a transporter to cross the BBB.
  • a method of disrupting the osmotic pressure of the BBB using a method such as bradkinin or HIFU (Hign density fucues ultrasound). It may also include the use of delivery systems such as receptor-mediated transcytosis of glucose and amino acid transporters, insulin or transferrin, or blocking the active efflux transporter of glycoproteins in cells.
  • a method such as bradkinin or HIFU (Hign density fucues ultrasound). It may also include the use of delivery systems such as receptor-mediated transcytosis of glucose and amino acid transporters, insulin or transferrin, or blocking the active efflux transporter of glycoproteins in cells.
  • compositions of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate. , microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • the composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components.
  • the pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration and rectal administration etc. can be administered.
  • parenteral administration intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration and rectal administration etc. can be administered.
  • oral compositions should be formulated to coat the active agent or to protect it from degradation in the stomach.
  • the pharmaceutical composition may be administered by any device capable of transporting the active agent to a target cell.
  • a suitable dosage of the composition according to the present invention varies depending on factors such as formulation method, administration mode, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity of the patient, usually Thus, a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention.
  • the daily dosage of the pharmaceutical composition of the present invention is 0.0001-100 mg/kg.
  • the term “pharmaceutically effective amount” refers to an amount sufficient to prevent or treat cancer or infectious diseases.
  • the pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. or it may be prepared by incorporation into a multi-dose container.
  • the formulation may be in the form of a solution, suspension, or emulsion in oil or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.
  • the present invention relates to a composition for combination therapy comprising immune cells.
  • the composition may further include a chemotherapeutic agent, wherein the chemotherapeutic agent is maytansinoid, auristatin (including MMAE, MMAF), aminopterin, actinomycin, bleomycin, thali Somycin, camptothecin, N8-acetyl spermidine, 1-(2 chloroethyl)-1,2-dimethyl sulfonyl hydrazide, esperamicin, etoposide, 6-mercaptopurine, dolastatin, tricho Tecene, calicheamicin, taxol, taxane, paclitaxel, docetaxel, methotrexate, vincristine, vinblastine, doxorubicin, melphalan, mitomycin A, mitomycin C, chlorambucil, duo Carmycin, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytar
  • the composition comprises FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137(4-1BB), VISTA, CD258(LIGHT), MARCO, CD134(OX40) ), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2 , KDR, PDGFRa, and may be an antibody or antigen-binding fragment thereof targeting one or more selected from the group consisting of NRP1.
  • the present invention relates to a composition for combination therapy comprising an antibody or antigen-binding fragment thereof and at least one selected from the group consisting of the following.
  • immune cells comprising an antigen receptor (CAR) comprising an scFv for an antibody against an immune checkpoint inhibitor as an extracellular domain; and
  • CAR antigen receptor
  • the present invention also relates to a composition for combination therapy comprising the bispecific or multispecific antibody and at least one selected from the group consisting of:
  • immune cells comprising an antigen receptor (CAR) comprising an scFv fragment for an antibody against an immune checkpoint inhibitor as an extracellular domain; and
  • CAR antigen receptor
  • the immune cells are capable of inducing a desired cancer therapeutic effect by inducing immune treatment, for example, immunity, for example, T cells, NK cells, cytokine-induced killer cells (CIK), activated cells.
  • immune treatment for example, immunity, for example, T cells, NK cells, cytokine-induced killer cells (CIK), activated cells.
  • the antibody against the checkpoint inhibitor is an antibody that targets an immune checkpoint inhibitor other than TIGIT, for example, the antibody against the checkpoint inhibitor is FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), MARCO, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, It may be an antibody or antigen-binding fragment thereof that binds to CD226 (DNAM1), CD96, CD200, CD200R, transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa and NRP1, but is not limited thereto.
  • CD226 DNAM1
  • CD96 CD200
  • CD200R transferrin receptor
  • c-Met EGFR
  • HER2, KDR PDGFRa and NRP1
  • the immune checkpoint inhibitor refers to an agent capable of inducing T cell activation by blocking the T cell inhibitory signal at a site where antigen presenting cells (APCs) and immune cells, for example, T cells meet.
  • the immune checkpoint inhibitor is, for example, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), CD258 (LIGHT), MARCO, CD134 (OX40), CD28, It may be a drug targeting CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200 or CD200R, but is not limited thereto.
  • Each of the first component and the second component to be administered in combination may be administered simultaneously.
  • each of the first component and the second component to be administered in combination may be administered separately at a predetermined time interval.
  • the second component may be separately administered before or after administration of the first component among the subject of the combined administration.
  • Companion diagnostics is a type of molecular diagnostic technique for predicting the patient's responsiveness to a specific drug treatment. Considering the sensitivity, it is possible to present a standard that allows the treatment of a drug suitable for the patient.
  • the sample refers to a biological subject obtained from a tissue or body fluid of a patient.
  • Sources of tissue samples include solid tissue, such as those derived from frozen and/or preserved organs, tissue samples, biopsies, or aspirates; blood or any blood component (eg, serum, plasma); bone marrow or any component of bone marrow; It can be body fluids such as urine, cerebrospinal fluid, whole blood, plasma and serum.
  • a sample may comprise a non-cellular fraction (eg, urine, plasma, serum or other non-cellular body fluid).
  • a sample may include a bodily fluid, such as blood (eg, whole blood).
  • the sample may be a whole blood sample, a whole bone marrow sample, a whole peripheral blood sample, or a whole tumor sample obtained from a patient.
  • the "pre" sample is a sample that is substantially free of components (eg, cells) that have been removed or isolated from the sample.
  • the sample eg, a blood sample
  • a “pre” sample such as a whole tissue sample or a whole tumor sample, substantially retains the microenvironment from the tissue of origin, eg, may substantially retain the structure of the tumor or immune microenvironment.
  • a sample, such as a tumor sample can be processed (eg, ground, minced, blended, milled, etc.) into smaller pieces and diluted (eg, with a physiologically compatible buffer or medium).
  • Confirmation of the expression of the gene is a process of confirming the presence and level of expression of the gene, and may be performed by polymerase chain reaction (PCR), and in some cases transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerization Competitive RT-PCR, Real-time RT-PCR, RNase protection assay (RPA), Northern blotting, can be performed using a DNA chip.
  • PCR polymerase chain reaction
  • RT-PCR transcription polymerase reaction
  • RPA RNase protection assay
  • Northern blotting can be performed using a DNA chip.
  • PCR Polymerase chain reaction
  • Multiplex PCR real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), inverse polymerase chain reaction chain reaction: IPCR), vectorette PCR, and TAIL-PCR (thermal asymmetric interlaced PCR) may be performed.
  • DD-PCR differential display PCR
  • RACE rapid amplification of cDNA ends
  • IPCR inverse polymerase chain reaction chain reaction
  • vectorette PCR vectorette PCR
  • TAIL-PCR thermo asymmetric interlaced PCR
  • Analysis methods for this include Western blot, ELISA (enzyme linked immunosorbent assay, ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immune diffusion method, and rocket Immunoelectrophoresis, tissue immunostaining, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), a method using a protein chip, etc. may be included. .
  • mice were immunized with recombinant human TIGIT-His protein, and hybridoma clones were generated by fusion with the spleen and SP2/0 myeloid cells.
  • candidate antibodies clones having binding affinity to recombinant human TIGIT-His protein were first selected by ELISA using a hybridoma culture medium. Clones having binding affinity from T cells were secondarily selected by flow cytometry. The flow cytometry results are shown in FIG. 1 .
  • the efficacy of the candidate antibody to inhibit the binding of TIGIT to its counterpart CD155 was investigated. Specifically, the efficacy test was performed using the TIGIT/CD155 Blockade Bioassay (Promega) kit.
  • TIGIT/CD155 Blockade Bioassay Promega
  • candidate antibodies When Jurkat Effector cells expressing human TIGIT and CD226 and having a luciferase reporter and CHO-K1 aAPC expressing CD155 were co-cultured, candidate antibodies During treatment, the antibody binds to TIGIT to block the binding to CD155, and a method capable of measuring the activated luciferase signal by binding of CD155 to CD226 was used.
  • Table 1 shows the results of sequence analysis of the VH, VL, and CDR regions of the selected clones.
  • the VH and VL sequences of each selected candidate antibody were cloned in the form of chimeric antibodies using human IgG1 heavy chain and human kappa light chain as constant regions.
  • the chimeric candidate antibody was produced using the Expi293 transient expression system, and the quantitative binding ability of each purified chimeric antibody was analyzed by ELISA.
  • ELISA recombinant human TIGIT-His protein was O/N coated on a 96-well ELISA plate at 4° C. and blocked with 5% non-fat skim milk for 1 hr at RT.
  • Antibodies were subjected to 1/2 serial dilution from each concentration of 500ng or 200ng, treated for 2hr at RT, and detected with anti-hIgG-HRP, and the Oncomed-313M32 antibody was produced as a positive control and compared. The results of comparing antibody production through cloning are shown in FIG. 3 .
  • the selected chimeric-candidate antibody clones 19D, 58C, 58E, 62F, 63F and 75G were produced, and binding to antigen was confirmed.
  • the binding affinity of the chimeric-candidate antibody clones 58E, 62F, 63F and 75G was higher than that of the control, Oncomed-313M32 antibody. More specifically, the candidate antibody 58E was about 13 times higher than the control group, 62F was about 30 times, 63F was about 6 times, and 75G was about 30 times higher.
  • Human PBMCs were isolated, activated with anti-hCD3/28, treated with purified chimeric-candidate antibody, and the binding force between each CD4 T cell and CD8 T cell was confirmed by flow cytometry. Flow cytometry results are shown in FIG. 5 .
  • the chimeric-candidate antibodies 58C, 58E, 62F, 63F and 75G showed higher avidity than the control (Oncomed-313M32 antibody) in all CD4/CD8 T cells.
  • Example 2 Using the antigen binding affinity analysis conditions of Example 1, the efficacy of the chimeric-candidate antibody to inhibit the binding of TIGIT to its counterpart CD155 was investigated. A chimeric-candidate antibody selected from hybridoma was produced, purified, and treated. The results confirming the inhibitory efficacy of the chimeric-candidate antibody are shown in FIG. 6 .
  • Humanized antibodies against clones 19D, 62F, 63F and 75G were prepared from the results of analysis of the characteristics and functions of hybridoma and chimeric-candidate antibodies. The results of sequence analysis of the prepared humanized antibody are shown in Table 2.
  • Humanized antibody sequences for candidate antibody clones 19D, 62F, 63F and 75G were prepared from the hybridoma and chimeric-candidate antibody characteristics and function analysis results, and cloned into human IgG1 heavy chain and kappa light chain, respectively. After each humanized candidate antibody was produced and purified, it was confirmed by SDS-PAGE staining. In addition, antibodies were produced and purified using the Expi293 transient expression system, and quantitative binding capacity of each purified humanized antibody was analyzed by ELISA method. ELISA was performed in the same manner as in Example 3-(1), and the antibody was treated by serial dilution of 1/2 from the concentration of 500ng each. The results of SDS-PAGE and ELISA are shown in FIG. 7 .
  • humanized candidate antibody clones 19D, 62F, 63F and 75G were produced, and binding to antigen was confirmed.
  • the binding affinity of the humanized candidate antibody was the lowest in 19D among the 4 clones. It was confirmed that the humanized candidate antibody clones 62F, 63F and 75G were 33-fold, 13-fold and 36-fold higher than clone 19D, which had the lowest binding affinity, respectively.
  • the binding ability of the humanized candidate antibody was confirmed by flow cytometry using artificial effector T cells expressing TIGIT. Flow cytometry results are shown in FIG. 9 .
  • the humanized candidate antibody clones 19D, 62F, 63F and 75G all showed a higher binding ratio than ebioscience's antibody for flow cytometry.
  • the efficacy of the humanized candidate antibody to inhibit the binding of TIGIT to its counterpart CD155 was investigated using the antigen binding affinity assay conditions of Example 1. The results of confirming the inhibitory efficacy are shown in FIG. 10 .
  • the inhibitory efficacy of the humanized candidate antibodies 62F, 75G and 63F was excellent when compared to the positive control group (ebioscience's function grade TIGIT antibody, Oncomed-313M32 antibody). It was confirmed that the humanized candidate antibody 19D had a similar inhibitory efficacy to that of OMP-313M32.
  • a tumor model was created using a cell line expressing hPD-L1 in mouse colon cancer MC38 in hTIGIT/hPD-1 double knock-in mice and the anticancer efficacy was analyzed.
  • Humanized candidate antibodies 62F, 63F or 75G were administered to the tumor model 5 times at a concentration of 10 mg/kg at 3-day intervals, and the size of the tumor was analyzed.
  • Each humanized candidate antibody consisted of a group of 5 animals, and a group in which the humanized candidate antibody and the humanized PD-1 2B3W antibody were co-administered was also included.
  • the humanized PD-1 2B3W antibody comprises a heavy chain and a light chain represented by the following sequence.
  • the humanized PD-1 2B3W antibody was used as a candidate antibody selected for the development of a new PD-1 antibody.
  • the results of analyzing the tumor size following administration of the humanized candidate antibody alone or in combination are shown in FIG. 11 , and the average value of each group is graphed and shown in FIG. 12 .
  • the humanized candidate antibody and humanized PD-1 2B3W antibody combined administration group significantly reduced the size of the tumor compared to the single administration group.
  • tumors were removed from all subjects in the group administered with the humanized candidate antibody 75G (anti-TIGIT) and humanized PD-1 2B3W antibody (anti-PD-1).
  • the ratio of CD8 T cells increased in all groups after administration of the humanized candidate antibody three times (post-bleed). In particular, it increased at a higher rate in the combined administration than in the single administration.
  • intratumoral TIL tumor infiltrated lymphocyte
  • liver toxicity ie, ALT, AST, BUN and T-BIL levels were measured
  • Candidate antibodies T21.01, T21.08, T21.09, T21.10, T21.11, T21.12 and T21.13 were obtained, and more specific heavy and light chain information is shown in Table 3.
  • T21.01, T21.08 and T21.09 contain the heavy chain of the non-humanized hybridoma of the 58E antibody.
  • T21.10, T21.11, T21.12, T21.13, T21.14, T21.16 and T21.33 also contain the heavy chain of the 58E humanized antibody.
  • T21.01, T21.10 and T21.11 include the light chains of the non-humanized hybridoma of the 58E antibody
  • T21.08, T21.09, T21.12, T21.13, T21.14, T21.16 and T21.33 contain humanized light chain sequences.
  • T21.14, T21.16 and T21.33 also contain the heavy chain and humanized T21L01 light chain of the 58E humanized antibody.
  • the characteristics of each antibody were analyzed by SDS-PAGE, SEC-HPLC and SPR.
  • the SDS-PAGE and SEC-HPLC results are shown in FIG. 15
  • the SPR results are shown in FIG. 16 .
  • candidate antibodies obtained through gene engineering were purified and separated using the Expi293F transient expression system, and were stained after electrophoresis by SDS-PAGE method to reduce the total antibody size in non-reducing and reducing conditions. The size of each heavy chain and light chain of the antibody was compared. In addition, the purity of the purified antibody was confirmed by SEC-HPLC analysis.
  • T21.01, T21.09, T21.11 and T21.13 had higher antigen affinity than the candidate antibody 58E.
  • T21.01, T21.09, T21.11 and T21.13 that were judged to have high antigen affinity were selected.
  • T21.14, T21.16 and T21.33 having similar affinity to T21.11 were selected, and humanized T21L01.01 and T21L01.03 respectively. and the light chain sequence of T21L01.20.
  • TIGIT/CD155 Blockade Bioassay Promega
  • Jurkat Effector cells expressing human TIGIT and CD226 and having a luciferase reporter and CHO-K1 aAPC expressing CD155 were co-cultured.
  • the affinity-improved candidate antibody was treated, the antibody bound to TIGIT to block the binding to CD155, and the luciferase signal activated by binding of CD155 to CD226 was measured.
  • Measurement results for each candidate antibody are shown in FIG. 17A, and a graph was prepared based on FIG. 17A and shown in FIG. 17B.
  • the candidate antibody 58E and the candidate antibody with improved affinity had superior inhibitory efficacy than the commercially available antibody Oncomed-313M32.
  • the T21.11 clone had a higher inhibitory effect than the candidate antibody 58E.
  • Example 8 Affinity engineering and efficacy evaluation of humanized candidate antibody 62F (hu62F)
  • CDR amino acids into which random sequences were to be introduced were selected.
  • the selected CDR amino acids are shown in Table 5.
  • Primers for randomization targeting the amino acids in Table 5 were designed, and 22 types of primers to implement the library were produced by IDT technology (www.IDTDNA.com). The prepared primer is shown in FIG. 19 .
  • hu62F scFv was cloned into pYD5 vector. It was constructed in the form of VH(G4S)3-VL scFv on pYD5 plasmid. The cleavage map of the produced plasmid pYD5 is shown in FIG. 20, and it was synthesized by requesting IDT technology. V5-tag was spliced after the c-terminal EcoR1 site of each scFv, and sequences homologous to both ends of the pYD5 vector cut with Nhe1 and EcoR1 restriction enzymes were synthesized at both ends of the synthetic gene.
  • the synthesized insert gene and linearized vector were cloned using the In-Fusion HD Cloning Kit (Clontech), and the sequencing primer was confirmed using T7. After transfection of the cloned plasmid into Stellar Competent Cells (Clontech), midi-prep with ZymoPUREII Plasmid Midiprep kit (Zymo research, Cat# D4201) to obtain more than 15 ug of DNA.
  • 1st PCR uses hu62F pYD5 as a template and puts 100ng per 50ul of PCR reaction volume, dNTP (invitrogen), 2uM forward and reverse primer, 5X Phusion HF buffer (Thermo scientific), 1.5ul DMSO, 2unit Phusion high fidelity polymerse (Thermo scientific, cat#F530L) was added, and the remaining volume was filled with distilled water.
  • the PCR reaction was carried out in the same manner as in the table above, and the DNA amplified by electrophoresis was confirmed by making a 1.5% agarose gel.
  • 2nd PCR was performed using fragments 1 and 2 obtained in 1st PCR. Specifically, a total of 400 ng of DNA was added to fragments 1 and 2 obtained in 1 st PCR so that the molar ratio is 1:1, 10mM each dNTP (invtrogen), 5X Phusion HF buffer (Thermo scientific), 2unit Phusion high fidelity polymerase ( Thermo scientific, cat#F530L) was added, and the remaining volume was filled with distilled water (Invitrogen, cat#10977) to a total volume of 50ul.
  • a schematic diagram and reaction conditions of 2nd PCR are shown in FIG. 23 .
  • 2nd PCR results are shown in FIG. 24 .
  • each PCR product was extracted with a DNA cleanup kit (Geneaid, cat#DFM025) and the concentration was measured with a spectrophotometer (Thermo, Model: Multiskan Go). did
  • Each insert DNA obtained through 2nd PCR was prepared to be 5ug or more.
  • pYD5 vector was cut with Nhe1 (NEB, cat# R3131L) and EcoR1 (NEB, cat# R3101S) and extracted with a DNA cleanup kit (Geneaid, cat#DFM025).
  • Pellet Paint®Co Precipitant Novagen, Cat#690493 was used to concentrate the final volume to 1 and 5ul or less, respectively.
  • yeast transformation was performed as shown in FIG. 25 . Specifically, yeast competent cells were streaked with EBY100 yeast strain on a YPD plate and cultured at 30°C for 2 days, then one yeast colony was picked and cultured overnight at 30°C and 250rpm in 5ml YPD media. The cultured yeast cells were subcultured on a 50ml scale and grown under the same conditions for about 6 hours until the measured OD value was about 1.0 to 1.5, and then 0.5ml of sterilized Tris DTT solution (0.39g 1,4 dithiothreitol in 1ml 1M). Tris pH8.0 buffer) was added and incubated for 15 minutes.
  • Tris DTT solution (0.39g 1,4 dithiothreitol in 1ml 1M). Tris pH8.0 buffer
  • yeast cells were centrifuged, washed with cold E buffer (1.2 g Tris base, 92.4 g sucrose, 1M MgCl 2 in distilled water), and resuspensioned with E buffer to make a total volume of 300 ⁇ l to prepare competent cells. 50ul of the prepared competent cells, 5ug of concentrated insert DNA, and 1ug of vector were placed in an electro cuvette and electroporated at 0.54kV and 25uF conditions. After that, 1ml of YPD media was immediately added, and incubated at 30°C and 250rpm for 1 hour.
  • E buffer 1.2 g Tris base, 92.4 g sucrose, 1M MgCl 2 in distilled water
  • yeast cells cultured in YPD were treated with 10 ml SDCAA media (20 g glucose, 14.7 g sodium citrate, 4.3 g citric acid monohydrate, 6.7 g yeast nitrogen base, 5 g bacto casamino acid in 1 L distilled water) containing 100 ⁇ g/ml kanamycin antibiotic.
  • SDCAA media 20 g glucose, 14.7 g sodium citrate, 4.3 g citric acid monohydrate, 6.7 g yeast nitrogen base, 5 g bacto casamino acid in 1 L distilled water.
  • SDCAA::Km plate 100ug/ml kanamycin, 20g glucose, 14.7g sodium citrate, 4.3g citric acid monohydrate, 6.7g yeast nitrogen base, 5g bacto
  • casamino acid 16g bacto agar
  • Transformed yeast cells were resuspensed in SDCAA media and cultured in 100ml SDCAA media for 24 hours so that the initial O.D was 0.1. After subculture in the same way, 100ml SGCAA media (20g galactose) containing 100ug/ml kanamycin antibiotic so that the initial O.D is 1. , 14.7 g sodium citrate, 4.3 g citric acid monohydrate, 6.7 g yeast nitrogen base, 5 g bacto casamino acid in 1 L distilled water), followed by induction at 30 °C and 250 rpm for 16 to 20 hours.
  • each library and TIGIT-antigen were stained and flow cytometric analysis to improve antigen binding, and cells with increased TIGIT-antigen binding than hu62F were isolated.
  • the cells with increased TIGIT-antigen avidity compared to hu62F isolated from the library were separated into single clones and expressed on the surface of each yeast, and the binding ability with TIGIT-antigen was analyzed by flow cytometry.
  • the heavy chain is a plasmid without the effector function of Fc by introducing the pOptivec (Invitrogen)AAA (L234A, L235A, K322A) mutation, an animal cell expression vector, and the light chain is pcDNA3.3 (Invitrogen).
  • plasmid was fabricated and synthesized by IDT.
  • HC was synthesized by adding sequences homologous to both ends of the pOptivec (AAA) vector cut with EcoR1 and Nhe1 restriction enzymes, and pcDNA3.3 vector cut with EcoR1 and BsiW1 restriction enzymes for LC at both ends of the synthetic gene.
  • the cleavage maps of the pOptivec (AAA) vector and pcDNA3.3 vector are shown in FIG. 28 .
  • Each individual clone was cloned by introducing a mutation site derived from the hu6H8.3 backbone, respectively.
  • the synthesized insert gene and linearized vector were cloned by inserting each insert gene using the In Fusion®HD Cloning Kit (Clontech), and sequencing primers were confirmed using CmV Forward and EMCV IRES reverse primers.
  • the plasmid into which the designed humanized antibody gene was introduced was expressed in the form of IgG using the Expi293 expression system (Invitrogen), which was obtained by AktaPure (GE healthcare), AktaPrime purifier (GE healthcare) and MabselectSURE column (GE healthcare, Cat#11-0034). -95) was used for purification.
  • the purified antibody was buffer-changed with PBS through a desalting column (GE healthcare, Cat#17-1408-01), and the concentration was measured through Multiskan GO (Thermo).
  • the yield was calculated based on the concentration measurement results. The results of measuring the concentration and calculating the yield are shown in Table 10.
  • the purified binding strength improvement candidate antibodies were stained after electrophoresis by the SDS-PAGE method to compare the total antibody size in non-reducing, and the size of each heavy chain and light chain of the antibody in reducing conditions. The purity of the purified antibody was confirmed.
  • the produced IgG antibody was analyzed using a size exclusion column (Tosoh, TSKgel G3000 SWXL, 7.8 ⁇ 300mm, Part No.0008541, Column No.023D08819D) by HPLC (Agilent Technologies, 1260 infinity II LC system).
  • a size exclusion column Tosoh, TSKgel G3000 SWXL, 7.8 ⁇ 300mm, Part No.0008541, Column No.023D08819D
  • HPLC Alent Technologies, 1260 infinity II LC system.
  • 10X Phosphate Buffered Saline buffer wellgene, Cat#LB204 02
  • the analysis method was flow rate 1ml/min, 280nm wavelength band was analyzed for 20min, and 20ul of the sample was injected.
  • a gel filtration standard BIO RAD, Cat. #151-1901
  • HPLC results of the produced IgG antibody are shown in FIGS. 30a to c.
  • the purified candidate antibodies to improve binding were analyzed by SEC-HPLC to analyze the qualitative and quantitative analysis of the purified antibody.
  • a CM5 chip (GE Healthcare, Cat#BR-1005-30) immobilized with an anti-human Fc antibody was prepared using a human capture kit (GE healthcare, Cat#BR-1008-39). After diluting the produced antibody to 1ug/mL, it was flowed to the CM5 chip at a flow rate of 10uL/min for 60 seconds to fix the antibody, and TIGIT-His(Sino) was added to 100, 50, 25, 12.5, 6.25, 3.125nM The association time was 150 seconds and the dissociation time was 240 seconds.
  • the binding force was analyzed by fitting the sensorgram measured through the Biacore T200 (GE healthcare) equipment to a 1:1 binding model.
  • Table 11 The results of analyzing the antigen-binding ability of the produced antibodies are shown in Table 11.
  • the results of analyzing the antigen-antibody binding capacity of Hu62F and T02.10 using Biacore are shown in FIG. 31 .
  • T02.08 and T02.10 had higher antigen-binding ability than Hu62F.
  • the T02.10 antibody had about 27 times higher antigen-binding ability than that of Hu62F.
  • the antibody-antigen avidity (ka) of T02.10 was increased when compared to hu62F, whereas the degree of antibody-antigen dissociation (kd) was decreased.
  • the bonding strength was increased.

Abstract

The present invention relates to: an anti-TIGIT (T cell immunoglobulin and ITIM domain) antibody or an antigen-binding fragment thereof; a nucleic acid encoding same; a recombinant expression vector containing the nucleic acid; a host cell which is transinfected with the recombinant expression vector; a method for preparing the antibody or the antigen-binding fragment thereof; a bi- or multi-specific antibody containing the antibody or the antigen-binding fragment thereof; an immune cell-engaging bi- or multi-specific antibody containing at least one among an scFv of the antibody and an scFv of the antibody binding to the immune cell activation antigen; an antibody-drug conjugate (ADC) in which the antibody or the antigen-binding fragment thereof is bound to a drug; a chimeric antigen receptor (CAR) containing the scFv of the anti-TIGIT antibody as an antigen-binding site of an extracellular domain; an immune cell into which the chimeric antigen receptor has been introduced; a composition for a combination therapy including the immune cell; a composition for a combination therapy including the antibody or the antigen-binding fragment thereof; a composition for treating cancer or infectious diseases; and a method for treating cancer or infectious diseases.

Description

항-TIGIT 항체 및 이의 용도Anti-TIGIT antibodies and uses thereof
본 발명은 항-TIGIT (T cell immunoglobulin and ITIM domain) 항체 또는 이의 항원 결합 단편, 이를 코딩하는 핵산, 상기 핵산을 포함하는 재조합 발현벡터, 상기 재조합 발현벡터로 형질 감염된 숙주세포, 상기 항체 또는 이의 항원 결합 단편의 제조방법, 상기 항체 또는 이의 항원 결합 단편을 포함하는 이중 또는 다중 특이적 항체, 상기 항체의 scFv 및 면역세포 활성화 항원에 결합하는 항체의 scFv를 하나 이상 포함하는, 면역세포 인게이징 (immune cell engage) 이중 특이적 또는 다중 특이적 항체, 상기 항체 또는 이의 항원 결합 단편이 약물에 결합된 항체-약물 접합체(ADC), 상기 항-TIGIT 항체의 scFv를 세포 외 도메인의 항원 결합 부위로 포함하는 키메라 항원 수용체(CAR), 상기 키메라 항원 수용체가 도입되어 있는 면역세포, 상기 면역세포를 포함하는 병용 치료용 조성물, 상기 항체 또는 이의 항원 결합 단편을 포함하는 병용 치료용 조성물, 암 또는 감염질환 치료용 조성물, 암 또는 감염질환 치료방법에 관한 것이다. The present invention provides an anti-TIGIT (T cell immunoglobulin and ITIM domain) antibody or antigen-binding fragment thereof, a nucleic acid encoding the same, a recombinant expression vector containing the nucleic acid, a host cell transfected with the recombinant expression vector, the antibody or antigen thereof A method for producing a binding fragment, a dual or multispecific antibody comprising the antibody or antigen-binding fragment thereof, an scFv of the antibody and one or more scFv of an antibody binding to an immune cell activating antigen, immune cell engaging (immune) cell engage) a bispecific or multispecific antibody, an antibody-drug conjugate (ADC) in which the antibody or antigen-binding fragment thereof is bound to a drug, and the scFv of the anti-TIGIT antibody as an antigen-binding site of the extracellular domain A chimeric antigen receptor (CAR), an immune cell into which the chimeric antigen receptor is introduced, a composition for combination therapy comprising the immune cell, a composition for combination therapy comprising the antibody or antigen-binding fragment thereof, for the treatment of cancer or infectious disease It relates to a composition, a method for treating cancer or an infectious disease.
TIGIT (Ig 및 ITIM 도메인을 갖는 T 세포 면역 수용체)은 활성화된 T 세포 및 NK 세포 상에서 주로 발현되는 면역조정 수용체이다. TIGIT은 VSIG9; VSTM3; 및 WUCAM으로도 알려져 있다. TIGIT의 구조는 1개의 세포외 면역글로불린 도메인, 유형 1 막횡단 영역 및 2개의 ITIM 모티프를 포함한다. TIGIT은 T 세포 상에서의 양성 (CD226) 및 음성 (TIGIT) 면역조정 수용체 및 APC 상에서 발현된 리간드 (CD155 및 CD112)로 이루어진 공동-자극성 네트워크의 일부를 형성한다 (Boles KS, et al., 2009 Eur J Immunol, 39:695-703).TIGIT (T cell immune receptor with Ig and ITIM domains) is an immunomodulatory receptor expressed primarily on activated T cells and NK cells. TIGIT is VSIG9; VSTM3; and WUCAM. The structure of TIGIT contains one extracellular immunoglobulin domain, a type 1 transmembrane region and two ITIM motifs. TIGIT forms part of a co-stimulatory network consisting of positive (CD226) and negative (TIGIT) immunomodulatory receptors on T cells and ligands (CD155 and CD112) expressed on APCs ( Boles KS, et al., 2009 Eur J Immunol, 39:695-703) .
면역 체크포인트(checkpoint) 분자로서, TIGIT는 그의 리간드인 CD155 및 CD112와 결합될 때 면역 세포에서 억제 신호전달을 개시한다. CD155에 대한 TIGIT의 결합 친화도(Kd: ~1 nM)는 CD112 보다 훨씬 높고, TIGIT:CD112 상호작용이 억제 신호를 매개하는 데 기능적으로 관련이 있는지 여부는 여전히 밝혀져야 할 것으로 남아있다. 공동 자극 수용체 CD226(DNAM-1)은 동일한 리간드에 낮은 친화도(Kd: ~100 nM)로 결합하지만, 양성 신호를 전달한다(Bottino C, et al., 2003 J Exp Med 198:557-67). 또한, "TIGIT-유사" 수용체 CD96(Tactile)도 동일한 경로에서 유사하게 억제 역할을 한다(Chan CJ, et al., 2014 Nat. Immunol 15:431-8).As an immune checkpoint molecule, TIGIT initiates inhibitory signaling in immune cells when bound to its ligands CD155 and CD112. The binding affinity of TIGIT for CD155 (Kd: ˜1 nM) is much higher than that of CD112, and whether the TIGIT:CD112 interaction is functionally involved in mediating inhibitory signaling remains to be determined. The costimulatory receptor CD226 (DNAM-1) binds the same ligand with low affinity (Kd: -100 nM), but transmits a positive signal ( Bottino C, et al., 2003 J Exp Med 198:557-67 ). . In addition, the “TIGIT-like” receptor CD96 (Tactile) also plays a similar inhibitory role in the same pathway ( Chan CJ, et al., 2014 Nat. Immunol 15:431-8 ).
암 및 바이러스 감염의 경우, TIGIT 신호전달의 활성화는 면역 세포 기능장애를 촉진하여, 암 성장 또는 늘어난 바이러스 감염을 초래한다. 치료제에 의한 TIGIT-매개 억제 신호전달의 억제는 T 세포, NK 세포 및 수지상세포(DCs)를 비롯한 면역 세포의 기능적 활성을 회복시켜, 암 또는 만성 바이러스 감염에 대한 면역성을 향상시킬 수 있다.In the case of cancer and viral infections, activation of TIGIT signaling promotes immune cell dysfunction, resulting in cancer growth or increased viral infection. Inhibition of TIGIT-mediated inhibitory signaling by therapeutic agents can restore functional activity of immune cells, including T cells, NK cells and dendritic cells (DCs), thereby enhancing immunity against cancer or chronic viral infection.
이러한 기술적 배경하에서, 본 발명자들은 신규 항-TIGIT 항체를 개발하고자 예의 노력한 결과, 우수한 항체의 특성 및 효능 등을 확인하고, 목적하는 암 또는 감염질환 치료에 사용될 수 있음을 확인함으로써, 본 발명을 완성하였다.Under this technical background, the present inventors completed the present invention by confirming that, as a result of diligent efforts to develop a novel anti-TIGIT antibody, excellent properties and efficacy of the antibody, and it can be used for the treatment of a desired cancer or infectious disease, did.
본 발명의 목적은 TIGIT (T cell immunoglobulin and ITIM domain)에 대한 신규 항체 또는 이의 항원 결합 단편을 제공하는데 있다.An object of the present invention is to provide a novel antibody or antigen-binding fragment thereof against TIGIT (T cell immunoglobulin and ITIM domain).
본 발명의 다른 목적은 상기 항체 또는 이의 항원 결합 단편을 코딩하는 핵산을 제공하는데 있다.Another object of the present invention is to provide a nucleic acid encoding the antibody or antigen-binding fragment thereof.
본 발명의 다른 목적은 상기 핵산을 포함하는 재조합 발현벡터 또는 상기 재조합 발현벡터로 형질감염된 숙주세포를 제공하는데 있다. Another object of the present invention is to provide a recombinant expression vector containing the nucleic acid or a host cell transfected with the recombinant expression vector.
본 발명의 다른 목적은 TIGIT에 특이적으로 결합하는 항체 또는 이의 항원 결합단편의 제조방법을 제공하는 데 있다.Another object of the present invention is to provide a method for producing an antibody or antigen-binding fragment thereof that specifically binds to TIGIT.
본 발명의 다른 목적은 상기 항체 또는 이의 항원 결합단편을 포함하는 이중 또는 다중특이적 항체를 제공하는 데 있다.Another object of the present invention is to provide a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof.
본 발명의 다른 목적은 상기 항체 또는 이의 항원 결합 단편을 포함하는 면역세포 인게이징(immune cell engage) 이중특이적 또는 다중특이적 항체를 제공하는 데 있다.Another object of the present invention is to provide an immune cell engaging bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof.
본 발명의 다른 목적은 상기 항체 또는 이의 항원 결합단편이 약물에 결합된 항체-약물 접합체 (ADC)를 제공하는 데 있다.Another object of the present invention is to provide an antibody-drug conjugate (ADC) in which the antibody or antigen-binding fragment thereof is bound to a drug.
본 발명의 다른 목적은 상기 항-TIGIT 항체의 scFv를 세포외도메인의 항원 결합 부위로 포함하는 키메라 항원 수용체(CAR), 상기 키메라 항원 수용체가 도입되어 있는 면역세포, 상기 면역세포를 포함하는 병용 치료용 조성물을 제공하는 데 있다.Another object of the present invention is a chimeric antigen receptor (CAR) comprising the scFv of the anti-TIGIT antibody as an antigen-binding site of an extracellular domain, an immune cell into which the chimeric antigen receptor is introduced, and combination therapy comprising the immune cell To provide a composition for
본 발명의 다른 목적은 상기 항체 또는 이의 항원 결합 단편 또는 상기 면역세포 인게이징(immune cell engage) 이중특이적 또는 다중특이적 항체를 포함하는 병용 치료용 조성물을 제공하는 데 있다.Another object of the present invention is to provide a composition for combination therapy comprising the antibody or antigen-binding fragment thereof or the immune cell engaging bispecific or multispecific antibody.
본 발명의 다른 목적은 항체 또는 이의 항원 결합단편, 상기 항체 또는 이의 항원 결합단편을 포함하는 이중 또는 다중특이적 항체, 상기 항체 또는 이의 항원 결합단편을 포함하는 항체-약물 접합체, 상기 항체 또는 이의 항원 결합단편을 포함하는 키메라 항원 수용체, 상기 키메라 항원 수용체를 포함하는 암 치료용 조성물 또는 암 예방 또는 치료방법을 제공하는 데 있다.Another object of the present invention is an antibody or antigen-binding fragment thereof, a bi- or multispecific antibody comprising the antibody or antigen-binding fragment thereof, an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof, the antibody or antigen thereof To provide a chimeric antigen receptor comprising a binding fragment, a composition for treating cancer comprising the chimeric antigen receptor, or a method for preventing or treating cancer.
본 발명의 다른 목적은 항체 또는 이의 항원 결합단편, 상기 항체 또는 이의 항원 결합단편을 포함하는 이중 또는 다중특이적 항체, 상기 항체 또는 이의 항원 결합단편을 포함하는 항체-약물 접합체, 상기 항체 또는 이의 항원 결합단편을 포함하는 키메라 항원 수용체, 상기 키메라 항원 수용체를 포함하는 감염질환 치료용 조성물 또는 감염질환 예방 또는 치료방법을 제공하는 데 있다.Another object of the present invention is an antibody or antigen-binding fragment thereof, a bi- or multispecific antibody comprising the antibody or antigen-binding fragment thereof, an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof, the antibody or antigen thereof To provide a chimeric antigen receptor comprising a binding fragment, a composition for treating an infectious disease comprising the chimeric antigen receptor, or a method for preventing or treating an infectious disease.
본 발명의 다른 목적은 상기 항체 또는 이의 항원 결합단편; 상기 항체 또는 이의 항원 결합단편을 포함하는 이중 또는 다중특이적 항체; 상기 항체 또는 이의 항원 결합단편을 포함하는 항체-약물 접합체; 상기 항체 또는 이의 항원 결합단편을 포함하는 키메라 항원 수용체; 또는 상기 키메라 항원 수용체를 포함하는 면역세포;를 이를 필요로 하는 개체에 투여하는 단계를 포함하는, 암 예방 또는 치료 방법을 제공하는 데 있다.Another object of the present invention is the antibody or antigen-binding fragment thereof; a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof; an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof; a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof; Or an immune cell comprising the chimeric antigen receptor; to provide a method for preventing or treating cancer, comprising administering to an individual in need thereof.
본 발명의 다른 목적은 상기 항체 또는 이의 항원 결합단편; 상기 항체 또는 이의 항원 결합단편을 포함하는 이중 또는 다중특이적 항체; 상기 항체 또는 이의 항원 결합단편을 포함하는 항체-약물 접합체; 상기 항체 또는 이의 항원 결합단편을 포함하는 키메라 항원 수용체; 또는 상기 키메라 항원 수용체를 포함하는 면역세포;를 이를 필요로 하는 개체에 투여하는 단계를 포함하는, 감염질환 예방 또는 치료 방법을 제공하는 데 있다.Another object of the present invention is the antibody or antigen-binding fragment thereof; a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof; an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof; a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof; Or an immune cell comprising the chimeric antigen receptor; to provide a method for preventing or treating an infectious disease, comprising administering to an individual in need thereof.
상기 목적을 달성하기 위하여, 본 발명은 다음을 포함하는 TIGIT (T cell immunoglobulin and ITIM domain)에 특이적으로 결합하는 항체 또는 이의 항원 결합단편을 제공한다: In order to achieve the above object, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to TIGIT (T cell immunoglobulin and ITIM domain) comprising:
서열번호 1, 9, 17, 25 및 33로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 중쇄 CDR1, heavy chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 9, 17, 25 and 33;
서열번호 2, 10, 18, 26, 34 및 45로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 중쇄 CDR2, a heavy chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 10, 18, 26, 34 and 45;
서열번호 3, 11, 19, 27 및 35로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 중쇄 CDR3, 및a heavy chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11, 19, 27 and 35, and
서열번호 4, 12, 20, 28, 36, 41, 46 및 79로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 경쇄 CDR1, a light chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 12, 20, 28, 36, 41, 46 and 79;
서열번호 5, 13, 21, 29, 37, 42 및 47로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 경쇄 CDR2 및a light chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37, 42 and 47;
서열번호 6, 14, 22, 30, 38 및 43으로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 경쇄 CDR3.A light chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 14, 22, 30, 38 and 43.
본 발명은 또한, 서열번호 83 내지 89로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 중쇄 가변영역; 및 서열번호 90 및 91로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 경쇄 가변영역;을 포함하는, TIGIT (T cell immunoglobulin and ITIM domain)에 특이적으로 결합하는 항체 또는 이의 항원 결합단편을 제공한다.The present invention also provides a heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 83 to 89; And it provides an antibody or antigen-binding fragment thereof that specifically binds to TIGIT (T cell immunoglobulin and ITIM domain), including; and a light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 90 and 91 .
본 발명은 상기 항체 또는 이의 항원 결합단편을 코딩하는 핵산을 제공한다.The present invention provides a nucleic acid encoding the antibody or antigen-binding fragment thereof.
본 발명은 또한, 상기 핵산을 포함하는 재조합 발현벡터를 제공한다.The present invention also provides a recombinant expression vector comprising the nucleic acid.
본 발명은 또한, 상기 재조합 발현벡터로 형질감염된 숙주세포를 제공한다.The present invention also provides a host cell transfected with the recombinant expression vector.
본 발명은 또한, 숙주세포를 배양하여 항체를 생성하는 단계; 및 생성된 항체를 분리 및 정제하는 단계를 포함하는, TIGIT에 특이적으로 결합하는 항체 또는 이의 항원 결합단편의 제조방법을 제공한다.The present invention also comprises the steps of culturing a host cell to produce an antibody; And it provides a method for producing an antibody or antigen-binding fragment thereof that specifically binds to TIGIT, comprising the step of isolating and purifying the produced antibody.
본 발명은 또한, 상기 항체 또는 이의 항원 결합단편을 포함하는 이중 또는 다중특이적 항체를 제공한다. The present invention also provides a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof.
본 발명은 또한, 상기 항체의 scFv 및 면역세포 활성화 항원에 결합하는 항체의 scFv를 하나 이상 포함하는, 면역세포 인게이징(immune cell engage) 이중특이적 또는 다중특이적 항체를 제공한다.The present invention also provides an immune cell engage bispecific or multispecific antibody comprising at least one scFv of the antibody and an scFv of the antibody that binds to an immune cell activating antigen.
본 발명은 또한, 상기 항체 또는 이의 항원 결합단편이 약물에 결합된 항체-약물 접합체 (ADC)를 제공한다.The present invention also provides an antibody-drug conjugate (ADC) wherein the antibody or antigen-binding fragment thereof is bound to a drug.
본 발명은 또한, 항원 결합 부위를 포함하는 세포외도메인, 트랜스 멤브레인 도메인 및 세포내 신호전달 도메인을 포함하는 키메라 항원 수용체(CAR)로, 상기 세포외도메인의 항원 결합 부위는 상기 항체의 scFv인 것을 특징으로 하는 키메라 항원 수용체를 제공한다.The present invention also provides a chimeric antigen receptor (CAR) comprising an extracellular domain comprising an antigen-binding site, a transmembrane domain and an intracellular signaling domain, wherein the antigen-binding site of the extracellular domain is an scFv of the antibody. A chimeric antigen receptor is provided.
본 발명은 또한, 상기 키메라 항원 수용체(CAR)를 포함하는 면역세포를 제공한다.The present invention also provides an immune cell comprising the chimeric antigen receptor (CAR).
본 발명은 또한, 상기 면역세포를 포함하는 병용 치료용 조성물을 제공한다.The present invention also provides a composition for combination therapy comprising the immune cells.
본 발명은 또한, 상기 항체 또는 이의 항원 결합단편과 다음으로 구성된 군에서 선택된 하나 이상을 포함하는 병용 치료용 조성물을 제공한다:The present invention also provides a composition for combination therapy comprising the antibody or antigen-binding fragment thereof and at least one selected from the group consisting of:
(i) 면역세포; (i) immune cells;
(ii) 면역관문 억제제에 대한 항체에 대한 scFv 단편을 세포외도메인으로 포함하는 항원 수용체(CAR)를 포함하는 면역세포; 및(ii) immune cells comprising an antigen receptor (CAR) comprising an scFv fragment for an antibody against an immune checkpoint inhibitor as an extracellular domain; and
(iii) 면역관문억제제 (Immune checkpoint inhibitor).(iii) Immune checkpoint inhibitors.
본 발명은 또한, 상기 이중특이적 또는 다중특이적 항체 및 다음으로 구성된 군에서 선택된 하나 이상을 포함하는 병용 치료용 조성물을 제공한다:The present invention also provides a composition for combination therapy comprising the bispecific or multispecific antibody and at least one selected from the group consisting of:
(i) 면역세포; (i) immune cells;
(ii) 면역관문 억제제에 대한 항체에 대한 scFv 단편을 세포외도메인으로 포함하는 항원 수용체(CAR)를 포함하는 면역세포; 및(ii) immune cells comprising an antigen receptor (CAR) comprising an scFv fragment for an antibody against an immune checkpoint inhibitor as an extracellular domain; and
(iii) 면역관문억제제 (Immune checkpoint inhibitor).(iii) Immune checkpoint inhibitors.
본 발명은 또한, 상기 항체 또는 이의 항원 결합단편, 상기 항체 또는 이의 항원 결합단편을 포함하는 이중 또는 다중특이적 항체, 상기 항체 또는 이의 항원 결합단편을 포함하는 항체-약물 접합체, 상기 항체 또는 이의 항원 결합단편을 포함하는 키메라 항원 수용체 또는 상기 키메라 항원 수용체가 도입되어 있는 면역세포를 포함하는, 암 치료용 조성물을 제공한다. The present invention also relates to the antibody or antigen-binding fragment thereof, a bi- or multispecific antibody comprising the antibody or antigen-binding fragment thereof, an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof, the antibody or antigen thereof It provides a composition for treating cancer, comprising a chimeric antigen receptor containing a binding fragment or immune cells into which the chimeric antigen receptor is introduced.
본 발명은 또한, 상기 항체 또는 이의 항원 결합단편, 상기 항체 또는 이의 항원 결합단편을 포함하는 이중 또는 다중특이적 항체, 상기 항체 또는 이의 항원 결합단편을 포함하는 항체-약물 접합체, 상기 항체 또는 이의 항원 결합단편을 포함하는 키메라 항원 수용체 또는 상기 키메라 항원 수용체가 도입되어 있는 면역세포를 포함하는, 감염질환 치료용 조성물을 제공한다. The present invention also relates to the antibody or antigen-binding fragment thereof, a bi- or multispecific antibody comprising the antibody or antigen-binding fragment thereof, an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof, the antibody or antigen thereof It provides a composition for treating infectious diseases, comprising a chimeric antigen receptor containing a binding fragment or immune cells into which the chimeric antigen receptor is introduced.
본 발명은 또한, 상기 항체 또는 이의 항원 결합단편; 상기 항체 또는 이의 항원 결합단편을 포함하는 이중 또는 다중특이적 항체; 상기 항체 또는 이의 항원 결합단편을 포함하는 항체-약물 접합체; 상기 항체 또는 이의 항원 결합단편을 포함하는 키메라 항원 수용체; 또는 상기 키메라 항원 수용체를 포함하는 면역세포;를 이를 필요로 하는 개체에 투여하는 단계를 포함하는, 암 예방 또는 치료 방법을 제공한다.The present invention also provides the antibody or antigen-binding fragment thereof; a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof; an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof; a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof; Or an immune cell comprising the chimeric antigen receptor; provides a method for preventing or treating cancer, comprising administering to an individual in need thereof.
본 발명은 또한, 상기 항체 또는 이의 항원 결합단편; 상기 항체 또는 이의 항원 결합단편을 포함하는 이중 또는 다중특이적 항체; 상기 항체 또는 이의 항원 결합단편을 포함하는 항체-약물 접합체; 상기 항체 또는 이의 항원 결합단편을 포함하는 키메라 항원 수용체; 또는 상기 키메라 항원 수용체를 포함하는 면역세포;를 이를 필요로 하는 개체에 투여하는 단계를 포함하는, 감염질환 예방 또는 치료 방법을 제공한다The present invention also provides the antibody or antigen-binding fragment thereof; a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof; an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof; a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof; Or an immune cell comprising the chimeric antigen receptor; provides a method for preventing or treating an infectious disease, comprising administering to an individual in need thereof
본 발명에 따른 항-TIGIT 항체 또는 이의 항원 결합단편은 TIGIT에 대한 우수한 결합력을 나타내며, 목적하는 종양 또는 암, 감염질환의 예방 또는 치료에 유용하게 사용될 수 있다. The anti-TIGIT antibody or antigen-binding fragment thereof according to the present invention exhibits excellent binding ability to TIGIT, and can be usefully used for the prevention or treatment of a desired tumor, cancer, or infectious disease.
도 1은 flow cytometry를 통해 후보 클론의 항원결합력을 분석한 결과를 나타낸 도이다. 1 is a diagram showing the results of analyzing the antigen-binding ability of a candidate clone through flow cytometry.
도 2는 TIGIT/CD155 Blockade Bioassay를 통해 후보항체의 TIGIT 및 CD155 결합 억제능을 확인한 결과를 나타낸 도이다.2 is a diagram showing the results of confirming the TIGIT and CD155 binding inhibitory ability of the candidate antibody through the TIGIT/CD155 Blockade Bioassay.
도 3은 클로닝을 통한 chimeric 항체의 생산을 비교한 결과를 나타낸 도이다.3 is a diagram showing the results of comparing the production of chimeric antibodies through cloning.
도 4는 HPLC 및 SPR을 통해 chimeric 후보항체의 순도 및 결합력을 분석한 결과를 나타낸 도이다.4 is a diagram showing the results of analyzing the purity and binding affinity of the chimeric candidate antibody through HPLC and SPR.
도 5는 Flow cytometry를 통해 chimeric 후보항체의 CD4 T 세포 및 CD8 T 세포에 대한 결합력을 확인한 결과를 나타낸 도이다.5 is a diagram showing the results of confirming the binding ability of the chimeric candidate antibody to CD4 T cells and CD8 T cells through flow cytometry.
도 6은 TIGIT/CD155 Blocking Assay를 통해 chimeric 후보항체의 TIGIT 및 CD155 결합 억제능을 확인한 결과를 나타낸 도이다.6 is a diagram showing the results of confirming the TIGIT and CD155 binding inhibitory ability of the chimeric candidate antibody through the TIGIT/CD155 Blocking Assay.
도 7은 SDS-PAGE 및 ELISA를 통해 인간화 후보항체의 정제 여부 및 정량적 결합력을 분석한 결과를 나타낸 도이다.7 is a diagram showing the results of analysis of whether the humanized candidate antibody was purified and quantitative binding ability through SDS-PAGE and ELISA.
도 8은 HPLC 및 SPR을 통해 인간화 후보항체의 순도 및 결합력을 분석한 결과를 나타낸 도이다.8 is a diagram showing the results of analyzing the purity and binding affinity of the humanized candidate antibody through HPLC and SPR.
도 9는 Flow cytometry를 통해 인간화 후보항체의 artificial effector T 세포에 대한 결합력을 확인한 결과를 나타낸 도이다.9 is a view showing the results of confirming the binding ability of the humanized candidate antibody to artificial effector T cells through flow cytometry.
도 10은 TIGIT/CD155 Blocking Assay를 통해 인간화 후보항체의 TIGIT 및 CD155 결합 억제능을 확인한 결과를 나타낸 도이다.10 is a diagram showing the results of confirming the TIGIT and CD155 binding inhibitory ability of the humanized candidate antibody through the TIGIT/CD155 Blocking Assay.
도 11은 인간화 후보항체 투여에 따른 종양 마우스 모델의 종양 크기를 측정한 결과를 나타낸 도이다.11 is a diagram showing the results of measuring the tumor size of a tumor mouse model according to administration of a humanized candidate antibody.
도 12는 인간화 후보항체 투여에 따른 종양 마우스 모델의 종양 크기 측정 결과에 기초하여, 각 그룹의 평균값을 계산한 것이다.12 shows the calculation of the average value of each group based on the tumor size measurement result of the tumor mouse model following administration of the humanized candidate antibody.
도 13은 인간화 후보항체 투여 후 희생된 종양 마우스 모델의 혈액으로부터 CD8 T세포의 비율을 분석한 결과를 나타낸 도이다.13 is a diagram showing the results of analyzing the ratio of CD8 T cells from the blood of a tumor mouse model sacrificed after administration of a humanized candidate antibody.
도 14는 인간화 후보항체 투여 후 희생된 종양 마우스 모델의 혈액을 이용하여 간 독성을 측정한 결과를 나타낸 도이다.14 is a diagram showing the results of measuring liver toxicity using blood from a tumor mouse model sacrificed after administration of a humanized candidate antibody.
도 15는 SDS-PAGE 및 SEC-HPLC를 통해 친화도가 개선된 gene engineered 후보항체의 특성을 분석한 결과를 나타낸 도이다.15 is a diagram showing the results of analyzing the characteristics of a gene engineered candidate antibody with improved affinity through SDS-PAGE and SEC-HPLC.
도 16은 SPR을 통해 친화도가 개선된 gene engineered 후보항체의 특성을 분석한 결과를 나타낸 도이다.16 is a diagram showing the results of analyzing the characteristics of a gene engineered candidate antibody with improved affinity through SPR.
도 17a는 TIGIT/CD155 Blocking Assay를 통해 친화도가 개선된 gene engineered 후보항체의 TIGIT 및 CD155 결합 억제능을 확인한 결과를 나타낸 도이다.17A is a diagram showing the results of confirming the TIGIT and CD155 binding inhibition ability of a gene engineered candidate antibody with improved affinity through the TIGIT/CD155 Blocking Assay.
도 17b는 상기 TIGIT 및 CD155 결합 억제능 확인 결과를 기반으로 작성한 그래프이다.Figure 17b is a graph prepared based on the TIGIT and CD155 binding inhibitory ability confirmation results.
도 18은 인간화 후보항체 62F(hu62F)의 서열을 분석하여 정의한 6종의 CDR을 나타낸 도이다.18 is a diagram showing six CDRs defined by analyzing the sequence of the humanized candidate antibody 62F (hu62F).
도 19는 CDR 라이브러리 제작을 위해 디자인된 primer 22종의 서열을 나타낸 도이다.19 is a diagram showing the sequences of 22 primers designed for CDR library construction.
도 20은 제작된 플라스미드 pYD5의 개열지도를 나타낸 도이다.20 is a diagram showing a cleavage map of the prepared plasmid pYD5.
도 21은 CDR region에 대한 PCR(1st PCR)의 모식도 및 실험 조건을 나타낸 도이다.21 is a schematic diagram and experimental conditions of PCR (1 st PCR) for the CDR region.
도 22는 CDR region에 대한 PCR 결과를 나타낸 도이다.22 is a diagram showing PCR results for a CDR region.
도 23은 2nd PCR의 모식도 및 반응 조건을 나타낸 도이다.23 is a diagram showing a schematic diagram and reaction conditions of 2nd PCR.
도 24는 2nd PCR 결과를 나타낸 도이다.24 is a diagram showing 2nd PCR results.
도 25는 Yeast transformation의 실험 과정을 나타낸 도이다.25 is a diagram illustrating an experimental process of yeast transformation.
도 26은 FACS를 통해 yeast cell 표면에 발현된 scFv를 staining한 결과를 나타낸 도이다.26 is a diagram showing the results of staining the scFv expressed on the yeast cell surface through FACS.
도 27은 FACS를 통해 affinity 향상 후보 개별 클론의 항원결합력을 분석한 결과를 나타낸 도이다.27 is a diagram showing the results of analyzing the antigen-binding capacity of individual clones for affinity enhancement through FACS.
도 28은 pOptivec(AAA) vector 및 pcDNA3.3 vector의 개열지도를 나타낸 도이다.28 is a diagram showing a cleavage map of the pOptivec (AAA) vector and pcDNA3.3 vector.
도 29는 SDS-PAGE를 통해 항원 결합력 향상 후보항체의 특성을 분석한 결과를 나타낸 도이다.29 is a diagram showing the results of analyzing the characteristics of the antigen-binding ability enhancing candidate antibody through SDS-PAGE.
도 30은 Size exclusion chromatography를 통해 생산된 IgG 항체를 분석한 결과를 나타낸 도이다.30 is a diagram showing the results of analyzing the IgG antibody produced through size exclusion chromatography.
도 31은 Biacore를 이용하여 인간화 항체 Hu62F 및 T02.10의 항원-항체 결합력을 분석한 결과를 나타낸 도이다. Figure 31 is a diagram showing the results of analyzing the antigen-antibody binding force of the humanized antibodies Hu62F and T02.10 using Biacore.
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로, 본 명세서에서 사용된 명명법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art.
항-TIGIT 항체anti-TIGIT antibody
본 발명은 일 관점에서, 다음을 포함하는 TIGIT (T cell immunoglobulin and ITIM domain)에 특이적으로 결합하는 항체 또는 이의 항원 결합단편에 관한 것이다: 서열번호 1, 9, 17, 25 및 33로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 중쇄 CDR1, 서열번호 2, 10, 18, 26, 34 및 45로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 중쇄 CDR2, 서열번호 3, 11, 19, 27 및 35로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 중쇄 CDR3, 및 서열번호 4, 12, 20, 28, 36, 41, 46 및 79로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 경쇄 CDR1, 서열번호 5, 13, 21, 29, 37, 42 및 47로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 경쇄 CDR2 및 서열번호 6, 14, 22, 30, 38 및 43으로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 경쇄 CDR3.In one aspect, the present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to TIGIT (T cell immunoglobulin and ITIM domain) comprising: a group consisting of SEQ ID NOs: 1, 9, 17, 25 and 33 A heavy chain CDR1 comprising at least one amino acid sequence selected from, a heavy chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 10, 18, 26, 34 and 45, SEQ ID NOs: 3, 11, 19, 27 and a heavy chain CDR3 comprising at least one amino acid sequence selected from the group consisting of 35, and a light chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 12, 20, 28, 36, 41, 46 and 79, the sequence a light chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37, 42 and 47 and at least one amino acid selected from the group consisting of SEQ ID NOs: 6, 14, 22, 30, 38 and 43 light chain CDR3 comprising the sequence.
본 명세서에서 사용된 용어, "항체(antibody)"는 TIGIT에 특이적으로 결합하는 항-TIGIT 항체를 의미한다. 본 발명의 범위에는 TIGIT에 특이적으로 결합하는 완전한 항체 형태뿐 아니라, 상기 항체 분자의 항원 결합 단편도 포함된다.As used herein, the term “antibody” refers to an anti-TIGIT antibody that specifically binds to TIGIT. The scope of the present invention includes not only complete antibody forms that specifically bind TIGIT, but also antigen-binding fragments of the antibody molecule.
완전한 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 가지는 구조이며, 각각의 경쇄는 중쇄와 다이설파이드 결합으로 연결되어 있다. A complete antibody has a structure having two full-length light chains and two full-length heavy chains, each light chain connected to the heavy chain by a disulfide bond.
본 명세서에서 사용되는 용어, "중쇄"는 항원에 특이성을 부여하기 위한 충분한 가변영역 서열을 갖는 아미노산 서열을 포함하는 가변영역 도메인 VH 및 3개의 불변영역 도메인 CH1, CH2 및 CH3을 포함하는 전체길이 중쇄 및 이의 단편을 모두 의미한다. 또한, 본 명세서에서 사용되는 용어, "경쇄"는 항원에 특이성을 부여하기 위한 충분한 가변영역 서열을 갖는 아미노산 서열을 포함하는 가변영역 도메인 VL 및 불변영역 도메인 CL을 포함하는 전체길이 경쇄 및 이의 단편을 모두 의미한다.As used herein, the term “heavy chain” refers to a full-length heavy chain comprising a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and three constant region domains CH1, CH2 and CH3. and fragments thereof. In addition, as used herein, the term "light chain" refers to a full-length light chain comprising a variable region domain VL and a constant region CL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and fragments thereof. all means
상기 전체 항체는 IgA, IgD, IgE, IgM 및 IgG의 아형(subtype)을 포함하며, 특히 IgG는 IgG1, IgG2, IgG3 및 IgG4를 포함한다. 중쇄 불변영역은 감마(γ), 뮤(μ), 알파(α), 델타(δ)및 엡실론(ε) 타입을 가지고 서브클래스로 감마1(γ), 감마2(γ), 감마3(γ), 감마4(γ), 알파1(α1) 및 알파2(α2)를 가진다. 경쇄의 불변영역은 카파(κ) 및 람다(λ)타입을 가진다.The whole antibody includes subtypes of IgA, IgD, IgE, IgM and IgG, in particular, IgG includes IgG1, IgG2, IgG3 and IgG4. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types and subclasses gamma 1 (γ), gamma 2 (γ), gamma 3 (γ). ), gamma 4 (γ), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain has a kappa (κ) and a lambda (λ) type.
항체의 항원 결합 단편 또는 항체 단편이란 항원 결합 기능을 보유하고 있는 단편을 의미하며, Fab, F(ab'), F(ab')2 및 Fv 등을 포함한다. 항체 단편 중 Fab는 경쇄 및 중쇄의 가변영역과 경쇄의 불변영역 및 중쇄의 첫 번째 불변영역(CH1)을 가지는 구조로 1개의 항원 결합 부위를 가진다. Fab'는 중쇄 CH1 도메인의 C-말단에 하나 이상의 시스테인 잔기를 포함하는 힌지 영역(hinge-region)을 가진다는 점에서 Fab와 차이가 있다. F(ab')2는 Fab'의 힌지 영역의 시스테인 잔기가 다이설파이드 결합을 이루면서 생성된다. Antigen-binding fragment or antibody fragment of an antibody refers to a fragment having an antigen-binding function, and includes Fab, F(ab'), F(ab')2 and Fv. Among the antibody fragments, Fab has a structure having variable regions of light and heavy chains, constant regions of light chain and first constant region (CH1) of heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge-region comprising one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 is formed when a cysteine residue in the hinge region of Fab' forms a disulfide bond.
Fv는 중쇄 가변영역 및 경쇄 가변영역만을 가지고 있는 최소의 항체조각에 해당한다. 이중쇄 Fv(two-chain Fv)는 비공유결합으로 중쇄 가변영역과 경쇄 가변영역이 연결되어 있고, 단쇄 Fv(single-chain Fv, scFv)는 일반적으로 펩타이드 링커를 통하여 중쇄의 가변영역과 경쇄의 가변영역이 공유결합으로 연결되거나 또는 C-말단에서 바로 연결되어 있어서 이중쇄 Fv와 같이 다이머와 같은 구조를 이룰 수 있다. 이러한 항체 단편은 단백질 가수분해 효소를 이용하거나 (예를 들어, 완전한 형태의 항체를 파파인으로 제한 절단하면 Fab를 얻을 수 있고 펩신으로 절단하면 F(ab')2를 얻을 수 있다), 유전자 재조합 기술을 이용하여 제작할 수 있다.Fv corresponds to the smallest antibody fragment having only a heavy chain variable region and a light chain variable region. In a double-chain Fv (two-chain Fv), the heavy chain variable region and the light chain variable region are connected by a non-covalent bond, and single-chain Fv (scFv) is generally a heavy chain variable region and light chain variable region through a peptide linker. Since the regions are covalently linked or linked directly at the C-terminus, they can form a dimer-like structure like a double-stranded Fv. These antibody fragments can be prepared by using proteolytic enzymes (for example, by restriction digestion of the intact antibody with papain to obtain Fab, and by digestion with pepsin to obtain F(ab')2), or by genetic recombination technology. can be produced using
"Fv" 단편은 완전한 항체 인식 및 결합 부위를 함유하는 항체 단편이다. 이러한 영역은 1개의 중쇄 가변 도메인과 1개의 경쇄 가변 도메인이 결합된 이량체이다.An “Fv” fragment is an antibody fragment that contains a complete antibody recognition and binding site. This region is a dimer in which one heavy chain variable domain and one light chain variable domain are bound.
"Fab" 단편은 경쇄의 가변 및 불변 도메인과, 중쇄의 가변 및 제1 불변 도메인(CH1)을 포함한다. F(ab')2 항체 단편은 일반적으로 Fab'단편 C-말단에 존재하는 힌지 영역의 시스테인에 의해 공유적으로 연결된 한 쌍의 Fab' 단편을 포함한다.A "Fab" fragment comprises the variable and constant domains of a light chain and the variable and first constant domains (CH1) of a heavy chain. F(ab')2 antibody fragments generally comprise a pair of Fab' fragments covalently linked by cysteines in the hinge region present at the C-terminus of the Fab' fragment.
"단일쇄 Fv (scFv)" 항체 단편은 항체의 VH 및 VL 도메인을 포함하는 단일 폴리펩티드 쇄로 이루어진 구조체이다. scFv가 항원 결합을 위해 목적하는 구조를 형성할 수 있도록 하는 폴리펩티드 링커를 VH 도메인과 VL 도메인 사이에 추가로 포함할 수 있다.A “single chain Fv (scFv)” antibody fragment is a construct consisting of a single polypeptide chain comprising the VH and VL domains of an antibody. It may further comprise a polypeptide linker between the VH domain and the VL domain that enables the scFv to form the desired structure for antigen binding.
하나의 실시예에서, 본 발명의 항체는 단일클론 항체, 다특이적 항체, 인간 항체, 인간화 항체, 키메라 항체, scFv, Fab 단편, F(ab')2 단편, 다이설파이드-결합 Fvs (sdFv) 및 항-이디오타입(항-Id) 항체 또는 상기 항체들의 에피토프-결합 단편 등을 포함하나, 이에 한정되는 것은 아니다. In one embodiment, the antibody of the invention is a monoclonal antibody, multispecific antibody, human antibody, humanized antibody, chimeric antibody, scFv, Fab fragment, F(ab')2 fragment, disulfide-bonded Fvs (sdFv) and anti-idiotypic (anti-Id) antibodies or epitope-binding fragments of the antibodies, and the like.
상기 중쇄 불변영역은 감마(γ), 뮤(μ), 알파(α), 델타(δ), 또는 엡실론(ε) 중의 어느 한 이소타입으로부터 선택될 수 있다. 예를 들어, 불변영역은 감마1 (IgG1), 감마 2 (IgG2), 감마 3 (IgG3) 또는 감마 4 (IgG4)이다. 경쇄 불변영역은 카파 또는 람다 형일 수 있다.The heavy chain constant region may be selected from any one isotype of gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε). For example, the constant region is gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3) or gamma 4 (IgG4). The light chain constant region may be kappa or lambda type.
상기 단일클론 항체는 실질적으로 동질적 항체 집단으로부터 수득한 항체, 즉 집단을 차지하고 있는 개개의 항체가 미량으로 존재할 수 있는 가능한 천연 발생적 돌연변이를 제외하고는 동일한 것을 지칭한다. 단일클론 항체는 고도로 특이적이어서, 단일 항원 부위에 대항하여 유도된다. 전형적으로 상이한 결정인자(에피토프)에 대해 상이한 항체를 포함하는 통상의 (폴리클로날) 항체와는 대조적으로, 각각의 단일클론 항체는 항원 상의 단일 결정인자에 대해 지시된다.Said monoclonal antibody refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to conventional (polyclonal) antibodies, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
"에피토프(epitope)"는 항체가 특이적으로 결합할 수 있는 단백질 결정부위(determinant)를 의미한다. 에피토프는 통상 화학적으로 활성인 표면 분자군, 예를 들어 아미노산 또는 당 측쇄로 구성되며, 일반적으로 특정한 3차원의 구조적 특징뿐만 아니라 특정한 전하 특성을 갖는다. 입체적 에피토프 및 비입체적 에피토프는 변성 용매의 존재하에서 전자에 대한 결합은 소실되지만 후자에 대해서는 소실되지 않는 점에서 구별된다."Epitope" refers to a protein determinant to which an antibody can specifically bind. Epitopes are usually composed of a group of chemically active surface molecules, such as amino acids or sugar side chains, and generally have specific three-dimensional structural characteristics as well as specific charge properties. Conformational and non-steric epitopes are distinguished in that binding to the former is lost but not to the latter in the presence of a denaturing solvent.
상기 "인간화" 형태의 비-인간(예: 마우스) 항체는 비-인간 면역글로불린으로부터 유래된 최소 서열을 함유하는 키메라 항체이다. 대부분의 경우, 인간화 항체는 수용자의 초가변영역으로부터의 잔기를 목적하는 특이성, 친화성 및 능력을 보유하고 있는 비-인간 종(공여자 항체), 예를 들어 마우스, 랫트, 토끼 또는 비-인간 영장류의 초가변영역으로부터의 잔기로 대체시킨 인간 면역글로불린 (수용자 항체)이다.Such "humanized" forms of non-human (eg, mouse) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are of a non-human species (donor antibody) that retain the desired specificity, affinity and ability for residues from the hypervariable region of the recipient, eg, mouse, rat, rabbit or non-human primate. It is a human immunoglobulin (recipient antibody) replaced with residues from the hypervariable region of
상기 "인간 항체"는 인간 면역글로불린으로부터 유래하는 분자로서 상보성 결정영역, 구조 영역을 포함한 항체를 구성하는 아미노산 서열 전체가 인간의 면역글로불린으로 구성되어 있는 것을 의미한다.The "human antibody" is a molecule derived from human immunoglobulin, and means that the entire amino acid sequence constituting the antibody, including the complementarity determining region and structural region, is composed of human immunoglobulin.
중쇄 및/또는 경쇄 일부가 특별한 종으로부터 유래되거나 특별한 항체 부류 또는 아부류에 속하는 항체 내의 상응하는 서열과 동일하거나 이와 상동성인 반면, 나머지 쇄(들)는 또 다른 종으로부터 유래되거나 또 다른 항체 부류 또는 아부류에 속하는 항체 내의 상응하는 서열과 동일하거나 이와 상동성인 "키메라" 항체(면역글로불린) 뿐 아니라 목적하는 생물학적 활성을 나타내는 상기 항체의 단편이 포함된다.A portion of the heavy and/or light chain is identical to or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remaining chain(s) are from another species or from another antibody class or subclass. Included are "chimeric" antibodies (immunoglobulins) that are identical to or homologous to the corresponding sequence in antibodies belonging to the subclass, as well as fragments of such antibodies that exhibit the desired biological activity.
본 발명에서 사용된 항체의 "가변영역"은 상보성 결정 영역 (CDR; 즉 CDR1, CDR2, 및 CDR3) 및 골격 영역 (FR)의 아미노산 서열을 포함하는 항체 분자의 경쇄 및 중쇄 부분을 의미한다. VH는 중쇄의 가변 도메인을 의미한다. VL은 경쇄의 가변 도메인을 의미한다.As used herein, the term "variable region" of an antibody refers to the light and heavy chain portions of an antibody molecule comprising the amino acid sequences of complementarity determining regions (CDRs; ie, CDR1, CDR2, and CDR3) and framework regions (FR). VH refers to the variable domain of a heavy chain. VL refers to the variable domain of the light chain.
"상보성 결정 영역(complement determining region, CDR)"은 항원 결합을 위해 필요한 존재인, 항체 가변 도메인의 아미노산 잔기를 의미한다. 각 가변 도메인은 전형적으로 CDR1, CDR2 및 CDR3로 확인된 3개의 CDR 영역을 갖는다. "Complementary determining region (CDR)" refers to amino acid residues of an antibody variable domain, which are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3.
본 발명에 따른 항-TIGIT 항체 또는 이의 항원 결합단편은 예를 들어, 다음을 포함할 수 있다:The anti-TIGIT antibody or antigen-binding fragment thereof according to the present invention may comprise, for example:
(i) 서열번호 1의 중쇄 CDR1, 서열번호 2의 중쇄 CDR2 및 서열번호 3의 중쇄 CDR3을 포함하는 중쇄 가변영역, 및 서열번호 4의 경쇄 CDR1, 서열번호 5의 경쇄 CDR2 및 서열번호 6의 경쇄 CDR3를 포함하는 경쇄 가변영역;(i) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 2 and the heavy chain CDR3 of SEQ ID NO: 3, and the light chain CDR1 of SEQ ID NO: 4, the light chain CDR2 of SEQ ID NO: 5 and the light chain of SEQ ID NO: 6 a light chain variable region comprising CDR3;
(ii) 서열번호 9의 중쇄 CDR1, 서열번호 10의 중쇄 CDR2 및 서열번호 11의 중쇄 CDR3을 포함하는 중쇄 가변영역, 및 서열번호 12의 경쇄 CDR1, 서열번호 13의 경쇄 CDR2 및 서열번호 14의 경쇄 CDR3를 포함하는 경쇄 가변영역; (ii) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 9, the heavy chain CDR2 of SEQ ID NO: 10 and the heavy chain CDR3 of SEQ ID NO: 11, and the light chain CDR1 of SEQ ID NO: 12, the light chain CDR2 of SEQ ID NO: 13 and the light chain of SEQ ID NO: 14 a light chain variable region comprising CDR3;
(iii) 서열번호 17의 중쇄 CDR1, 서열번호 18의 중쇄 CDR2 및 서열번호 19의 중쇄 CDR3을 포함하는 중쇄 가변영역, 및 서열번호 20의 경쇄 CDR1, 서열번호 21의 경쇄 CDR2 및 서열번호 22의 경쇄 CDR3를 포함하는 경쇄 가변영역;(iii) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 17, the heavy chain CDR2 of SEQ ID NO: 18 and the heavy chain CDR3 of SEQ ID NO: 19, and the light chain CDR1 of SEQ ID NO: 20, the light chain CDR2 of SEQ ID NO: 21 and the light chain of SEQ ID NO: 22 a light chain variable region comprising CDR3;
(iv) 서열번호 25의 중쇄 CDR1, 서열번호 26의 중쇄 CDR2 및 서열번호 27의 중쇄 CDR3을 포함하는 중쇄 가변영역, 및 서열번호 28의 경쇄 CDR1, 서열번호 29의 경쇄 CDR2 및 서열번호 30의 경쇄 CDR3를 포함하는 경쇄 가변영역;(iv) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 25, the heavy chain CDR2 of SEQ ID NO: 26 and the heavy chain CDR3 of SEQ ID NO: 27, and the light chain CDR1 of SEQ ID NO: 28, the light chain CDR2 of SEQ ID NO: 29 and the light chain of SEQ ID NO: 30 a light chain variable region comprising CDR3;
(v) 서열번호 33의 중쇄 CDR1, 서열번호 34의 중쇄 CDR2 및 서열번호 35의 중쇄 CDR3을 포함하는 중쇄 가변영역, 및 서열번호 36의 경쇄 CDR1, 서열번호 37의 경쇄 CDR2 및 서열번호 38의 경쇄 CDR3를 포함하는 경쇄 가변영역;(v) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 33, the heavy chain CDR2 of SEQ ID NO: 34 and the heavy chain CDR3 of SEQ ID NO: 35, and the light chain CDR1 of SEQ ID NO: 36, the light chain CDR2 of SEQ ID NO: 37 and the light chain of SEQ ID NO: 38 a light chain variable region comprising CDR3;
(vi) 서열번호 33의 중쇄 CDR1, 서열번호 34의 중쇄 CDR2 및 서열번호 35의 중쇄 CDR3을 포함하는 중쇄 가변영역, 및 서열번호 41의 경쇄 CDR1, 서열번호 42의 경쇄 CDR2 및 서열번호 43의 경쇄 CDR3를 포함하는 경쇄 가변영역;(vi) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 33, the heavy chain CDR2 of SEQ ID NO: 34 and the heavy chain CDR3 of SEQ ID NO: 35, and the light chain CDR1 of SEQ ID NO: 41, the light chain CDR2 of SEQ ID NO: 42 and the light chain of SEQ ID NO: 43 a light chain variable region comprising CDR3;
(vii) 서열번호 33의 중쇄 CDR1, 서열번호 45의 중쇄 CDR2 및 서열번호 35의 중쇄 CDR3을 포함하는 중쇄 가변영역, 및 서열번호 46의 경쇄 CDR1, 서열번호 47의 경쇄 CDR2 및 서열번호 38의 경쇄 CDR3를 포함하는 경쇄 가변영역; 또는(vii) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 33, the heavy chain CDR2 of SEQ ID NO: 45 and the heavy chain CDR3 of SEQ ID NO: 35, and the light chain CDR1 of SEQ ID NO: 46, the light chain CDR2 of SEQ ID NO: 47 and the light chain of SEQ ID NO: 38 a light chain variable region comprising CDR3; or
(viii) 서열번호 17의 중쇄 CDR1, 서열번호 18의 중쇄 CDR2 및 서열번호 19의 중쇄 CDR3을 포함하는 중쇄 가변영역, 및 서열번호 79의 경쇄 CDR1, 서열번호 21의 경쇄 CDR2 및 서열번호 22의 경쇄 CDR3를 포함하는 경쇄 가변영역.(viii) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 17, the heavy chain CDR2 of SEQ ID NO: 18 and the heavy chain CDR3 of SEQ ID NO: 19, and the light chain CDR1 of SEQ ID NO: 79, the light chain CDR2 of SEQ ID NO: 21 and the light chain of SEQ ID NO: 22 A light chain variable region comprising CDR3.
"골격 영역(FR)"은 CDR 잔기 이외의 가변 도메인 잔기이다. 각 가변 도메인은 전형적으로, FR1, FR2, FR3 및 FR4의 4개의 FR을 갖는다.A “framework region (FR)” is a variable domain residue other than CDR residues. Each variable domain typically has four FRs: FR1, FR2, FR3 and FR4.
항-TIGIT 항체의 TIGIT에 대한 결합 친화성은 10-5M 내지 10-12M 범위 내에 있다. 예를 들어, 항-TIGIT 항체의 TIGIT에 대한 결합 친화성은 10-6M 내지 10-12M, 10-7M 내지 10-12M, 10-8M 내지 10-12M, 10-9M 내지 10-12M, 10-5M 내지 10-11M, 10-6M 내지 10-11M, 10-7M 내지 10-11M, 10-8M 내지 10-11M, 10-9M 내지 10-11M, 10-10M 내지 10-11M, 10-5M 내지 10-10M, 10-6M 내지 10-10M, 10-7M 내지 10-10M, 10-8M 내지 10-10M, 10-9M 내지 10-10M, 10-5M 내지 10-9M, 10-6M 내지 10-9M, 10-7M 내지 10-9M, 10-8M 내지 10-9M, 10-5M 내지 10-8M, 10-6M 내지 10-8M, 10-7M 내지 10-8M, 10-5M 내지 10-7M, 10-6M 내지 10-7M 또는 10-5M 내지 10-6M이다.The binding affinity of the anti-TIGIT antibody to TIGIT is in the range of 10 -5 M to 10 -12 M. For example, the binding affinity of an anti-TIGIT antibody to TIGIT is 10 -6 M to 10 -12 M, 10 -7 M to 10 -12 M, 10 -8 M to 10 -12 M, 10 -9 M to 10 -12 M, 10 -5 M to 10 -11 M, 10 -6 M to 10 -11 M, 10 -7 M to 10 -11 M, 10 -8 M to 10 -11 M, 10 -9 M to 10 -11 M, 10 -10 M to 10 -11 M, 10 -5 M to 10 -10 M, 10 -6 M to 10 -10 M, 10 -7 M to 10 -10 M, 10 -8 M to 10 -10 M, 10 -9 M to 10 -10 M, 10 -5 M to 10 -9 M, 10 -6 M to 10 -9 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -9 M, 10 -5 M to 10 -8 M, 10 -6 M to 10 -8 M, 10 -7 M to 10 -8 M, 10 -5 M to 10 -7 M, 10 -6 M to 10 -7 M or 10 -5 M to 10 -6 M.
상기 TIGIT에 결합하는 항체 또는 이의 항원 결합단편은 서열번호 7, 15, 23, 31, 39, 48, 50, 52, 54, 56 및 82로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 중쇄 가변영역을 포함할 수 있다. 또한, 상기 TIGIT에 결합하는 항체 또는 이의 항원 결합단편은 서열번호 8, 16, 24, 32, 40, 44, 49, 51, 53, 55, 57, 80, 81, 101, 102 및 103으로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 경쇄 가변영역을 포함할 수 있다.The antibody or antigen-binding fragment thereof binding to TIGIT is a heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 7, 15, 23, 31, 39, 48, 50, 52, 54, 56 and 82 may include In addition, the TIGIT-binding antibody or antigen-binding fragment thereof is a group consisting of SEQ ID NOs: 8, 16, 24, 32, 40, 44, 49, 51, 53, 55, 57, 80, 81, 101, 102 and 103 It may include a light chain variable region comprising one or more amino acid sequences selected from.
본 발명에 따른 구체적 실시예에서, 다음을 포함할 수 있다:In specific embodiments according to the present invention, it may include:
서열번호 7의 중쇄 가변영역 및 서열번호 8의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 7 and a light chain variable region of SEQ ID NO: 8;
서열번호 15의 중쇄 가변영역 및 서열번호 16의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 15 and a light chain variable region of SEQ ID NO: 16;
서열번호 23의 중쇄 가변영역 및 서열번호 24의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 23 and a light chain variable region of SEQ ID NO: 24;
서열번호 31의 중쇄 가변영역 및 서열번호 32의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 31 and a light chain variable region of SEQ ID NO: 32;
서열번호 39의 중쇄 가변영역 및 서열번호 40의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 39 and a light chain variable region of SEQ ID NO: 40;
서열번호 39의 중쇄 가변영역 및 서열번호 44의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 39 and a light chain variable region of SEQ ID NO: 44;
서열번호 48의 중쇄 가변영역 및 서열번호 49의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 48 and a light chain variable region of SEQ ID NO: 49;
서열번호 50의 중쇄 가변영역 및 서열번호 51의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 50 and a light chain variable region of SEQ ID NO: 51;
서열번호 52의 중쇄 가변영역 및 서열번호 53의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO: 53;
서열번호 54의 중쇄 가변영역 및 서열번호 55의 경쇄 가변영역; a heavy chain variable region of SEQ ID NO: 54 and a light chain variable region of SEQ ID NO: 55;
서열번호 56의 중쇄 가변영역 및 서열번호 57의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 56 and a light chain variable region of SEQ ID NO: 57;
서열번호 23의 중쇄 가변영역 및 서열번호 80의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 23 and a light chain variable region of SEQ ID NO: 80;
서열번호 23의 중쇄 가변영역 및 서열번호 81의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 23 and a light chain variable region of SEQ ID NO: 81;
서열번호 82의 중쇄 가변영역 및 서열번호 80의 경쇄 가변영역; a heavy chain variable region of SEQ ID NO: 82 and a light chain variable region of SEQ ID NO: 80;
서열번호 82의 중쇄 가변영역 및 서열번호 81의 경쇄 가변영역; a heavy chain variable region of SEQ ID NO: 82 and a light chain variable region of SEQ ID NO: 81;
서열번호 82의 중쇄 가변영역 및 서열번호 101의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 82 and a light chain variable region of SEQ ID NO: 101;
서열번호 82의 중쇄 가변영역 및 서열번호 102의 경쇄 가변영역; 또는a heavy chain variable region of SEQ ID NO: 82 and a light chain variable region of SEQ ID NO: 102; or
서열번호 82의 중쇄 가변영역 및 서열번호 103의 경쇄 가변영역.The heavy chain variable region of SEQ ID NO: 82 and the light chain variable region of SEQ ID NO: 103.
본 발명은 다른 관점에서, 다음을 포함하는 TIGIT (T cell immunoglobulin and ITIM domain)에 특이적으로 결합하는 항체 또는 이의 항원 결합단편에 관한 것이다:In another aspect, the present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to TIGIT (T cell immunoglobulin and ITIM domain) comprising:
서열번호 83 내지 89로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 중쇄 가변영역; 및a heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 83 to 89; and
서열번호 90 및 91로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 경쇄 가변영역.A light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 90 and 91.
본 발명에 따른 구체적 실시예에서, 다음을 포함할 수 있다:In specific embodiments according to the present invention, it may include:
서열번호 83의 중쇄 가변영역 및 서열번호 53의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 83 and a light chain variable region of SEQ ID NO: 53;
서열번호 84의 중쇄 가변영역 및 서열번호 53의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 84 and a light chain variable region of SEQ ID NO: 53;
서열번호 85의 중쇄 가변영역 및 서열번호 53의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 85 and a light chain variable region of SEQ ID NO: 53;
서열번호 86의 중쇄 가변영역 및 서열번호 53의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 86 and a light chain variable region of SEQ ID NO: 53;
서열번호 52의 중쇄 가변영역 및 서열번호 90의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO: 90;
서열번호 52의 중쇄 가변영역 및 서열번호 91의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO: 91;
서열번호 87의 중쇄 가변영역 및 서열번호 53의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 87 and a light chain variable region of SEQ ID NO: 53;
서열번호 88의 중쇄 가변영역 및 서열번호 53의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 88 and a light chain variable region of SEQ ID NO: 53;
서열번호 89의 중쇄 가변영역 및 서열번호 53의 경쇄 가변영역; 또는a heavy chain variable region of SEQ ID NO: 89 and a light chain variable region of SEQ ID NO: 53; or
서열번호 84의 중쇄 가변영역 및 서열번호 91의 경쇄 가변영역.The heavy chain variable region of SEQ ID NO: 84 and the light chain variable region of SEQ ID NO: 91.
본 발명에 따른 구체적 실시예에서, 본 발명의 항체 또는 이의 항원 결합 단편은 단쇄 Fvs (scFv), 단쇄 항체, Fab, F(ab'), 다이설파이드-결합 Fvs (sdFv)을 포함할 수 있다. In a specific embodiment according to the present invention, the antibody or antigen-binding fragment thereof of the present invention may include single chain Fvs (scFv), single chain antibody, Fab, F(ab'), disulfide-bonded Fvs (sdFv).
scFv는 항체의 VH 및 VL 도메인을 포함하는 단일 폴리펩티드 쇄로 이루어진 구조체로, 항체 단편이다. An scFv is an antibody fragment, which is a construct consisting of a single polypeptide chain comprising the VH and VL domains of an antibody.
본 발명에 따른 구체적 실시예에서, 상기 scFv는 중쇄 가변영역과 경쇄 가변영역이 링커를 통해 연결된 것일 수 있으며, 바람직하게는 폴리펩티드 링커로 VH 도메인과 VL 도메인 사이가 연결된 것일 수 있다.In a specific embodiment according to the present invention, the scFv may have a heavy chain variable region and a light chain variable region linked through a linker, preferably a polypeptide linker between the VH domain and the VL domain.
상기 링커는 펩타이드 링커일 수 있으며, 약 10-25 aa 길이를 가질 수 있다. 예를 들어, 글리신 및/또는 세린과 같은 친수성 아미노산이 포함될 수 있으나, 이에 제한되는 것은 아니다.The linker may be a peptide linker and may have a length of about 10-25 aa. For example, hydrophilic amino acids such as glycine and/or serine may be included, but are not limited thereto.
구체적으로, 상기 링커는 예를 들어, (GS)n, (GGS)n, (GSGGS)n 또는 (GnS)m (n, m은 각각 1 내지 10)을 포함할 수 있으나, 상기 링커는 예를 들어 (GnS)m (n, m은 각각 1 내지 10)일 수 있다. 구체적으로, 상기 링커는 GGGGS를 포함할 수 있다. Specifically, the linker may include, for example, (GS) n , (GGS) n , (GSGGS) n or (G n S) m (n and m are each 1 to 10), but the linker is For example, it may be (G n S) m (n and m are each 1 to 10). Specifically, the linker may include GGGGS.
"파지 디스플레이"는 변이체 폴리펩티드를 파지, 예를 들어 섬유상 파지 입자의 표면 상에 외피 단백질의 적어도 일부와의 융합 단백질로서 디스플레이하는 기술이다. 파지 디스플레이의 유용성은 무작위화 단백질 변이체의 큰 라이브러리를 대상으로 하여, 표적 항원과 고친화도로 결합하는 서열을 신속하고도 효율적으로 분류할 수 있다는 사실에 있다. 펩티드 및 단백질 라이브러리를 파지 상에 디스플레이하는 것은 특이적 결합 특성을 지닌 폴리펩티드를 알아보기 위해 수 백만개의 폴리펩티드를 스크리닝하는데 사용되어 왔다."Phage display" is a technique for displaying a variant polypeptide as a fusion protein with at least a portion of an envelope protein on the surface of a phage, eg, a filamentous phage particle. The usefulness of phage display resides in the fact that, by targeting a large library of randomized protein variants, it is possible to quickly and efficiently sort sequences that bind to a target antigen with high affinity. Displaying peptide and protein libraries on phage has been used to screen millions of polypeptides for polypeptides with specific binding properties.
파지 디스플레이 기술은 특정 리간드(예: 항원)와 결합하는 신규 단백질을 생성 및 선별하기 위한 강력한 도구를 제공하였다. 파지 디스플레이 기술을 사용하여, 단백질 변이체의 큰 라이브러리를 생성시키고, 표적 항원과 고 친화성으로 결합하는 서열을 신속하게 분류할 수 있다. 변이체 폴리펩티드를 암호화하는 핵산을 바이러스성 외피 단백질, 예를 들어 유전자 III 단백질 또는 유전자 VIII 단백질을 암호화하는 핵산 서열과 융합시킨다. 단백질 또는 폴리펩티드를 암호화하는 핵산 서열을 유전자 III 단백질의 일부를 암호화하는 핵산 서열과 융합시킨 1가 파지 디스플레이 시스템이 개발되었다. 1가 파지 디스플레이 시스템에서는, 유전자 융합물이 저수준으로 발현되고 야생형 유전자 III 단백질이 또한 발현되어 입자 감염성이 유지된다.Phage display technology has provided a powerful tool for generating and screening novel proteins that bind specific ligands (eg antigens). Phage display technology can be used to generate large libraries of protein variants and rapidly sort sequences that bind target antigens with high affinity. A nucleic acid encoding a variant polypeptide is fused with a nucleic acid sequence encoding a viral envelope protein, such as a gene III protein or a gene VIII protein. A monovalent phage display system has been developed in which a nucleic acid sequence encoding a protein or polypeptide is fused with a nucleic acid sequence encoding a portion of a gene III protein. In a monovalent phage display system, the gene fusion is expressed at low levels and the wild-type gene III protein is also expressed to maintain particle infectivity.
섬유상 파지 표면 상에서의 펩티드의 발현과 E. coli의 주변세포질에서의 기능성 항체 단편의 발현을 입증하는 것이 항체 파지 디스플레이 라이브러리를 개발하는 데에 있어 중요하다. 항체 또는 항원 결합성 폴리펩티드의 라이브러리는 수 많은 방식, 예를 들어 무작위 DNA 서열을 삽입함으로써 단일 유전자를 변경시키는 방법 또는 관련 유전자 계열을 클로닝하는 방법으로 제조하였다. 라이브러리를 대상으로 하여, 목적하는 특징을 수반한 항체 또는 항원 결합성 단백질의 발현에 관하여 스크리닝할 수 있다.Proving the expression of peptides on the surface of filamentous phage and functional antibody fragments in the periplasm of E. coli is important for developing antibody phage display libraries. Libraries of antibodies or antigen-binding polypeptides have been prepared in a number of ways, for example, by altering a single gene by inserting a random DNA sequence or by cloning a related gene family. The library can be screened for expression of an antibody or antigen-binding protein accompanied by a desired characteristic.
파지 디스플레이 기술은 목적하는 특징을 지닌 항체를 제조하기 위한 통상적인 하이브리도마 및 재조합 방법에 비해 몇 가지 이점을 지니고 있다. 이러한 기술은 동물을 사용하지 않고서도 짧은 시간에 다양한 서열을 지닌 큰 항체 라이브러리를 생성시킬 수 있도록 한다. 하이브리도마의 제조나 인간화 항체의 제조는 수 개월의 제조기간을 필요로 할 수 있다. 또한, 면역이 전혀 요구되지 않기 때문에, 파지 항체 라이브러리는 독성이거나 항원성이 낮은 항원에 대해서도 항체를 생성시킬 수 있다. 파지 항체 라이브러리를 또한 사용하여 신규한 치료적 항체를 생성 및 확인할 수 있다.Phage display technology has several advantages over conventional hybridoma and recombinant methods for producing antibodies with desired characteristics. This technology allows the generation of large antibody libraries with various sequences in a short time without using animals. Preparation of hybridomas or humanized antibodies may require a production period of several months. In addition, since no immunization is required, the phage antibody library can generate antibodies against antigens that are toxic or of low antigenicity. Phage antibody libraries can also be used to generate and identify novel therapeutic antibodies.
파지 디스플레이 라이브러리를 사용하여 면역시킨, 비-면역시킨 인간, 생식세포계 서열 또는 미감작 B 세포 Ig 레퍼토리 (repertory)로부터 인간 항체를 생성시키는 기술을 사용할 수 있다. 각종 림프계 조직을 사용하여, 미감작 또는 비면역 항원 결합성 라이브러리를 제조할 수 있다. Techniques for generating human antibodies from immunized, non-immunized human, germline sequences or naïve B cell Ig repertoires using phage display libraries can be used. A variety of lymphoid tissues can be used to prepare naïve or non-immune antigen-binding libraries.
파지 디스플레이 라이브러리로부터 고친화성 항체를 확인 및 분리할 수 있는 기술은 치료용 신규 항체 분리에 중요하다. 라이브러리로부터 고친화성 항체를 분리하는 것은 라이브러리의 크기, 세균성 세포 중에서의 생산 효율 및 라이브러리의 다양성에 좌우될 수 있다. 라이브러리의 크기는 항체 또는 항원 결합성 단백질의 부적절한 폴딩과 정지 코돈의 존재로 인한 비효율적 생산에 의해 감소된다. 세균성 세포에서의 발현은 항체 또는 항원 결합성 도메인이 적절하게 폴딩되지 않는 경우에는 억제될 수 있다. 발현은 가변/불변 계면의 표면이나 선별된 CDR 잔기에서의 잔기를 교대로 돌연변이 시킴으로써 개선시킬 수 있다. 골격 영역의 서열은 세균성 세포에서 항체 파지 라이브러리를 생성시키는 경우에 적절한 폴딩을 제공하기 위한 하나의 요소이다.A technology capable of identifying and isolating high-affinity antibodies from phage display libraries is important for isolating novel therapeutic antibodies. Isolation of high affinity antibodies from a library may depend on the size of the library, production efficiency in bacterial cells, and diversity of the library. The size of the library is reduced by improper folding of the antibody or antigen-binding protein and inefficient production due to the presence of stop codons. Expression in bacterial cells can be inhibited if the antibody or antigen binding domain does not fold properly. Expression can be improved by alternately mutating residues on the surface of the variable/constant interface or at selected CDR residues. The sequence of the framework region is one element to provide for proper folding when generating antibody phage libraries in bacterial cells.
고친화성 항체 분리에서 항체 또는 항원 결합성 단백질의 다양한 라이브러리를 생성시키는 것이 중요하다. CDR3 영역은 이들이 종종 항원 결합에 참여하는 것으로 밝혀졌다. 중쇄 상의 CDR3 영역은 크기, 서열 및 구조적 입체 형태 면에서 상당히 다양하므로, 이를 이용하여 다양한 라이브러리를 제조할 수 있다.In high-affinity antibody isolation, it is important to generate diverse libraries of antibodies or antigen-binding proteins. The CDR3 regions have been found to often participate in antigen binding. Since the CDR3 regions on the heavy chain vary considerably in size, sequence and structural conformation, they can be used to prepare a variety of libraries.
또한, 각 위치에서 20개 아미노산 모두를 사용하여 가변 중쇄 및 경쇄의 CDR 영역을 무작위화함으로써 다양성을 발생시킬 수 있다. 20개의 모든 아미노산을 사용하면 다양성이 큰 변이체 항체 서열이 생성되고 신규한 항체를 확인할 기회가 증가할 수 있다.In addition, diversity can be generated by randomizing the CDR regions of the variable heavy and light chains using all 20 amino acids at each position. The use of all 20 amino acids can result in highly diverse variant antibody sequences and increase the chances of identifying novel antibodies.
본 발명의 항체 또는 항체 단편은 TIGIT을 특이적으로 인식할 수 있는 범위 내에서, 본 명세서에 기재된 본 발명의 항-TIGIT 항체의 서열뿐만 아니라, 이의 생물학적 균등물도 포함할 수 있다. 예를 들면, 항체의 결합 친화도 및/또는 기타 생물학적 특성을 보다 더 개선시키기 위하여 항체의 아미노산 서열에 추가적인 변화를 줄 수 있다. 이러한 변형은 예를 들어, 항체의 아미노산 서열 잔기의 결실, 삽입 및/또는 치환을 포함한다. 이러한 아미노산 변이는 아미노산 곁사슬 치환체의 상대적 유사성, 예컨대, 소수성, 친수성, 전하, 크기 등에 기초하여 이루어진다. 아미노산 곁사슬 치환체의 크기, 모양 및 종류에 대한 분석에 의하여, 아르기닌, 라이신과 히스티딘은 모두 양전하를 띤 잔기이고; 알라닌, 글라이신과 세린은 유사한 크기를 가지며; 페닐알라닌, 트립토판과 타이로신은 유사한 모양을 갖는다는 것을 알 수 있다. 따라서, 이러한 고려 사항에 기초하여, 아르기닌, 라이신과 히스티딘; 알라닌, 글라이신과 세린; 그리고 페닐알라닌, 트립토판과 타이로신은 생물학적으로 기능 균등물이라 할 수 있다.The antibody or antibody fragment of the present invention may include not only the sequence of the anti-TIGIT antibody of the present invention described herein, but also a biological equivalent thereof to the extent that it can specifically recognize TIGIT. For example, additional changes may be made to the amino acid sequence of an antibody to further improve its binding affinity and/or other biological properties. Such modifications include, for example, deletions, insertions and/or substitutions of amino acid sequence residues of the antibody. Such amino acid variations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. According to the analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine can be said to be biologically functional equivalents.
상술한 생물학적 균등 활성을 갖는 변이를 고려한다면, 본 발명의 항체 또는 이를 코딩하는 핵산 분자는 서열번호에 기재된 서열과 실질적인 동일성(substantial identity)을 나타내는 서열도 포함하는 것으로 해석된다. 상기의 실질적인 동일성은, 상기한 본 발명의 서열과 임의의 다른 서열을 최대한 대응되도록 얼라인하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인된 서열을 분석한 경우에, 최소 90%의 상동성, 가장 바람직하게는 최소 95%의 상동성, 96% 이상, 97% 이상, 98% 이상, 99% 이상의 상동성을 나타내는 서열을 의미한다. 서열비교를 위한 얼라인먼트 방법은 당업계에 공지되어 있다. NCBI Basic Local Alignment Search Tool(BLAST)은 NBCI 등에서 접근 가능하며, 인터넷 상에서 blastp, blasm, blastx, tblastn 및 tblastx와 같은 서열 분석 프로그램과 연동되어 이용할 수 있다. BLAST는 www.ncbi.nlm.nih.gov/BLAST/에서 접속 가능하다. 이 프로그램을 이용한 서열 상동성 비교 방법은 www.ncbi.nlm.nih.gov/BLAST/blast_ help.html에서 확인할 수 있다.Considering the above-described mutations having the biological equivalent activity, the antibody or nucleic acid molecule encoding the same of the present invention is interpreted to include a sequence showing substantial identity to the sequence set forth in SEQ ID NO:. The substantial identity is at least 90% when the sequence of the present invention and any other sequences are aligned as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. refers to a sequence exhibiting homology, most preferably at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, at least 99% homology. Alignment methods for sequence comparison are known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) can be accessed from NBCI, etc. BLAST can be accessed at www.ncbi.nlm.nih.gov/BLAST/. A method for comparing sequence homology using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.
이에 기초하여, 본 발명의 항체 또는 그의 항원 결합 단편은 명세서에 기재된 명시된 서열 또는 전체와 비교하여 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% 또는 그 이상의 상동성을 가질 수 있다. 이러한 상동성은 당업계에 공지된 방법에 의한 서열 비교 및/또는 정렬에 의해 결정될 수 있다. 예를 들어, 서열 비교 알고리즘(즉, BLAST 또는 BLAST 2.0), 수동 정렬, 육안 검사를 이용하여 본 발명의 핵산 또는 단백질의 퍼센트 서열 상동성을 결정할 수 있다. Based thereon, the antibody or antigen-binding fragment thereof of the present invention is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% compared to the specified sequence or all of the sequences described in the specification. , 99% or more homology. Such homology can be determined by sequence comparison and/or alignment by methods known in the art. For example, a sequence comparison algorithm (ie, BLAST or BLAST 2.0), manual alignment, or visual inspection can be used to determine the percent sequence homology of a nucleic acid or protein of the invention.
본 발명은 다른 관점에서, 상기 항체 또는 그의 항원 결합 단편을 코딩하는 핵산에 관한 것이다. 본 발명의 항체 또는 그의 항원 결합 단편을 코딩하는 핵산을 분리하여 항체 또는 그의 항원 결합 단편을 재조합적으로 생산할 수 있다. In another aspect, the present invention relates to a nucleic acid encoding the antibody or antigen-binding fragment thereof. An antibody or antigen-binding fragment thereof can be recombinantly produced by isolating a nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention.
"핵산"은 DNA(gDNA 및 cDNA) 및 RNA 분자를 포괄적으로 포함하는 의미를 가지며, 핵산에서 기본 구성단위인 뉴클레오타이드는 자연의 뉴클레오타이드 뿐만 아니라, 당 또는 염기 부위가 변형된 유사체(analogue)도 포함한다. 본 발명의 중쇄 및 경쇄 가변영역을 코딩하는 핵산의 서열은 변형될 수 있다. 상기 변형은 뉴클레오타이드의 추가, 결실 또는 비보존적 치환 또는 보존적 치환을 포함한다."Nucleic acid" has a meaning comprehensively encompassing DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acids, include natural nucleotides as well as analogues in which sugar or base regions are modified. . The sequences of the nucleic acids encoding the heavy and light chain variable regions of the present invention may be modified. Such modifications include additions, deletions or non-conservative substitutions or conservative substitutions of nucleotides.
본 발명에 따른 구체적인 실시예에서, 상기 TIGIT에 결합하는 항체 또는 이의 항원 결합단편을 코딩하는 핵산은 서열번호 58 내지 78로 구성된 군에서 선택된 중쇄 가변영역 또는 경쇄 가변영역을 코딩하는 핵산을 포함할 수 있다. In a specific embodiment according to the present invention, the nucleic acid encoding the TIGIT-binding antibody or antigen-binding fragment thereof may include a nucleic acid encoding a heavy chain variable region or a light chain variable region selected from the group consisting of SEQ ID NOs: 58 to 78. have.
본 발명에 따른 구체적 실시예에서, 다음을 포함할 수 있다:In specific embodiments according to the present invention, it may include:
서열번호 58의 중쇄 가변영역 코딩 핵산 및 서열번호 59의 경쇄 가변영역 코딩 핵산;a nucleic acid encoding a heavy chain variable region of SEQ ID NO: 58 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 59;
서열번호 60의 중쇄 가변영역 코딩 핵산 및 서열번호 61의 경쇄 가변영역 코딩 핵산;a nucleic acid encoding a heavy chain variable region of SEQ ID NO: 60 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 61;
서열번호 62의 중쇄 가변영역 코딩 핵산 및 서열번호 63의 경쇄 가변영역 코딩 핵산;a nucleic acid encoding a heavy chain variable region of SEQ ID NO: 62 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 63;
서열번호 64의 중쇄 가변영역 코딩 핵산 및 서열번호 65의 경쇄 가변영역 코딩 핵산;a nucleic acid encoding a heavy chain variable region of SEQ ID NO: 64 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 65;
서열번호 66의 중쇄 가변영역 코딩 핵산 및 서열번호 67의 경쇄 가변영역 코딩 핵산;a nucleic acid encoding a heavy chain variable region of SEQ ID NO: 66 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 67;
서열번호 66의 중쇄 가변영역 코딩 핵산 및 서열번호 68의 경쇄 가변영역 코딩 핵산;a nucleic acid encoding a heavy chain variable region of SEQ ID NO: 66 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 68;
서열번호 69의 중쇄 가변영역 코딩 핵산 및 서열번호 70의 경쇄 가변영역 코딩 핵산;a nucleic acid encoding a heavy chain variable region of SEQ ID NO: 69 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 70;
서열번호 71의 중쇄 가변영역 코딩 핵산 및 서열번호 72의 경쇄 가변영역 코딩 핵산;a nucleic acid encoding a heavy chain variable region of SEQ ID NO: 71 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 72;
서열번호 73의 중쇄 가변영역 코딩 핵산 및 서열번호 74의 경쇄 가변영역 코딩 핵산;a nucleic acid encoding a heavy chain variable region of SEQ ID NO: 73 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 74;
서열번호 75의 중쇄 가변영역 코딩 핵산 및 서열번호 76의 경쇄 가변영역 코딩 핵산; 또는a nucleic acid encoding a heavy chain variable region of SEQ ID NO: 75 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 76; or
서열번호 77의 중쇄 가변영역 코딩 핵산 및 서열번호 78의 경쇄 가변영역 코딩 핵산.A nucleic acid encoding a heavy chain variable region of SEQ ID NO: 77 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 78.
본 발명에 따른 다른 구체적 실시예에서, 상기 TIGIT에 결합하는 항체 또는 이의 항원 결합단편을 코딩하는 핵산은 서열번호 64, 92 내지 98로 구성된 군에서 선택된 중쇄 가변영역; 또는 서열번호 65, 99 및 100으로 구성된 군에서 선택된 경쇄 가변영역을 코딩하는 핵산을 포함할 수 있다. In another specific embodiment according to the present invention, the nucleic acid encoding the antibody or antigen-binding fragment thereof binding to TIGIT comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs: 64, 92 to 98; Or it may include a nucleic acid encoding a light chain variable region selected from the group consisting of SEQ ID NOs: 65, 99 and 100.
본 발명에 따른 구체적 실시예에서, 다음을 포함할 수 있다:In specific embodiments according to the present invention, it may include:
서열번호 92의 중쇄 가변영역 코딩 핵산 및 서열번호 65의 경쇄 가변영역 코딩 핵산;a nucleic acid encoding a heavy chain variable region of SEQ ID NO: 92 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 65;
서열번호 93의 중쇄 가변영역 코딩 핵산 및 서열번호 65의 경쇄 가변영역 코딩 핵산;a nucleic acid encoding a heavy chain variable region of SEQ ID NO: 93 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 65;
서열번호 94의 중쇄 가변영역 코딩 핵산 및 서열번호 65의 경쇄 가변영역 코딩 핵산;a nucleic acid encoding a heavy chain variable region of SEQ ID NO: 94 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 65;
서열번호 95의 중쇄 가변영역 코딩 핵산 및 서열번호 65의 경쇄 가변영역 코딩 핵산;a nucleic acid encoding a heavy chain variable region of SEQ ID NO: 95 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 65;
서열번호 64의 중쇄 가변영역 코딩 핵산 및 서열번호 99의 경쇄 가변영역 코딩 핵산;a nucleic acid encoding a heavy chain variable region of SEQ ID NO: 64 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 99;
서열번호 64의 중쇄 가변영역 코딩 핵산 및 서열번호 100의 경쇄 가변영역 코딩 핵산;a nucleic acid encoding a heavy chain variable region of SEQ ID NO: 64 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 100;
서열번호 96의 중쇄 가변영역 코딩 핵산 및 서열번호 65의 경쇄 가변영역 코딩 핵산;a nucleic acid encoding a heavy chain variable region of SEQ ID NO: 96 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 65;
서열번호 97의 중쇄 가변영역 코딩 핵산 및 서열번호 65의 경쇄 가변영역 코딩 핵산;a nucleic acid encoding a heavy chain variable region of SEQ ID NO: 97 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 65;
서열번호 98의 중쇄 가변영역 코딩 핵산 및 서열번호 65의 경쇄 가변영역 코딩 핵산; 또는a nucleic acid encoding a heavy chain variable region of SEQ ID NO: 98 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 65; or
서열번호 93의 중쇄 가변영역 코딩 핵산 및 서열번호 100의 경쇄 가변영역 코딩 핵산.A nucleic acid encoding a heavy chain variable region of SEQ ID NO: 93 and a nucleic acid encoding a light chain variable region of SEQ ID NO: 100.
상기 항체를 암호화하는 DNA는 통상적인 분자생물학적 수법을 사용하여 (예를 들어, 항체와 중쇄와 경쇄를 암호화하는 DNA와 특이적으로 결합할 수 있는 올리코뉴클레오타이드 프로브를 사용함으로써) 용이하게 분리 또는 합성할 수 있으며, 핵산을 분리하고, 이를 복제 가능한 벡터 내로 삽입하여 추가로 클로닝하거나(DNA의 증폭) 또는 추가로 발현시킨다. 이를 바탕으로, 본 발명은 또 다른 관점에서 상기 핵산을 포함하는 재조합 발현 벡터에 관한 것이다.DNA encoding the antibody is easily isolated or synthesized using conventional molecular biological techniques (eg, by using an oligonucleotide probe capable of specifically binding to DNA encoding the antibody and the heavy and light chains) Alternatively, the nucleic acid is isolated and inserted into a replicable vector for further cloning (amplification of DNA) or further expression. Based on this, the present invention relates to a recombinant expression vector comprising the nucleic acid from another aspect.
본 명세서에서 사용되는 용어, "벡터"는 숙주세포에서 목적 유전자를 발현시키기 위한 수단으로, 플라스미드 벡터, 코즈미드 벡터, 박테리오파지 벡터, 아데노바이러스 벡터, 레트로바이러스 벡터, 아데노-연관 바이러스 벡터와 같은 바이러스 벡터 등을 포함한다. 벡터의 성분으로는 일반적으로 다음 중의 하나 이상이 포함되지만, 그에 제한되지 않는다: 신호 서열, 복제 기점, 하나 이상의 항생제 내성 마커 유전자, 증강인자 요소, 프로모터, 전사 종결 서열. 항체를 코딩하는 핵산은 프로모터 및 전사 종결 서열 등과 같이 작동적으로 연결되어 있다.As used herein, the term "vector" is a means for expressing a target gene in a host cell, and a viral vector such as a plasmid vector, a cosmid vector, a bacteriophage vector, an adenoviral vector, a retroviral vector, and an adeno-associated viral vector. etc. Components of a vector generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more antibiotic resistance marker genes, an enhancer element, a promoter, a transcription termination sequence. Nucleic acids encoding antibodies are operatively linked, such as promoters and transcription termination sequences.
"작동적으로 연결"은 핵산 발현조절서열(예: 프로모터, 시그널 서열 또는 전사조절인자 결합 위치의 어레이)과 다른 핵산 서열사이의 기능적인 결합을 의미하며, 이에 따라 상기 조절서열은 상기 다른 핵산 서열의 전사 및/또는 해독을 조절하게 된다."Operably linked" refers to a functional association between a nucleic acid expression control sequence (eg, an array of promoter, signal sequence or transcriptional regulator binding sites) and another nucleic acid sequence, such that the control sequence is the other nucleic acid sequence to regulate transcription and/or translation of
원핵세포를 숙주로 하는 경우에는, 전사를 진행시킬 수 있는 강력한 프로모터(예컨대, tac 프로모터, lac 프로모터, lacUV5 프로모터, lpp 프로모터, pLλ프로모터, pRλ프로모터, rac5 프로모터, amp 프로모터, recA 프로모터, SP6 프로모터, trp 프로모터 및 T7 프로모터 등), 해독의 개시를 위한 라이보좀 결합 자리 및 전사/해독 종결 서열을 포함하는 것이 일반적이다. 또한, 예를 들어, 진핵 세포를 숙주로 하는 경우에는, 포유동물 세포의 지놈으로부터 유래된 프로모터(예: 메탈로티오닌 프로모터, β액틴 프로모터, 사람 헤로글로빈 프로모터 및 사람 근육 크레아틴 프로모터) 또는 포유동물 바이러스로부터 유래된 프로모터(예: 아데노바이러스 후기 프로모터, 백시니아 바이러스 7.5K 프로모터, SV40프로모터, 사이토메갈로바이러스(CMV) 프로모터, HSV의 tk 프로모터, 마우스 유방종양 바이러스(MMTV) 프로모터, HIV의 LTR 프로모터, 몰로니 바이러스의 프로모터엡스타인바 바이러스(EBV)의 프로모터 및 로우스 사코마 바이러스(RSV)의 프로모터)가 이용될 수 있으며, 전사 종결 서열로서 폴리아데닐화 서열을 일반적으로 갖는다.When using a prokaryotic cell as a host, a strong promoter capable of propagating transcription (eg, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter), a ribosome binding site for initiation of translation, and a transcription/translation termination sequence. Further, for example, when a eukaryotic cell is used as a host, a promoter derived from the genome of a mammalian cell (eg, metallotionine promoter, β-actin promoter, human hegglobin promoter, and human muscle creatine promoter) or mammalian Promoters derived from viruses (eg, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, Moloney virus promoter Epstein Barr virus (EBV) promoter and Loose Sacoma virus (RSV) promoter) can be used, and generally has a polyadenylation sequence as a transcription termination sequence.
경우에 따라서, 벡터는 그로부터 발현되는 항체의 정제를 용이하게 하기 위하여 다른 서열과 융합될 수도 있다. 융합되는 서열은, 예컨대 글루타티온 S-트랜스퍼라제(Pharmacia, USA), 말토스 결합 단백질(NEB, USA), FLAG(IBI, USA) 및 6x His(hexahistidine; Quiagen, USA) 등이 있다.Optionally, the vector may be fused with other sequences to facilitate purification of the antibody expressed therefrom. The sequence to be fused includes, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA).
상기 벡터는 선택표지로서 당업계에서 통상적으로 이용되는 항생제 내성 유전자를 포함하며, 예를 들어 암피실린, 겐타마이신, 카베니실린, 클로람페니콜, 스트렙토마이신, 카나마이신, 게네티신, 네오마이신 및 테트라사이클린에 대한 내성 유전자가 있다.The vector contains an antibiotic resistance gene commonly used in the art as a selection marker, and for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline. There is a resistance gene.
본 발명은 또 다른 관점에서, 상기 재조합 발현 벡터로 형질감염된 숙주세포에 관한 것이다. 본 발명의 항체를 생성시키기 위해 사용된 숙주세포는 원핵생물, 효모 또는 고등 진핵생물 세포일 수 있으며, 이에 제한되는 것은 아니다. In another aspect, the present invention relates to a host cell transfected with the recombinant expression vector. The host cell used to produce the antibody of the present invention may be, but is not limited to, a prokaryotic, yeast or higher eukaryotic cell.
에스케리치아 콜라이(Escherichia coli), 바실러스 서브틸리스(Bacillus subtilus) 및 바실러스 투링기엔시스(Bacillus thuringiensis)와 같은 바실러스 속 균주, 스트렙토마이세스(Streptomyces), 슈도모나스(Pseudomonas)(예를 들면, 슈도모나스 푸티다(Pseudomonas putida)), 프로테우스미라빌리스(Proteus mirabilis) 및 스타필로코쿠스(Staphylococcus)(예를 들면, 스타필로코쿠스 카르노수스(Staphylocus carnosus))와 같은 원핵 숙주세포를 이용할 수 있다.Escherichia coli ( Escherichia coli ), Bacillus subtilus ( Bacillus subtilus ) and Bacillus thuringiensis , such as Bacillus genus strains, Streptomyces , Pseudomonas ( Pseudomonas ) (for example, Pseudomonas putida ), Proteus mirabilis ) and Staphylococcus ) (eg, Staphylocus carnosus ), such as prokaryotic host cells can be used.
다만, 동물 세포에 대한 관심이 가장 크며, 유용한 숙주 세포주의 예는 COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO/-DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2/0, NS-0, U20S 또는 HT1080일 수 있으나, 이에 제한되는 것은 아니다.However, animal cells are of greatest interest, and examples of useful host cell lines include COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO/-DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2/0, NS-0 , but may be U20S or HT1080, but is not limited thereto.
본 발명은 또 다른 관점에서, 상기 숙주세포를 배양하여 항체를 생성하는 단계; 및 생성된 항체를 분리 및 정제하는 단계를 포함하는, TIGIT에 특이적으로 결합하는 항체 또는 이의 항원 결합단편의 제조방법에 관한 것이다. In another aspect, the present invention comprises the steps of culturing the host cell to generate an antibody; And it relates to a method for producing an antibody or antigen-binding fragment thereof that specifically binds to TIGIT, comprising the step of isolating and purifying the produced antibody.
상기 숙주세포는 각종 배지에서 배양할 수 있다. 시판용 배지 중 제한없이 배양 배지로서 사용할 수 있다. 당업자에게 공지되어 있는 기타 모든 필수 보충물이 적당한 농도로 포함될 수도 있다. 배양 조건, 예를 들어 온도, pH 등이 발현을 위해 선별된 숙주세포와 함께 이미 사용되고 있고, 이는 당업자에게 명백할 것이다.The host cells may be cultured in various media. Among commercially available media, it can be used as a culture medium without limitation. All other essential supplements known to those skilled in the art may be included in appropriate concentrations. Culture conditions, such as temperature, pH, etc., are already used with the host cells selected for expression and will be apparent to those skilled in the art.
상기 항체 또는 그의 항원 결합 단편의 회수는 예를 들어 원심분리 또는 한외여과에 의해 불순물을 제거하고, 그 결과물을 예를 들어 친화 크로마토그래피 등을 이용하여 정제할 수 있다. 추가의 기타 정제 기술 예를 들어 음이온 또는 양이온 교환 크로마토그래피, 소수성 상호 작용 크로마토그래피, 히드록실아파타이트 크로마토그래피 등이 사용될 수 있다.For the recovery of the antibody or antigen-binding fragment thereof, impurities may be removed by, for example, centrifugation or ultrafiltration, and the resultant product may be purified using, for example, affinity chromatography. Additional other purification techniques may be used, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, and the like.
이중 또는 다중특이적 항체bispecific or multispecific antibody
본 발명은 다른 관점에서, 상기 항체 또는 이의 항원 결합단편을 포함하는 이중 또는 다중특이적 항체에 관한 것이다. In another aspect, the present invention relates to a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof.
이중특이적 항체는 하나 이상의 타겟에 결합능 또는 길항능을 가지는 항체를 의미하며, 2개의 서로 다른 타겟에 대한 결합능 또는 길항능을 가지는 항체가 결합된 형태 또는 한 타겟에 대한 결합능을 가지는 항체와 다른 타겟에 대한 길항능을 가지는 물질이 결합되어 있는 항체를 의미한다.Bispecific antibody refers to an antibody having binding ability or antagonistic ability to one or more targets, and a form in which an antibody having binding ability or antagonistic ability to two different targets is bound, or an antibody having binding ability to one target and a different target It refers to an antibody to which a substance having antagonistic ability is bound.
다중특이적 항체는 적어도 3 이상의 상이한 항원에 대해 결합 특이성을 가지는 항체를 의미한다. 다중특이적(multi-specific) 항체는 삼중특이적(Tri-specific) 이상의 항체, 예를 들어 삼중특이적(Tri-specific) 항체, 사중특이적(Tetra-specific) 항체 또는 그 이상의 타겟을 표적하는 항체를 포함할 수 있다. A multispecific antibody refers to an antibody having binding specificities for at least three different antigens. A multi-specific antibody is a tri-specific antibody or more, for example, a tri-specific antibody, a tetra-specific antibody, or a target that targets more. may include antibodies.
이중특이적 또는 다중특이적 항체에 속하는 항체들은 scFv 기반 항체, Fab 기반 항체 및 IgG 기반 항체 등으로 구분할 수 있다. 이중특이적 또는 다중특이적 항체의 경우 두 개 이상의 신호를 동시에 억제 또는 증폭시킬 수 있기 때문에 하나의 신호를 억제/증폭하는 경우보다 더욱 효과적일 수 있으며, 각각의 신호를 각각의 신호억제제로 처리했을 경우와 비교하면, 저용량 투약이 가능하며, 동일한 시간 및 공간에서의 두 개 이상의 신호를 억제/증폭시킬 수 있다.Antibodies belonging to bispecific or multispecific antibodies may be classified into scFv-based antibodies, Fab-based antibodies, and IgG-based antibodies. In the case of a bispecific or multispecific antibody, since two or more signals can be simultaneously inhibited or amplified, it can be more effective than when one signal is inhibited/amplified, and each signal must be treated with a respective signal inhibitor. Compared with the case, low dose administration is possible, and it is possible to suppress/amplify two or more signals in the same time and space.
이중특이적 또는 다중특이적 항체의 제조 방법은 널리 공지되어 있다. 전통적으로, 이중특이적 항체의 재조합 생산은 두 개 이상의 중쇄가 상이한 특이성을 가지는 조건에서 두 개 이상의 면역글로불린 중쇄/경쇄 쌍의 공동 발현을 근간으로 한다.Methods for making bispecific or multispecific antibodies are well known. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two or more immunoglobulin heavy/light chain pairs under conditions in which the two or more heavy chains have different specificities.
scFv를 기반으로 하는 이중특이적 또는 다중특이적 항체의 경우, 상이한 scFv들의 VL과 VH를 각기 서로 조합하여 혼성 scFv를 heterodimeric 형태로 제조하여 디아바디(diabody)를 만들 수 있고, 상이한 scFv를 서로 연결해서 tendem ScFv를 제조할 수 있으며, Fab의 CH1과 CL을 각각의 scFv의 말단에 발현시켜 heterodimeric 미니항체(miniantibody)를 제조할 수 있고, Fc의 homodimeric 도메인인 CH3 도메인의 일부 아미노산을 치환하여 'knob into hole' 형태의 heterodimeric 구조로 변경시켜, 이들 변경된 CH3 도메인을 상이한 각각의 scFv 말단에 발현시킴으로써 heterodimeric scFv 형태의 미니바디(minibody)를 제조할 수 있다.In the case of an scFv-based bispecific or multispecific antibody, a diabody can be made by preparing a hybrid scFv in a heterodimeric form by combining the VL and VH of different scFvs, and linking different scFvs with each other Thus, tendem ScFv can be prepared, and heterodimeric miniantibodies can be prepared by expressing CH1 and CL of Fab at the ends of each scFv. By changing to a heterodimeric structure in the ' into hole' form, these altered CH3 domains are expressed at different scFv ends, thereby preparing a heterodimeric scFv-type minibody.
Fab을 기반으로 하는 이중특이적 또는 다중특이적 항체의 경우, 특정 항원에 대한 개별 Fab'를 이황화결합 또는 매개체를 이용해서 서로 조합하여 heterodimeric Fab 형태로 제조할 수 있고, 특정 Fab의 중쇄 또는 경쇄의 말단에 상이한 항원에 대한 scFv를 발현시킴으로써 항원 결합가(valency)를 2개로 하거나, Fab과 scFv 사이에 경첩부위(hinge region)를 둠으로써 homodimeric 형태로 4개의 항원결합가를 가지도록 제조할 수 있다. 또한, Fab의 경쇄 말단과 중쇄 말단에 상이한 항원에 대한 scFv를 융합시킴으로써 항원에 대한 결합가를 3개로 만든 이중표적 바이바디(bibody), Fab의 경쇄 말단과 중쇄 말단에 상이한 scFv를 각각 융합시킴으로써 항원에 대한 결합가를 3개로 가지도록 한 삼중표적 바이바디, 상이한 Fab 3개를 화학적으로 접합시킴으로써 수득할 수 있다.In the case of a bispecific or multispecific antibody based on a Fab, individual Fab's directed against a specific antigen can be combined with each other using a disulfide bond or a mediator to form a heterodimeric Fab, and the heavy or light chain of a specific Fab can be It can be prepared to have two antigen valencies by expressing scFvs for different antigens at the ends, or to have four antigen valencies in homodimeric form by providing a hinge region between Fab and scFv. In addition, by fusing scFvs for different antigens to the light and heavy chain ends of the Fab, a dual-target bibody with three antigen binding values, and different scFvs to the light and heavy chain ends of the Fab are fused to the antigen. It can be obtained by chemically conjugating three different Fabs, a triple-targeted bibody having three valencies.
IgG를 기반으로 하는 이중특이적 또는 다중특이적 항체의 경우, 트리온파마(Trion Pharma)사에 의해 마우스와 렛트 하이브리도마를 다시 교잡함으로써, 하이브리드 하이브리도마, 일명 쿼드로마(quadromas)를 제조하여 이중특이적 항체를 생산하는 방법이 알려져 있다. 또한, 경쇄부분은 공유하면서, 상이한 중쇄에 대해서 Fc의 CH3 homodimeric 도메인의 일부 아미노산을 변형시켜 heterodimeric 형태로 제작한 이른 바 'Holes and Knob' 형태로 이중특이적 항체를 제조할 수 있다. heterodimeric 형태의 이중특이적 항체 이외에, 상이한 2종의 scFv를 IgG의 경쇄와 중쇄의 가변 도메인 대신 constant 도메인에 각각 융합 발현시켜 homodimeric 형태의 (scFv)4-IgG로 제조할 수 있다. 또한, 임클론(ImClone)사는 인간 VEGFR-2에 대한 키메릭 단클론항체인 IMC-1C11을 기반으로하여, 이 항체의 경쇄 아미노 말단에 마우스 혈소판유도성장인자수용체-알파(Platelet-derived Growth Factor Receptor-α)에 대한 single variable domain만을 융합시켜 이중특이적 항체를 제작하여 보고하였다. 또한, 단백질 카이네이즈 A(protein kinase A, PKA) R 서브유닛의 dimerization and docking domain(DDD)과 PKA의 anchoring domain을 이용한 이른 바 'dock and lock(DNL)' 방법을 통해서 CD20에 대한 다수의 항원결합가를 지니는 항체로 제작할 수 있다.In the case of bispecific or multispecific antibodies based on IgG, hybrid hybridomas, also known as quadromas, were prepared by re-crossing mouse and rat hybridomas by Trion Pharma. Thus, a method for producing a bispecific antibody is known. In addition, a bispecific antibody can be prepared in the so-called 'Holes and Knob' form, which is produced in a heterodimeric form by modifying some amino acids of the CH3 homodimeric domain of Fc for different heavy chains while sharing the light chain portion. In addition to the heterodimeric form of the bispecific antibody, two different scFvs can be fused to the constant domains instead of the light and heavy chain variable domains of IgG to produce homodimeric (scFv)4-IgG. In addition, ImClone Inc. is based on IMC-1C11, a chimeric monoclonal antibody against human VEGFR-2, and mouse platelet-derived growth factor receptor-α at the amino terminus of the light chain of this antibody. ), a bispecific antibody was produced and reported by fusion of only a single variable domain. In addition, through the so-called 'dock and lock (DNL)' method using the dimerization and docking domain (DDD) of the protein kinase A (PKA) R subunit and the anchoring domain of PKA, a large number of antigen-binding properties for CD20 It can be prepared with an antibody having
매우 다양한 재조합 항체 포맷, 예를 들면, 2가 이상, 3가 이상 또는 4가 이상의 이중특이적 또는 다중특이적 항체가 개발되었다. 예를 들어, 국제특허출원 공개 제WO2001/077342호, 제WO2009/080251호, 제WO2009/080252호, 제WO2009/080253호, 제WO2009/080254호, 제WO2010/112193호, 제WO2010/115589호, 제WO2010/136172호, 제WO2010/145792호, 제WO2010/145793호 및 제WO2011/117330호에 기재된 2가 이상, 3가 이상 또는 4가 이상의　항체도 포함한다. 2가 이상, 3가 이상 또는 4가 이상의 항체는 각각 2개 이상의 결합 도메인, 3개 이상의 결합 도메인 또는 4개 이상의 결합 도메인이 항체 분자에 존재한다는 것을 표시한다.A wide variety of recombinant antibody formats have been developed, eg, bispecific or multispecific antibodies, which are bispecific, trivalent or tetravalent or higher. For example, WO2001/077342, WO2009/080251, WO2009/080252, WO2009/080253, WO2009/080254, WO2010/112193, WO2010/115589, Also included are antibodies described in WO2010/136172, WO2010/145792, WO2010/145793 and WO2011/117330, which are bivalent, trivalent or tetravalent or higher. A bivalent, trivalent or more tetravalent antibody indicates that two or more binding domains, three or more binding domains or four or more binding domains, respectively, are present in the antibody molecule.
구체적 실시예에서, 본 발명에 따른 이중 또는 다중특이적 항체는 상기 항-TIGIT 항체 또는 항원 결합단편을 구체적으로 IgG 완전한 항체 또는 이의 단편 형태 예를 들어 단일쇄 Fv, VH　도메인 및/또는 VL　도메인, Fab 또는 (Fab)2의 형태로 포함할 수 있다. In a specific embodiment, the bi- or multispecific antibody according to the present invention comprises the anti-TIGIT antibody or antigen-binding fragment, specifically in the form of a complete IgG antibody or fragment thereof, for example, single-chain Fv, V H domain and/or V L It may be included in the form of a domain, Fab or (Fab) 2 .
또한, 상기 TIGIT을 타겟으로 하는 항체와 다른 타겟에 결합하는 항체 또는 이의 항원 결합단편 예를 들어, 상기 항체는 FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137(4-1BB), VISTA, CD258(LIGHT), MARCO, CD134(OX40), CD28, CD278(ICOS), CD27, CD154(CD40L), CD357(GITR), CD30, DR3, CD226(DNAM1), CD96, CD200, CD200R, 트랜스페린 수용체 (Transferrin receptor), c-Met, EGFR, HER2, KDR, PDGFRa 및 NRP1으로 구성된 군에서 선택된 하나 이상을 포함할 수 있다. 상기 항체 또는 이의 항원 결합단편은 구체적으로 IgG 완전한 항체 또는 이의 단편 형태 예를 들어 단일쇄 Fv, VH　도메인 및/또는 VL　도메인, Fab 또는 (Fab)2의 형태로 포함할 수 있다. In addition, the antibody or antigen-binding fragment thereof that binds to a different target than the antibody targeting the TIGIT, for example, the antibody is FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), MARCO, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96 , CD200, CD200R, transferrin receptor (Transferrin receptor), c-Met, EGFR, HER2, KDR, may include one or more selected from the group consisting of PDGFRa and NRP1. The antibody or antigen-binding fragment thereof may specifically include a complete IgG antibody or fragment thereof, for example, in the form of a single chain Fv, V H domain and/or V L domain, Fab or (Fab) 2 .
본 발명에 따른 이중 또는 다중특이적 항체를 통해 TIGIT 이외에, 다른 타겟에 의해 유도되거나 매개된 추가적 결합 특이성을 확보할 수 있다.Through the bi- or multispecific antibody according to the present invention, it is possible to secure additional binding specificity induced or mediated by other targets in addition to TIGIT.
예를 들어, 본 발명에 따른 이중특이적 항체는 TIGIT 와 FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137(4-1BB), VISTA, CD258(LIGHT), MARCO, CD134(OX40), CD28, CD278(ICOS), CD27, CD154(CD40L), CD357(GITR), CD30, DR3, CD226(DNAM1), CD96, CD200, CD200R, 트랜스페린 수용체 (Transferrin receptor), c-Met, EGFR, HER2, KDR, PDGFRa 및 NRP1로 구성된 군에서 선택된 하나를 동시에 타겟으로 할 수 있다. For example, the bispecific antibody according to the present invention comprises TIGIT and FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT) , MARCO, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c One selected from the group consisting of -Met, EGFR, HER2, KDR, PDGFRa and NRP1 may be simultaneously targeted.
예를 들어, 본 발명에 따른 다중특이적 항체는 TIGIT 와 FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137(4-1BB), VISTA, CD258(LIGHT), MARCO, CD134(OX40), CD28, CD278(ICOS), CD27, CD154(CD40L), CD357(GITR), CD30, DR3, CD226(DNAM1), CD96, CD200, CD200R, 트랜스페린 수용체 (Transferrin receptor), c-Met, EGFR, HER2, KDR, PDGFRa 및 NRP1으로 구성된 군에서 선택된 둘 이상을 동시에 타겟으로 할 수 있다.For example, the multispecific antibody according to the present invention is TIGIT and FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT) , MARCO, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c Two or more selected from the group consisting of -Met, EGFR, HER2, KDR, PDGFRa and NRP1 may be simultaneously targeted.
면역세포 인게이징(immune cell engage) 이중특이적 또는 다중특이적 항체Immune cell engage bispecific or multispecific antibody
본 발명은 또 다른 관점에서 항체의 scFv 및 면역세포 활성화 항원에 결합하는 항체의 scFv를 하나 이상 포함하는, 면역세포 인게이징(immune cell engage) 이중특이적 또는 다중특이적 항체에 관한 것이다. In another aspect the present invention relates to an immune cell engage bispecific or multispecific antibody comprising an scFv of an antibody and one or more scFvs of an antibody that bind to an immune cell activating antigen.
상기 면역세포 인게이징 이중특이적 또는 다중특이적 항체를 통해 세포독성 T 세포와 암 타겟 세포 사이의 세포 용해성 시냅스를 일시적으로 유도하여 독성물을 방출하도록 한다. Through the immune cell engaging bispecific or multispecific antibody, a cytolytic synapse is temporarily induced between a cytotoxic T cell and a cancer target cell to release a toxic substance.
하나의 실시예에서, 상기 면역세포 활성화 항원은 예를 들어 다음에서 선택될 수 있으며, 이에 결합하는 항체 또는 이의 항원 결합 단편이 면역세포 인게이저(immune cell engager) 역할을 할 수 있다:In one embodiment, the immune cell activating antigen may be selected from, for example, an antibody or antigen-binding fragment thereof binding thereto may serve as an immune cell engager:
T 세포 활성화 항원은 CD3, TCRα, TCRβ, TCRγ, TCRξ, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2 또는 CD226;T cell activating antigens include CD3, TCRα, TCRβ, TCRγ, TCRξ, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2 or CD226;
NK세포 활성화 항원은 NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (e.g., CD16a, CD16b), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (SLAMF4 또는 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E 또는 CD160;NK cell activating antigens are NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (e.g., CD16a, CD16b), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E or CD160;
B세포 활성화 항원은 OX40, CD40 또는 CD70;B cell activating antigens include OX40, CD40 or CD70;
대식세포 활성화 항원은 CD2 아고니스트, CD40, CD70, TCR (Toll-like Receptor) 아고니스트, CD47, STING 또는 OX40L; 또는Macrophage activating antigens include CD2 agonists, CD40, CD70, Toll-like Receptor (TCR) agonists, CD47, STING or OX40L; or
수지상세포 활성화 항원은 CD2 아고니스트, OX40, OX40L, 41BB 아고니스트, TCR 아고니스트, CD47 아고니스트 또는 STING 아고니스트.The dendritic cell activating antigen is a CD2 agonist, OX40, OX40L, 41BB agonist, TCR agonist, CD47 agonist or STING agonist.
면역세포 인게이저(immune cell engager)는 미국특허출원공개 제2017/0368169호에 구체적으로 기재되어 있으며, 본 발명에 참조로서 도입될 수 있다. The immune cell engager is specifically described in US Patent Application Publication No. 2017/0368169, and may be incorporated herein by reference.
구체적으로, 상기 면역세포 인게이징 이중특이적 또는 다중특이적 항체는 직렬 scFv를 포함하고, 다음의 항원 및 암세포 상의 표면 항원에 결합할 수 있다. 상기 암세포 상의 표면 항원은 본 발명에 따른 항체가 표적하는 TIGIT이다:Specifically, the immune cell-engaging bispecific or multispecific antibody comprises a tandem scFv and is capable of binding to the following antigens and surface antigens on cancer cells. The surface antigen on said cancer cell is TIGIT which the antibody according to the invention targets:
CD3, TCRα, TCRβ, TCRγ, TCRξ 또는 CD226;CD3, TCRα, TCRβ, TCRγ, TCRξ or CD226;
NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (e.g., CD16a, CD16b), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (SLAMF4 또는 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E 또는 CD160;NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (e.g., CD16a, CD16b), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (SLAMF4 or 2B4), SLAMF7, KIR2DS2, SLAMF7, KIR2DS2 , KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E or CD160;
OX40, CD40 또는 CD70;OX40, CD40 or CD70;
CD2 아고니스트, CD40, CD70, TCR (Toll-like Receptor) 아고니스트, CD47, STING 또는 OX40L; 또는CD2 agonist, CD40, CD70, TCR (Toll-like Receptor) agonist, CD47, STING or OX40L; or
CD2 아고니스트, OX40, OX40L, 41BB 아고니스트, TCR 아고니스트, CD47 아고니스트 또는 STING 아고니스트.CD2 agonist, OX40, OX40L, 41BB agonist, TCR agonist, CD47 agonist or STING agonist.
상기 면역세포 인게이징 이중특이적 또는 다중특이적 항체는 예를 들어, VL(TIGIT)-VH(TIGIT )-VH(CD3 또는 CD16A)-VL(CD3 또는 CD16A), VH(TIGIT )-VL(TIGIT )-VH(CD3 또는 CD16A)-VL(CD3 또는 CD16A), VH(CD3 또는 CD16A)-VL(CD3 또는 CD16A)-VH(TIGIT )-VL(TIGIT ) 또는 VH(CD3 또는 CD16A)-VL(CD3 또는 CD16A)-VL(TIGIT )-VH(TIGIT ) 형태의 구조를 포함할 수 있다. Said immune cell engaging bispecific or multispecific antibody can be, for example, VL(TIGIT)-VH(TIGIT)-VH(CD3 or CD16A)-VL(CD3 or CD16A), VH(TIGIT)-VL(TIGIT) )-VH(CD3 or CD16A)-VL(CD3 or CD16A), VH(CD3 or CD16A)-VL(CD3 or CD16A)-VH(TIGIT )-VL(TIGIT ) or VH(CD3 or CD16A)-VL(CD3) Or CD16A)-VL(TIGIT )-VH(TIGIT ) may include a structure.
상기 scFv는 예를 들어, 서열번호 17 내지 24로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 중쇄 가변영역과 서열번호 25 내지 27로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 경쇄 가변영역을 포함하고, 상기 중쇄 가변영역과 경쇄 가변영역은 링커로 연결될 수 있다. The scFv includes, for example, a heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 17 to 24 and a light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 25 to 27 and the heavy chain variable region and the light chain variable region may be connected by a linker.
상기 링커는 펩타이드 링커일 수 있으며, 약 10-25 aa 길이를 가질 수 있다. 예를 들어, 글리신 및/또는 세린과 같은 친수성 아미노산이 포함될 수 있다. The linker may be a peptide linker and may have a length of about 10-25 aa. For example, hydrophilic amino acids such as glycine and/or serine may be included.
상기 링커는 예를 들어, (GS)n, (GGS)n, (GSGGS)n 또는 (GnS)m (n, m은 각각 1 내지 10)을 포함할 수 있으나, 상기 링커는 예를 들어 (GnS)m (n, m은 각각 1 내지 10)일 수 있다. 구체적으로, 상기 링커는 GGGGS를 포함할 수 있다. The linker may include, for example, (GS)n, (GGS)n, (GSGGS)n or (GnS)m (n and m are each 1 to 10), but the linker is, for example, (G n S) m (n and m are each 1 to 10). Specifically, the linker may include GGGGS.
상기 면역세포 인게이징 이중특이적 또는 다중특이적 항체의 예시로, CD3 및 CD19에 결합하는 블리나투모마브(blinatumomab)(Amgen); CD3 및 EpCAM에 결합하는 솔리토마브(solitomab)(Amgen); CD3 및 CEA에 결합하는 MEDI 565(MedImmune, Amgen); 및 CD3 및 PSMA에 결합하는 BAY2010112(Bayer, Amgen)를 포함한다. 예시적인 DART는 CD3 및 CD123에 결합하는 MGD006(Macrogenics); 및 CD3 및 gpA33에 결합하는 MGD007(Macrogenics)를 포함한다. 예시적인 TandAbs는 CD3 및 CD19에 결합하는 AFM11(Affimed Therapeutics); 및 CD30 및 CD16A에 결합하는 AFM13(Affimed Therapeutics)을 포함할 수 있다. Examples of the immune cell engaging bispecific or multispecific antibody include blinatumomab (Amgen) that binds to CD3 and CD19; solitomab (Amgen) that binds to CD3 and EpCAM; MEDI 565 (MedImmune, Amgen) that binds to CD3 and CEA; and BAY2010112 (Bayer, Amgen) that binds to CD3 and PSMA. Exemplary DARTs include MGD006 (Macrogenics) that binds CD3 and CD123; and MGD007 (Macrogenics), which binds to CD3 and gpA33. Exemplary TandAbs include AFM11 (Affimed Therapeutics) that binds to CD3 and CD19; and AFM13 (Affimed Therapeutics) that binds to CD30 and CD16A.
항체-약물 접합체 (ADC)Antibody-Drug Conjugates (ADCs)
본 발명은 또 다른 관점에서, 상기 항체 또는 이의 항원 결합단편이 약물에 결합된 항체-약물 접합체 (ADC)에 관한 것이다.In another aspect, the present invention relates to an antibody-drug conjugate (ADC) in which the antibody or antigen-binding fragment thereof is bound to a drug.
항체-약물 접합체는 타겟 암세포로 항암 약물을 전달하기 전까지 항암 약물이 항체에 안정적으로 결합되어 있어야 한다. 타겟으로 전달된 약물은 항체로부터 유리되어 타겟 세포의 사멸을 유도해야 한다. 이를 위해서는 약물이 항체에 안정적으로 결합함과 동시에 타겟 세포에서 유리될 때는 타겟 세포의 사멸을 유도할 충분한 세포독성을 가져야 한다. In the antibody-drug conjugate, the anticancer drug must be stably bound to the antibody until the anticancer drug is delivered to the target cancer cell. The drug delivered to the target must be released from the antibody to induce the death of the target cell. To this end, when the drug is stably bound to the antibody and released from the target cell, it must have sufficient cytotoxicity to induce the death of the target cell.
하나의 실시예에서, 상기 항체 또는 이의 항원 결합단편은 약물과 링커를 통하여 결합될 수 있다. 상기 링커는 항-TIGIT 항체와 약물 사이를 연결하는 부위로, 세포내 조건에서 절단 가능한 형태 즉, 세포 내 환경에서 항체에서 약물이 방출될 수 있도록 하며, 항체의 긴 반감기를 반영하여 항체가 전신 순환 중에는 안정하고, 링커와 약물의 결합이 항체의 안정성 및 약물 동태에 영향을 주지 않아야 한다. In one embodiment, the antibody or antigen-binding fragment thereof may be bound to a drug through a linker. The linker is a site linking the anti-TIGIT antibody and the drug, and allows the drug to be released from the antibody in a form that is cleavable under intracellular conditions, that is, in the intracellular environment, and reflects the long half-life of the antibody, allowing the antibody to circulate throughout the body It should be stable, and binding of the linker to the drug should not affect the stability and pharmacokinetics of the antibody.
상기 링커는 예를 들어 절단성 링커 또는 비절단성 링커를 포함할 수 있다. 절단성 링커의 경우 펩타이드 링커와 같이 세포 내 펩티다아제 또는 프로테아제 효소 예를 들어 리소좀 또는 엔도좀 프로테아제에 의해 절단될 수 있고, 비절단성 링커의 경우 예를 들어 티오에테르 링커는 항체가 세포내 가수분해에 의해 비선택적으로 분해된 후에 약물이 방출될 수 있다. The linker may include, for example, a cleavable linker or a non-cleavable linker. In the case of a cleavable linker, such as a peptide linker, it can be cleaved by an intracellular peptidase or protease enzyme such as a lysosomal or endosomal protease, and in the case of a non-cleavable linker, e.g. a thioether linker, where the antibody is cleaved by intracellular hydrolysis. The drug may be released after non-selective degradation.
하나의 실시예에서, 상기 절단성 링커는 펩타이드 링커를 포함할 수 있다. 상기 펩타이드 링커는 적어도 2개 이상의 아미노산 길이를 가진다. 예를 들어, Val-Cit, Val-Ala 또는 Val-Cit의 디펩티드 또는 Phe-Leu 또는 Gly-Phe-Leu-Gly가 포함될 수 있다. 링커의 예시는 국제특허출원공개 제WO2004/010957호에 구체적으로 기재되어 있으며, 본 발명에 참조로서 도입될 수 있다. In one embodiment, the cleavable linker may include a peptide linker. The peptide linker has a length of at least two or more amino acids. For example, dipeptides of Val-Cit, Val-Ala or Val-Cit or Phe-Leu or Gly-Phe-Leu-Gly may be included. Examples of linkers are specifically described in International Patent Application Publication No. WO2004/010957, which may be incorporated herein by reference.
상기 항체-약물 접합체는 표적 암세포의 항원에 ADC의 항체영역이 결합하여 ADC-항원 복합체를 형성한 후 엔도좀-리소좀 경로로 암세포 내부로 내포화 된다. 이 경우 세포 독성 약물의 세포 내 방출은 엔도솜/리소좀의 내부 환경에 의해 조절된다.The antibody-drug conjugate is encapsulated into the cancer cell through the endo-lysosomal pathway after the antibody region of the ADC binds to the antigen of the target cancer cell to form the ADC-antigen complex. In this case, the intracellular release of the cytotoxic drug is regulated by the internal environment of the endosome/lysosome.
하나의 실시예에서, 상기 절단성 링커 또는 비절단성 링커는 산성 불안정 링커 (acid-labile linker), 이황화 링커, 펩타이드 링커, 베타-글루쿠로나이드 (beta-glucuronide) 링커, 티오에테르기 또는 말레이미도카프로일기를 포함할 수 있다.In one embodiment, the cleavable linker or non-cleavable linker is an acid labile linker, disulfide linker, peptide linker, beta-glucuronide linker, thioether group or maleimido It may contain a caproyl group.
상기 절단성 링커는 pH 민감성으로, 특정 pH 값에서 가수분해에 민감할 수 있다. 일반적으로, pH 민감성 링커는 산성 조건에서 가수분해될 수 있음을 나타낸다. 예를 들어, 리소좀에서 가수분해될 수 있는 산성 불안정 링커 예를 들어, 하이드라존, 세미카바존, 티오세미카바존, 시스-아코니틱 아마이드 (cis-aconitic amide), 오르쏘에스테르, 아세탈, 케탈 등일 수 있다. The cleavable linker is pH sensitive and may be susceptible to hydrolysis at certain pH values. In general, it is indicated that pH sensitive linkers can be hydrolyzed under acidic conditions. For example, acid labile linkers capable of being hydrolyzed in the lysosome, such as hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketal or the like.
다른 실시예에서, 상기 링커는 환원 조건에서 절단될 수도 있으며, 예를 들어 이황화 링커가 이에 해당할 수 있다. SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) 및 SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)를 사용하여 다양한 이황화 결합이 형성될 수 있다. 이러한 이황화 링커는 세포내 글루타치온의 티올과 이황화 교환에 의해 분해될 수 있다.In another embodiment, the linker may be cleaved under reducing conditions, for example, a disulfide linker may correspond to this. SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl -alpha-methyl-alpha-(2-pyridyl-dithio)toluene) can be used to form various disulfide bonds. This disulfide linker can be cleaved by disulfide exchange with thiols of intracellular glutathione.
상기 약물 및/또는 약물-링커는 항체의 라이신을 통해 무작위로 접합되거나, 항체의 이황화 결합 사슬을 환원하였을 때 노출되는 시스테인을 통해 접합될 수 있다. 경우에 따라서, 유전공학적으로 제작된 태그 예를 들어, 펩타이드 또는 단백질에 존재하는 시스테인을 통해 링커-약물이 결합될 수 있다. 상기 유전공학적으로 제작된 태그 예를 들어, 펩타이드 또는 단백질은 예를 들어, 이소프레노이드 트랜스퍼라제에 의하여 인식될 수 있는 아미노산 모티프를 포함할 수 있다. 상기 펩타이드 또는 단백질은 펩타이드 또는 단백질의 카복시 말단에서 결실(deletion)을 가지거나, 펩타이드 또는 단백질의 카복시(C) 말단에 스페이서 유닛의 공유결합을 통한 부가를 갖는다. The drug and/or drug-linker may be randomly conjugated via a lysine of the antibody, or may be conjugated via a cysteine exposed when the disulfide bond chain of the antibody is reduced. In some cases, the linker-drug may be bound through a genetically engineered tag, for example, a cysteine present in a peptide or protein. The genetically engineered tag, for example, a peptide or protein, may include an amino acid motif that can be recognized by, for example, isoprenoid transferase. The peptide or protein has a deletion at the carboxy terminus of the peptide or protein, or has an addition through a covalent bond of a spacer unit to the carboxy (C) terminus of the peptide or protein.
상기 펩타이드 또는 단백질은 아미노산 모티프와 바로 공유결합되거나 스페이서 유닛과 공유결합되어 아미노산 모티프와 연결될 수 있다. 상기 아미노산 스페이서 유닛은 1 내지 20개의 아미노산으로 구성되며, 그 중에서 글리신(Glycine) 유닛이 바람직하다.The peptide or protein may be directly covalently bonded to an amino acid motif or may be covalently bonded to a spacer unit to be linked to the amino acid motif. The amino acid spacer unit is composed of 1 to 20 amino acids, and among them, a glycine unit is preferable.
상기 이소프레노이드 트랜스퍼라제는 예를 들어, 파네실트랜스퍼라제 (FTase, farnesyl protein transferase) 또는 게라닐게라닐트랜스퍼라제 (GGTase, geranylgeranyl transferase)일 수 있고, FTase 및 GGTase I은 앞서 언급한 화학식 1 중 CAAX 모티프를 인식할 수 있고, GGTase II는 XXCC, XCXC 또는 CXX 모티프(여기서 C는 시스테인이고, A는 지방족 아미노산이고, X는 이소프레노이드 트랜스퍼라제의 기질 특이성을 결정하는 아미노산이다)를 인식할 수 있다.The isoprenoid transferase may be, for example, farnesyl transferase (FTase, farnesyl protein transferase) or geranylgeranyl transferase (GGTase, geranylgeranyl transferase), and FTase and GGTase I are CAAX motif and GGTase II can recognize XXCC, XCXC or CXX motif, where C is cysteine, A is an aliphatic amino acid, and X is an amino acid that determines the substrate specificity of an isoprenoid transferase. have.
또 다른 실시예에서, 상기 링커는 리소좀에서 다수 존재하거나 또는 몇몇 종양세포에서 과발현되는 베타-글루쿠로니데이즈(β에 의해 인식되어 가수분해 되는 베타-글루쿠로나이드 링커를 포함할 수 있다. 펩타이드 링커와는 달리 친수성(hydrophilicity)이 커서 소수성의 성질이 높은 약물과 결합시 항체-약물 복합체의 용해도를 증가시킬 수 있는 장점을 지닌다.In another embodiment, the linker may include a beta-glucuronide linker that is recognized and hydrolyzed by beta-glucuronidase (β), which is present in many lysosomes or overexpressed in some tumor cells. Peptide Unlike the linker, it has the advantage of increasing the solubility of the antibody-drug complex when combined with a drug with high hydrophobicity due to its high hydrophilicity.
이와 관련하여, 국제특허출원공개 제WO2015/182984호에 개시된 베타-글루쿠로나이드 링커, 예를 들어 자가-희생기 (self-immolative group)를 포함하는 베타-글루쿠로나이드 링커를 사용할 수 있으며, 위 문헌은 참조로 도입된다. In this regard, the beta-glucuronide linker disclosed in International Patent Application Publication No. WO2015/182984, for example, a beta-glucuronide linker including a self-immolative group may be used. , the above documents are incorporated by reference.
경우에 따라서, 상기 링커는 예를 들어 비절단성 링커일 수 있으며, 세포 내에서 항체 가수분해 한 단계만을 통해 약물이 방출되어, 예를 들어 아미노산-링커-약물 복합체를 생산한다. 이러한 유형의 링커는 티오에테르기 또는 말레이미도카프로일기 (maleimidocaproyl)일 수 있고, 혈액 내 안정성을 유지할 수 있다.In some cases, the linker may be, for example, a non-cleavable linker, and the drug is released through only one step of antibody hydrolysis in the cell, for example, to produce an amino acid-linker-drug complex. This type of linker may be a thioether group or a maleimidocaproyl group, and may maintain stability in blood.
본 발명의 일 실시예에 따르면, 항체의 이황화 결합 사슬을 환원하였을 때 노출되는 시스테인을 통해 링커-약물이 무작위 결합 또는 서열 GGGGGGGCVIM을 가지는 항체 말단 결합 펩타이드를 도입하여 링커-약물이 결합될 수 있다. According to an embodiment of the present invention, the linker-drug may be bound through a cysteine exposed when the disulfide bond chain of the antibody is reduced, or the linker-drug may be bound by random linkage or by introducing an antibody terminal binding peptide having the sequence GGGGGGGCVIM.
상기 약물 (화학식 (1)의 D 포함)은 약리학적 효과를 나타내는 제제로 항체에 결합될 수 있으며, 구체적으로 화학요법제, 독소, 마이크로 RNA (miRNA), siRNA, shRNA 또는 방사성 동위원소일 수 있다. 상기 화학요법제는 예를 들어, 세포독성 제제 또는 면역억제제일 수 있다. 구체적으로 마이크로투불린 억제제, 유사분열 억제제, 토포이소머라아제 억제제 또는 DNA 인터컬레이터로서 기능할 수 있는 화학요법제를 포함할 수 있다. 또한, 면역조절 화합물, 항암제, 항바이러스제, 항박테리아제, 항진균제, 구충제 또는 이들의 조합을 포함할 수 있다.The drug (including D of Formula (1)) may be bound to an antibody as an agent exhibiting a pharmacological effect, and specifically may be a chemotherapeutic agent, a toxin, micro RNA (miRNA), siRNA, shRNA, or a radioactive isotope. . The chemotherapeutic agent may be, for example, a cytotoxic agent or an immunosuppressive agent. Specifically, it may include a microtubulin inhibitor, a mitosis inhibitor, a topoisomerase inhibitor, or a chemotherapeutic agent capable of functioning as a DNA intercalator. In addition, it may include an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an anthelmintic agent, or a combination thereof.
이러한 약물에는 예를 들어, 마이탄시노이드, 오리스타틴 (MMAE, MMAF 포함), 아미노프테린, 악티노마이신, 블레오마이신, 탈리소마이신, 캄프토쎄신, N8-아세틸 스퍼미딘, 1-(2 클로로에틸)-1,2-다이메틸 술포닐 하이드라자이드, 에스퍼라마이신, 에토포사이드, 6-머캅토퓨린, 돌라스타틴, 트리코테센, 칼리케아미신, 탁솔(taxol), 탁산, 파클리탁셀(paclitaxel), 도세탁셀(docetaxel), 메토트렉세이트, 빈크리스틴, 빈블라스틴, 독소루비신, 멜팔란, 미토마이신 A, 미토마이신 C, 클로람부실, 듀오카마이신, L-아스파라기나제(L-asparaginase), 머캡토퓨린(mercaptopurine), 티오구아닌(thioguanine), 하이드록시우레아(hydroxyurea), 시타라빈(cytarabine), 사이클로포스파미드(cyclophosphamide), 이포스파미드(ifosfamide), 니트로소우레아(nitrosourea), 시스플라틴(cisplatin), 카보플라틴(carboplatin), 미토마이신(mitomycin), 다카바진(dacarbazine), 프로카바진(procarbazine), 토포테칸(topotecan), 질소 머스터드(nitrogen mustard), 사이톡산(cytoxan), 에토포시드(etoposide),5-플루오로우라실(5-fluorouracil), CNU(bischloroethylnitrosourea), 이리노테칸(irinotecan), 캄포토테신(camptothecin), 블레오마이신(bleomycin), 이다루비신(idarubicin), 다우노루비신(daunorubicin), 닥티노마이신(dactinomycin), 플리카마이신(plicamycin), 미톡산트론(mitoxantrone), 아스파라기나제(asparaginase), 비노렐빈(vinorelbine), 클로로람부실(chlorambucil), 멜파란(melphalan), 카르무스틴(carmustine), 로무스틴(lomustine), 부설판(busuLfan), 트레오설판(treosulfan), 데카바진(decarbazine), 에토포시드(etoposide), 테니포시드(teniposide), 토포테칸(topotecan), 9-아미노캠프토테신(9-aminocamptothecin), 크리스나톨(crisnatol), 미토마이신 C(mitomycin C), 트리메트렉세이트(trimetrexate), 마이코페놀산(mycophenolic acid), 티아조퓨린(tiazofurin), 리바비린(ribavirin), EICAR(5-ethynyl-1-beta-Dribofuranosylimidazole-4-carboxamide), 하이드록시우레아(hydroxyurea), 데프록사민(deferoxamine), 플룩수리딘(floxuridine), 독시플루리딘(doxifluridine), 랄티트렉세드(raltitrexed), 시타라빈(cytarabine(ara C)), 시토신 아라비노시드(cytosine arabinoside), 플루다라빈(fludarabine), 타목시펜(tamoxifen), 라록시펜(raloxifene), 메게스트롤(megestrol), 고세렐린(goserelin), 류프롤리드 아세 테이트(leuprolide acetate), 플루타미드(flutamide), 바이칼루타마이드(bicalutamide), EB1089, CB1093, KH1060, 베르테포르핀(verteporfin), 프탈로시아닌(phthalocyanine), 광감작제 Pe4(photosensitizer Pe4), 데메톡시-하이포크레린 A(demethoxy-hypocrellin A), 인터페론-α(Interferon-α), 인터페론-γ(Interferon-γ), 종양 괴사 인자(tumor necrosis factor), 겜사이타빈(Gemcitabine), 벨케이드(velcade), 레발미드(revamid), 탈라미드(thalamid), 로바스타틴(lovastatin), 1-메틸-4-페닐피리디늄 이온(1-methyl-4-phenylpyridiniumion), 스타우로스포린(staurosporine), 악티노마이신 D(actinomycin D), 닥티노마이신(dactinomycin), 블레오마이신 A2(bleomycin A2), 블레오마이신 B2(bleomycinB2), 페플로마이신(peplomycin), 에피루비신(epirubicin), 피라루비신(pirarubicin), 조루비신(zorubicin), 마이토산트론(mitoxantrone), 베라파밀(verapamil) 및 탑시가르긴(thapsigargin), 핵산 분해 효소 및 세균이나 동식물 유래의 독소로 구성된 군에서 선택된 하나 이상일 수 있으나, 이에 한정되는 것은 아니다.Such drugs include, for example, maytansinoids, auristatins (including MMAE, MMAF), aminopterin, actinomycin, bleomycin, thalisomycin, camptothecin, N8-acetyl spermidine, 1-(2 Chloroethyl)-1,2-dimethyl sulfonyl hydrazide, esperamicin, etoposide, 6-mercaptopurine, dolastatin, trichothecene, calicheamicin, taxol, taxane, paclitaxel , docetaxel, methotrexate, vincristine, vinblastine, doxorubicin, melphalan, mitomycin A, mitomycin C, chlorambucil, duocarmycin, L-asparaginase (L-asparaginase), mercaptopurine (mercaptopurine), thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, Carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustard, cytoxan, etoposide ), 5-fluorouracil, CNU (bischloroethylnitrosourea), irinotecan, camptothecin, bleomycin, idarubicin, daunorubicin, Dactinomycin, plicamycin, mitoxantrone, asparaginase, vinorelbine, chlorambucil, melphalan, carmustine (carmustine), lomustine (lomustine), busulfan (bus) uLfan), treosulfan, decarbazine, etoposide, teniposide, topotecan, 9-aminocamptothecin, cristol ( crisnatol), mitomycin C, trimetrexate, mycophenolic acid, tiazofurin, ribavirin, EICAR (5-ethynyl-1-beta-Dribofuranosylimidazole) -4-carboxamide), hydroxyurea, deferoxamine, floxuridine, doxifluridine, raltitrexed, cytarabine (ara C) ), cytosine arabinoside, fludarabine, tamoxifen, raloxifene, megestrol, goserelin, leuprolide acetate acetate), flutamide, bicalutamide, EB1089, CB1093, KH1060, verteporfin, phthalocyanine, photosensitizer Pe4 (photosensitizer Pe4), demethoxy-hypocrerin A (demethoxy-hypocrellin A), interferon-α (Interferon-α), interferon-γ (Interferon-γ), tumor necrosis factor, gemcitabine, velcade, levalmid ( revamid), thalamid, lovastatin, 1-methyl-4-phenylpyridinium ion (1-methyl-4-phenylpyridini) umion), staurosporine, actinomycin D, dactinomycin, bleomycin A2, bleomycin A2, bleomycin B2, peplomycin, epiruby group consisting of epirubicin, pirarubicin, zorubicin, mitoxantrone, verapamil and thapsigargin, nucleases and toxins derived from bacteria, animals or plants It may be one or more selected from, but is not limited thereto.
경우에 따라서, 상기 약물은 링커 및 링커 시약 상의 친전자성 기와 공유결합을 형성하기 위해 반응할 수 있는 아민, 티올, 히드록실, 히드라지드, 옥심, 히드라진, 티오세미카바존, 히드라진 카르복실레이트, 및 아릴히드라지드기로 구성된 군에서 선택된 하나 이상의 친핵기를 포함할 수 있다.Optionally, the drug is an amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, capable of reacting to form a covalent bond with an electrophilic group on the linker and linker reagent; and one or more nucleophilic groups selected from the group consisting of an arylhydrazide group.
본 발명에 따른 구체적 실시예에서 MC-vc-PAB 링커를 통해, 본 발명에 따른 항체 또는 이의 항원 결합 단편을 약물 예를 들어, 오리스타틴 (MMAE)과 연결한 ADC를 제작하였다. 이러한 ADC는 목적하는 세포독성을 나타냄을 확인하였다. In a specific example according to the present invention, ADC was prepared in which the antibody or antigen-binding fragment thereof according to the present invention was linked to a drug, for example, auristatin (MMAE) through the MC-vc-PAB linker. It was confirmed that these ADCs exhibit the desired cytotoxicity.
키메라 항원 수용체(CAR)Chimeric Antigen Receptor (CAR)
본 발명은 다른 관점에서, 항원 결합 부위를 포함하는 세포외도메인, 트랜스 멤브레인 도메인 및 세포내 신호전달 도메인을 포함하는 키메라 항원 수용체(CAR)로, 상기 세포외도메인의 항원 결합 부위는 상기 항체의 scFv인 것을 특징으로 하는 키메라 항원 수용체에 관한 것이다. In another aspect, the present invention is a chimeric antigen receptor (CAR) comprising an extracellular domain comprising an antigen-binding site, a transmembrane domain and an intracellular signaling domain, wherein the antigen-binding site of the extracellular domain is the scFv of the antibody It relates to a chimeric antigen receptor, characterized in that.
키메라 항원 수용체(CAR)는 표적 항원 및 해당 항원을 발현하는 세포에 대하여 면역반응을 유도할 수 있도록 설계된 합성 컨스트럭트이다. CAR는 세포외도메인, 트랜스 멤브레인 도메인 및 세포내 신호전달 도메인을 포함한다. 암세포의 표면에 특이적으로 발현되는 암세포 표면 항원을 인식하는 수용체를 코딩하는 유전자를 면역세포에 도입하여, 암세포를 사멸시킬 수 있다. 암세포에서 특이적으로 발현하는 항원과 결합하는 수용체를 포함하는 면역세포를 통해, 암세포만 표적하여 면역반응을 일으킬 수 있다. 상기 CAR는 본 발명에 따른 항-TIGIT 항체의 scFv를 세포외도메인의 항원 인식 부위로 포함한다. Chimeric antigen receptors (CARs) are synthetic constructs designed to induce an immune response against a target antigen and cells expressing the antigen. The CAR comprises an extracellular domain, a transmembrane domain and an intracellular signaling domain. By introducing a gene encoding a receptor recognizing a cancer cell surface antigen specifically expressed on the surface of cancer cells into immune cells, cancer cells can be killed. Through immune cells containing a receptor that binds to an antigen specifically expressed in cancer cells, it is possible to induce an immune response by targeting only cancer cells. The CAR includes the scFv of the anti-TIGIT antibody according to the present invention as an antigen recognition site of an extracellular domain.
1세대 CAR에서는 암세포에서 특이적으로 발현하는 항원 인식 부위를 포함하는 세포외도메인, 트랜스 멤브레인 도메인 및 세포내 신호전달 도메인을 포함하고, 신호전달 도메인으로서 CD3ζ만을 이용하였는데, 암에 대한 치료 효과가 미미하였고, 지속시간이 짧다는 문제가 있었다. 이러한 1세대 CAR는 미국등록특허 제6,319,494호에 구체적으로 기재되어 있으며, 본 발명에 참조로서 도입된다. In the first-generation CAR, an extracellular domain including an antigen recognition site specifically expressed in cancer cells, a transmembrane domain, and an intracellular signaling domain were used, and only CD3ζ was used as the signaling domain, but the therapeutic effect on cancer was insignificant. And there was a problem that the duration was short. This first-generation CAR is specifically described in US Patent No. 6,319,494, which is incorporated herein by reference.
면역세포에 대한 반응성 향상을 위하여 보조 자극 도메인(CD28 또는 CD137/4-1BB)과 CD3ζ를 결합한 2세대 CAR가 제조되었는데 1세대 CAR와 비교하여 체내에 잔존하는 CAR 포함 면역세포의 수가 현저히 증가하였다. 2세대 CAR는 한 가지의 보조 자극 도메인을 이용한 것에 반해, 3세대 CAR에서는 두 가지 이상의 보조 자극 도메인을 이용하였다. 생체 내 CAR를 포함하는 면역세포의 확장 및 지속성 달성을 위해 보조 자극 도메인을 4-1BB, CD28 또는 OX40 등과 결합시킬 수 있다. 2세대 CAR는 미국등록특허 제7,741,465호, 제7,446,190호 또는 제9,212,229호에 구체적으로 기재되어 있고 3세대 CAR는 미국등록특허 제8,822,647호에 구체적으로 기재되어 있으며, 본 발명에 참조로서 도입된다. A second-generation CAR combining a co-stimulatory domain (CD28 or CD137/4-1BB) and CD3ζ was prepared to improve responsiveness to immune cells. Compared with the first-generation CAR, the number of CAR-containing immune cells remaining in the body significantly increased. The second generation CAR used one auxiliary stimulation domain, whereas the third generation CAR used two or more auxiliary stimulation domains. A co-stimulatory domain can be combined with 4-1BB, CD28 or OX40, etc. to achieve expansion and persistence of immune cells including CAR in vivo. The second-generation CAR is specifically described in U.S. Patent Nos. 7,741,465, 7,446,190 or 9,212,229, and the third-generation CAR is specifically described in U.S. Patent No. 8,822,647, which is incorporated herein by reference.
4세대 CAR에서는 IL-12 또는 IL-15와 같은 사이토카인을 암호화하는 추가 유전자를 포함하여, 사이토카인의 CAR 기반 면역단백질이 추가로 발현될 수 있도록 하고, 5세대 CAR는 면역세포 강화를 위해 인터루킨 리셉터 체인 예를 들어, IL-2Rβ를 추가로 포함한다. 4세대 CAR는 미국등록특허 제10,316,102호, 5세대 CAR는 미국등록특허 제10,336,810호에 구체적으로 기재되어 있으며, 본 발명에 참조로서 도입된다. In the fourth-generation CAR, additional genes encoding cytokines such as IL-12 or IL-15 are included to allow the expression of additional CAR-based immune proteins of cytokines, and the fifth-generation CAR includes interleukins to enhance immune cells. It further comprises a receptor chain such as IL-2Rβ. The 4th generation CAR is specifically described in US Patent No. 10,316,102, and the 5th generation CAR is specifically described in US Patent No. 10,336,810, which is incorporated herein by reference.
하나의 실시예에서, 상기 세포외도메인의 항원 결합 부위는 항체의 scFv이다. 항체의 VH 및 VL 도메인을 포함하는 scFv에서 VH 및 VL 도메인은 링커를 통해 연결될 수 있다. 서열번호 17 내지 24로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 중쇄 가변영역은 링커를 통해 서열번호 25 내지 27로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 경쇄 가변영역과 연결될 수 있다.In one embodiment, the antigen binding site of the extracellular domain is an scFv of an antibody. In an scFv comprising the VH and VL domains of an antibody, the VH and VL domains may be linked via a linker. The heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 17 to 24 may be linked to a light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 25 to 27 through a linker.
상기 링커는 펩타이드 링커일 수 있으며, 약 10-25 aa 길이를 가질 수 있다. 예를 들어, 글리신 및/또는 세린과 같은 친수성 아미노산이 포함될 수 있다. The linker may be a peptide linker and may have a length of about 10-25 aa. For example, hydrophilic amino acids such as glycine and/or serine may be included.
상기 링커는 예를 들어, (GS)n, (GGS)n, (GSGGS)n 또는 (GnS)m (n, m은 각각 1 내지 10)을 포함할 수 있으나, 상기 링커는 예를 들어 (GnS)m (n, m은 각각 1 내지 10)일 수 있다. 구체적으로, 상기 링커는 GGGGS를 포함할 수 있다. The linker may include, for example, (GS) n , (GGS) n , (GSGGS) n or (G n S) m (n and m are each 1 to 10), but the linker is, for example (G n S) m (n and m are each 1 to 10). Specifically, the linker may include GGGGS.
상기 트랜스 멤브레인 도메인은 천연 또는 합성 공급원으로부터 유래될 수 있다. 공급원이 천연인 경우, 도메인은 임의의 막 결합된 단백질 또는 트랜스 멤브레인 단백질로부터 유래될 수 있다. 상기 트랜스 멤브레인 도메인은 T-세포 수 용체, CD28, CD3 엡실론, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS의 알파, 베타 또는 제타 쇄를 포함할 수 있다. 상기 트랜스 멤브레인 도메인을 합성하는 경우 류신과 발린과 같은 소수성 잔기를 포함하거나, 페닐알라닌, 트립토판 및 발린 포함 펩타이드를 각 말단에 포함할 수 있다. 2개 내지 10개 아미노산 길이인 짧은 올리고- 또는 폴리펩티드 링커가 트랜스 멤브레인 도메인과 CAR의 세포질 신호전달 도메인 사이에 결합을 형성할 수 있다. 글리신-세린 펩타이드를 링커로 이용할 수 있다. The trans membrane domain may be derived from a natural or synthetic source. When the source is natural, the domain may be derived from any membrane bound protein or trans membrane protein. The transmembrane domain is the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS alpha, beta or zeta chains. When synthesizing the trans-membrane domain, hydrophobic residues such as leucine and valine may be included, or peptides including phenylalanine, tryptophan, and valine may be included at each end. A short oligo- or polypeptide linker of 2 to 10 amino acids in length can form a bond between the trans membrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine peptide may be used as a linker.
상기 신호 전달 도메인은 CAR이 위치된 면역세포의 정상 효과기 기능에 대한 활성화를 유도할 수 있다. 예를 들어 사이토카인의 분비를 통해 세포용해 활성화 또는 헬퍼 활성화를 유도할 수 있다. 상기 신호 전달 도메인은 효과기 기능 신호를 형질도입하기에 충분한 세포내 신호전달 도메인의 절단된 단편을 포함할 수 있다. The signal transduction domain can induce activation of the normal effector function of the immune cell in which the CAR is located. For example, it can induce cytolytic activation or helper activation through the secretion of cytokines. The signaling domain may comprise a truncated fragment of an intracellular signaling domain sufficient to transduce an effector function signal.
상기 신호 전달 도메인으로 항원 수용체 관여 후 신호 전달을 개시하기 위해 협력하여 작용하는 T 세포 수용체(TCR) 및 공동-수용체의 세포질이 포함될 수 있다. The cytoplasm of the T cell receptor (TCR) and the co-receptor, which act in concert to initiate signal transduction after antigen receptor engagement with the signal transduction domain, may be included.
TCR 단독을 통해 생성된 신호는 T 세포의 완전한 활성화를 위해 불충분하고 공동-자극 신호가 필요하다는 것이 또한 알려져 있다. 따라서, T 세포 활성화에 TCR을 통해 항원-의존성 일차 활성화를 개시하는 것 및 이차 또는 공동-자극 신호를 제공하도록 항원-의존성 방식으로 작용하는 것이 관여할 수 있다. 일차 세포질 신호전달 서열은 자극성 방식으로 또는 억제성 방식으로 TCR 복합체의 일차 활성화를 조절한다. 자극성 방식으로 작용하는 일차 세포질 신호전달 서열은 면역수용체 티로신-기반 활성화 모티프 또는 ITAM으로 서 공지된 신호전달 모티프를 함유할 수 있다. 일차 세포질 신호전달 서열을 함유하는 ITAM의 예로는 TCR 제타, FcR 감마, FcR 베타, CD3 감마, CD3 델타, CD3 엡실론, CD5, CD22, CD79a, CD79b, 및 CD66d를 포함할 수 있다. It is also known that signals generated via TCR alone are insufficient for full activation of T cells and that co-stimulatory signals are required. Thus, initiating antigen-dependent primary activation via TCRs and acting in an antigen-dependent manner to provide secondary or co-stimulatory signals may be involved in T cell activation. Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex in a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAMs containing primary cytoplasmic signaling sequences may include TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
경우에 따라서, CAR의 세포질 도메인은 CD3 제타쇄 부분 및 공동자극 신호전달 영역을 포함할 수 있다. 공동자극 신호전달 영역은 공동자극 분자의 세포내 도메인을 포함하는 CAR의 일 부분을 의미한다. 예를 들어, CD27, CD28, 4-1BB(CD137), OX40, CD30, CD40, PD-1, ICOS, 림프구 기능-관련된 항원-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, 및 CD83과 특이적으로 결합하는 리간드 등이 포함될 수 있다. CAR의 세포질 신호전달 부분 내의 세포질 신호전달 서열은 2개 내지 10개 아미노산 예를 들어, 글리신-세린 포함 펩타이드 링커를 통해 연결될 수 있다. Optionally, the cytoplasmic domain of the CAR may include a CD3 zeta chain portion and a costimulatory signaling region. Costimulatory signaling region refers to the portion of the CAR comprising the intracellular domain of a costimulatory molecule. For example, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, Lymphocyte Function-Related Antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7- H3, and a ligand that specifically binds to CD83, and the like may be included. Cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR may be linked via a peptide linker comprising 2 to 10 amino acids, eg, glycine-serine.
본 발명은 다른 관점에서, 상기 키메라 항원 수용체(CAR)가 도입된 면역세포에 관한 것이다. In another aspect, the present invention relates to an immune cell into which the chimeric antigen receptor (CAR) is introduced.
상기 면역세포는 면역을 유도하여 목적하는 암 치료 효과를 유발할 수 있는 것으로, 예를 들어, T 세포, NK 세포, 사이토카인 유도 살해세포(Cytokine Induced Killer cell, CIK), 활성화 세포독성 T 림프구(Cytotoxic T Lymphocyte, CTL), 마크로파지, 종양 조직 내 침투 T 세포 (Tumor-Infiltrating Lymphocytes, TIL) 및 수지상세포로 구성된 군에서 선택될 수 있으나, 이에 제한되는 것은 아니다. The immune cells are capable of inducing a desired cancer therapeutic effect by inducing immunity, for example, T cells, NK cells, cytokine-induced killer cells (CIK), activated cytotoxic T lymphocytes (Cytotoxic). T Lymphocyte, CTL), macrophages, tumor tissue infiltrating T cells (Tumor-Infiltrating Lymphocytes, TIL) and may be selected from the group consisting of dendritic cells, but is not limited thereto.
경우에 따라서, 상기 면역세포는 면역관문 억제제에 대한 항체에 대한 scFv가 세포외도메인의 항원 결합 부위로 포함된 키메라 항원 수용체(CAR)가 추가로 도입될 수 있다. In some cases, the immune cells may be additionally introduced with a chimeric antigen receptor (CAR) in which an scFv for an antibody against an immune checkpoint inhibitor is included as an antigen-binding site of an extracellular domain.
상기 면역관문 억제제에 대한 항체는 예를 들어 항체는 FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137(4-1BB), VISTA, CD258(LIGHT), MARCO, CD134(OX40), CD28, CD278(ICOS), CD27, CD154(CD40L), CD357(GITR), CD30, DR3, CD226(DNAM1), CD96, CD200, CD200R, 트랜스페린 수용체 (Transferrin receptor), c-Met, EGFR, HER2, KDR, PDGFRa 및 NRP1으로 구성된 군에서 선택된 하나 이상을 타겟하는 항체일 수 있다.The antibody against the checkpoint inhibitor is, for example, the antibody is FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), MARCO , CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met , EGFR, HER2, KDR, PDGFRa, and may be an antibody that targets one or more selected from the group consisting of NRP1.
치료용 조성물 및 치료 방법Therapeutic compositions and methods of treatment
본 발명은 또 다른 관점에서 항체 또는 이의 항원 결합단편; 상기 항체 또는 이의 항원 결합단편을 포함하는 이중 또는 다중특이적 항체; 상기 항체 또는 이의 항원 결합단편을 포함하는 항체-약물 접합체; 상기 항체 또는 이의 항원 결합단편을 포함하는 키메라 항원 수용체; 또는 상기 키메라 항원 수용체를 포함하는 면역세포;를 포함하는 암 예방 또는 치료용 조성물에 관한 것이다. In another aspect, the present invention provides an antibody or antigen-binding fragment thereof; a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof; an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof; a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof; Or immune cells comprising the chimeric antigen receptor; relates to a composition for preventing or treating cancer comprising.
본 발명은 예를 들어, (a) 본 발명에 따른 TIGIT에 대한 항체 또는 이의 항원 결합 단편, 상기 항체 또는 이의 항원 결합단편을 포함하는 이중 또는 다중특이적 항체, 상기 항체 또는 이의 항원 결합단편을 포함하는 항체-약물 접합체, 상기 항체 또는 이의 항원 결합단편을 포함하는 키메라 항원 수용체 또는 상기 키메라 항원 수용체를 포함하는 면역세포의 약제학적 유효량; 및 (b) 약제학적으로 허용되는 담체를 포함하는 암의 예방 또는 치료용 약제학적 조성물일 수 있다. 본 발명은 또한, 본 발명에 따른 TIGIT에 대한 항체 또는 이의 항원 결합 단편, 상기 항체 또는 이의 항원 결합단편을 포함하는 이중 또는 다중특이적 항체, 상기 항체 또는 이의 항원 결합단편을 포함하는 항체-약물 접합체, 상기 항체 또는 이의 항원 결합단편을 포함하는 키메라 항원 수용체 또는 상기 키메라 항원 수용체를 포함하는 면역세포를 이를 필요로 하는 개체에 투여하는 단계를 포함하는 암의 예방 또는 치료방법일 수 있다. The present invention includes, for example, (a) an antibody or antigen-binding fragment thereof against TIGIT according to the present invention, a bi- or multispecific antibody comprising the antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof a pharmaceutically effective amount of an antibody-drug conjugate, a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof, or an immune cell comprising the chimeric antigen receptor; And (b) may be a pharmaceutical composition for the prevention or treatment of cancer comprising a pharmaceutically acceptable carrier. The present invention also provides an antibody or antigen-binding fragment thereof against TIGIT according to the present invention, a bi- or multispecific antibody comprising the antibody or antigen-binding fragment thereof, and an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof , It may be a method of preventing or treating cancer comprising administering to an individual in need thereof, a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof, or an immune cell comprising the chimeric antigen receptor.
본 발명의 구체예에서, 상기 개체는 암이 발병할 것으로 예상되는 개체; 발병한 개체; 또는 완치판정을 받은 개체일 수 있으나, 이에 제한되지 않는다.In an embodiment of the present invention, the subject is a subject expected to develop cancer; affected individuals; Or it may be an individual who has been cured, but is not limited thereto.
본 발명은 또 다른 관점에서 상기 항체 또는 이의 항원 결합단편; 상기 항체 또는 이의 항원 결합단편을 포함하는 이중 또는 다중특이적 항체; 상기 항체 또는 이의 항원 결합단편을 포함하는 항체-약물 접합체; 상기 항체 또는 이의 항원 결합단편을 포함하는 키메라 항원 수용체; 또는 상기 키메라 항원 수용체를 포함하는 면역세포;를 포함하는 감염질환 예방 또는 치료용 조성물에 관한 것이다. The present invention in another aspect the antibody or antigen-binding fragment thereof; a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof; an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof; a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof; Or immune cells comprising the chimeric antigen receptor; relates to a composition for preventing or treating infectious diseases, including.
본 발명은 예를 들어, (a) 본 발명에 따른 TIGIT에 대한 항체 또는 이의 항원 결합 단편, 상기 항체 또는 이의 항원 결합단편을 포함하는 이중 또는 다중특이적 항체, 상기 항체 또는 이의 항원 결합단편을 포함하는 항체-약물 접합체, 상기 항체 또는 이의 항원 결합단편을 포함하는 키메라 항원 수용체 또는 상기 키메라 항원 수용체를 포함하는 면역세포의 약제학적 유효량; 및 (b) 약제학적으로 허용되는 담체를 포함하는 감염질환의 예방 또는 치료용 약제학적 조성물일 수 있다. 본 발명은 또한, 본 발명에 따른 TIGIT에 대한 항체 또는 이의 항원 결합 단편, 상기 항체 또는 이의 항원 결합단편을 포함하는 이중 또는 다중특이적 항체, 상기 항체 또는 이의 항원 결합단편을 포함하는 항체-약물 접합체, 상기 항체 또는 이의 항원 결합단편을 포함하는 키메라 항원 수용체 또는 상기 키메라 항원 수용체를 포함하는 면역세포를 이를 필요로 하는 개체에 투여하는 단계를 포함하는 감염질환의 예방 또는 치료방법일 수 있다. The present invention includes, for example, (a) an antibody or antigen-binding fragment thereof against TIGIT according to the present invention, a bi- or multispecific antibody comprising the antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof a pharmaceutically effective amount of an antibody-drug conjugate, a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof, or an immune cell comprising the chimeric antigen receptor; And (b) it may be a pharmaceutical composition for preventing or treating an infectious disease comprising a pharmaceutically acceptable carrier. The present invention also provides an antibody or antigen-binding fragment thereof against TIGIT according to the present invention, a bi- or multispecific antibody comprising the antibody or antigen-binding fragment thereof, and an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof , It may be a method for preventing or treating an infectious disease comprising administering to an individual in need thereof, a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof, or an immune cell comprising the chimeric antigen receptor.
본 발명의 구체예에서, 상기 개체는 감염질환이 발병할 것으로 예상되는 개체; 발병한 개체; 또는 완치판정을 받은 개체일 수 있으나, 이에 제한되지 않는다.In an embodiment of the present invention, the subject is an individual expected to develop an infectious disease; affected individuals; Or it may be an individual who has been cured, but is not limited thereto.
"예방"은 본 발명에 따른 조성물의 투여로 질병의 임상 증상을 억제시키거나 진행을 지연시키는 모든 행위를 의미하며, "치료"는 질병에 대한 임상 증상 발전의 억제, 질병에 대한 임상 증상 경감 또는 제거를 의미한다."Prevention" means any action that suppresses or delays the progression of clinical symptoms of a disease by administration of the composition according to the present invention, and "treatment" refers to suppression of the development of clinical symptoms for a disease, alleviation of clinical symptoms for a disease, or means removal.
상기 감염질환은 바이러스 감염을 의미할 수 있다. 상기 바이러스에 예를 들어, 아데노연관 바이러스, 아이치(Aichi) 바이러스, 호주 박쥐 리사바이러스(lyssavirus), BK 폴리오마바이러스, 바나(Banna) 바이러스, 바마포리스트(Barmah forest) 바이러스, 부니암웨라(Bunyamwera) 바이러스, 분야바이러스 라 크로세(Bunyavirus La Crosse), 분야바이러스 눈덧신토끼(Bunyavirus snowshoe hare), 세르코피테신 헤르페스바이러스(Cercopithecine herpesvirus), 찬디퓨라(Chandipura) 바이러스, 치쿤군야(Chikungunya) 바이러스, 코사바이러스 A(Cosavirus A), 카우폭스 바이러스, 콕사키바이러스(Coxsackievirus), 크리민-콩고(Crimean-Congo) 출혈열 바이러스, 뎅기열 바이러스, 도리(Dhori) 바이러스, 두그베(Dugbe) 바이러스, 두벤하게(Duvenhage) 바이러스, 동부말 뇌염 바이러스, 에볼라바이러스, 에코바이러스, 뇌심근염 바이러스, 엡스타인-바르 바이러스, 유럽 박쥐 리사바이러스, GB 바이러스 C/G형 간염 바이러스, 한탄 바이러스, 헨드라 바이러스, A형 간염 바이러스, B형 간염 바이러스, C형 간염바이러스, E형 간염 바이러스, 델타 간염 바이러스, 마두바이러스, 인간 아데노바이러스, 인간 아스트로바이러스, 인간 코로나바이러스, 인간 사이토메갈로바이러스, 인간 엔테로바이러스, 인간 헤르페스바이러스 1, 인간 헤르페스바이러스 2, 인간 헤르페스바이러스 6, 인간 헤르페스바이러스 7, 인간 헤르페스바이러스 8, 인간 면역결핍 바이러스, 인유두종 바이러스 1, 인유두종바이러스 2, 인유두종바이러스, 인간 파라인플루엔자, 인간 파보바이러스 B19, 인간 호흡기 세포융합 바이러스, 인간 리노바이러스, 인간 SARS 코로나바이러스, 인간 스푸마레트로바이러스, 인간 T-림프친화성 바이러스, 인간 토로바이러스, 인플루엔자 A 바이러스, 인플루엔자 B 바이러스, 인플루엔자 C 바이러스, 이스파한 바이러스, JC 폴리오마바이러스, 일본뇌염 바이러스, 쥬닌 아레나바이러스, KI 폴리오마바이러스, 쿤진(Kunjin) 바이러스, 라고스(Lagos) 박쥐 바이러스, 레이크 빅토리아 마르부르그바이러스, 란가트(Langat) 바이러스, 라싸 바이러스, 로드스데일(Lordsdale) 바이러스, 도약병 바이러스, 림프구성 맥락 수막염 바이러스, 마추포(Machupo) 바이러스, 마야로(Mayaro) 바이러스, MERS 코로나바이러스, 홍역 바이러스, 멘고(Mengo) 뇌심근염 바이러스, 메르켈 세포 폴리오마바이러스, 모콜라(Mokola) 바이러스, 전염성 연속종 바이러스, 원두증 바이러스, 볼거리 바이러스, 머레이 밸리(Murray valley) 뇌염 바이러스, 뉴욕 바이러스, 니파 바이러스, 노워크 바이러스, 용뇽(O'nyong-nyong) 바이러스, Orf 바이러스, 오로포우체(Oropouche) 바이러스, 피킨드(Pichinde) 바이러스, 폴리오바이러스, 푼타 토로 플레보바이러스, 퓨말라(Puumala) 바이러스, 광견병 바이러스, 리프트 밸리열 바이러스, 로사바이러스 A, 로스강 바이러스, 로타바이러스 A, 로타바이러스 B, 로타바이러스 C, 루벨라 바이러스, 사기야마(Sagiyama) 바이러스, 살리바이러스 A, 모래파리열 시실리안 바이러스, 삿포로 바이러스, 셈리키 삼림열 바이러스, 서울 바이러스, 시미안 포아미 바이러스, 시미안 바이러스 5, 신도비스 바이러스, 사우샘프터 바이러스, 세인트루이스 뇌막염 바이러스, 진드기 매개 파와산 바이러스, 토크 테노 바이러스, 토스카나 바이러스, 유쿠니에미(Uukuniemi) 바이러스, 백시니아 바이러스, 바리셀라-조로아스터 바이러스, 바리올라 바이러스, 베네수엘라 말뇌염 바이러스, 수포성 구내염 바이러스, 서부말 뇌염 바이러스, WU 폴리오마바이러스, 웨스트 나일 바이러스, 야바 원숭이 종양 바이러스, 야바-유사 질환 바이러스, 황열 바이러스 및 지카 바이러스가 포함될 수 있다.The infectious disease may mean a viral infection. Such viruses include, for example, adeno-associated virus, Aichi virus, Australian bat lyssavirus, BK polyomavirus, Banna virus, Barmah forest virus, Bunyamwera Virus, Bunyavirus La Crosse, Bunyavirus snowshoe hare, Cercopithecine herpesvirus, Chandipura virus, Chikungunya virus, Cosavirus A (Cosavirus A), cowpox virus, Coxsackievirus, Crimean-Congo hemorrhagic fever virus, Dengue virus, Dhori virus, Dugbe virus, Duvenhage Virus, Eastern encephalitis virus, Ebola virus, echovirus, encephalomyocarditis virus, Epstein-Barr virus, European bat lyssavirus, GB virus Hepatitis C/G virus, Hantan virus, Hendra virus, Hepatitis A virus, Hepatitis B virus, hepatitis C virus, hepatitis E virus, hepatitis delta virus, maduvirus, human adenovirus, human astrovirus, human coronavirus, human cytomegalovirus, human enterovirus, human herpesvirus 1, human herpesvirus 2, human herpesvirus 6, human herpesvirus 7, human herpesvirus 8, human immunodeficiency virus, human papillomavirus 1, human papillomavirus 2, human papillomavirus, human parainfluenza, human parvovirus B19, human respiratory syncytial virus, human rhinovirus, Human SARS coronavirus, human spumaretrovirus, human T-lymphotropic virus, human torovirus, influenza A virus, influenza B virus, influenza C virus, lice Spahan virus, JC polyomavirus, Japanese encephalitis virus, Junin Arenavirus, KI polyomavirus, Kunjin virus, Lagos bat virus, Lake Victoria Marburg virus, Langat virus, Lasa virus , Lordsdale virus, hoppy virus, lymphocytic choriomeningitis virus, Machupo virus, Mayaro virus, MERS coronavirus, measles virus, Mengo encephalomyocarditis virus, Merkel cell Polyomavirus, Mokola virus, Molecular virus, schizophrenia virus, mumps virus, Murray valley encephalitis virus, New York virus, Nipah virus, Norwalk virus, O'nyong-nyong Virus, Orf Virus, Oropouche Virus, Pichinde Virus, Poliovirus, Punta Toro Flevovirus, Puumala Virus, Rabies Virus, Rift Valley Virus, Rosavirus A, Ross River Virus, Rotavirus A, Rotavirus B, Rotavirus C, Rubella Virus, Sagiyama Virus, Salivirus A, Sandfly Sicilian Virus, Sapporo Virus, Semliki Forest Fever Virus, Seoul Virus, Simian pho Army Virus, Simian Virus 5, Sindobis Virus, Southampter Virus, St. Louis Meningitis Virus, Tick-borne Pawasan Virus, Toctenovirus, Tuscan Virus, Uukuniemi Virus, Vaccinia Virus, Varicella-Zoroaster virus, variola virus, Venezuelan equine encephalitis virus, bullous stomatitis virus, western equine encephalitis virus, WU polyomavirus, West Nile virus, yaba monkey tumor virus, yaba-like disease virus, yellow fever virus and Zika virus. .
상기 암은 예를 들어, 호지킨 림프종, 비-호지킨 림프종(예컨대, B 세포 림프종, 미만성 거대 B 세포 림프종, 여포성 림프종, 만성 림프구성 백혈병, 외투세포 림프종, 변연구역 B 세포 림프종, 버킷 림프종, 림프형질세포 림프종, 모상 세포 백혈병), 급성 골수성 백혈병, 만성 골수성 백혈병, 골수형성이상 증후군, 다발성 골수종 또는 급성 림프구성 백혈병을 포함한다. The cancer is, for example, Hodgkin's lymphoma, non-Hodgkin's lymphoma (eg, B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, marginal area B-cell lymphoma, Burkitt's lymphoma) , lymphoid cell lymphoma, hairy cell leukemia), acute myeloid leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, multiple myeloma or acute lymphocytic leukemia.
상기 암은 예를 들어, 난소암, 직장암, 위암, 고환암, 항문 부위 암, 자궁암, 결장암, 직장암, 신장 세포 암종, 간암, 폐의 비-소세포 암종, 소장 암, 식도암, 흑색종, 카포시 육종, 내분비계 암, 갑상선암, 부갑상선 암, 부신암, 골암, 췌장암, 피부암, 두경부암, 피부 또는 안내 악성 흑색종, 자궁암, 뇌줄기 신경아교종, 뇌하수체 선암, 표피모양 암, 자궁경부 편평세포 암의 암종, 난관 암종, 자궁내막 암종, 질 암종, 연조직 육종, 요도암, 외음부 암종, 음경암, 방광암, 신장암 또는 요관암, 신우 암종, 척추 종양, 중추신경계(CNS)의 신생물, 원발성 CNS 림프종, 종양 혈관신생, 상기 암들의 전이 병변 또는 상기 암의 조합을 포함한다.Said cancer is, for example, ovarian cancer, rectal cancer, gastric cancer, testicular cancer, anal region cancer, uterine cancer, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, small intestine cancer, esophageal cancer, melanoma, Kaposi's sarcoma, Endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, brain stem glioma, pituitary adenocarcinoma, epidermal cancer, carcinoma of the cervical squamous cell carcinoma, fallopian tube Carcinoma, endometrial carcinoma, vaginal carcinoma, soft tissue sarcoma, urethral cancer, vulvar carcinoma, penile cancer, bladder cancer, kidney or ureter cancer, renal pelvic carcinoma, spinal tumor, neoplasm of central nervous system (CNS), primary CNS lymphoma, tumor vascular neonatal, metastatic lesions of the cancers or combinations of the cancers.
상기 암은 예를 들어, 교모세포종, 폐암, 방광암, 구강암, 두경부 편평세포암, 담낭암 또는 자궁경부암일 수 있다. 특히, 교모세포종의 경우 상기 항체 또는 이의 항원 결합단편이 뇌 및 척추와 그 주변 순환계 사이에 존재하는 뇌의 모세혈관 내피세포 막내에 타이트 결합 (junction)에 의해 형성된 장벽인 혈액뇌장벽 (blood-brain barrier)을 통과할 필요가 있다. BBB 통과를 위해 전달체와 연결되어 사용될 수 있다. BBB를 통하여 약물을 전달하기 위해 예를 들면 브래드키닌 또는 HIFU(Hign density foucues ultrasound)과 같은 방법을 사용하여 BBB의 삼투압을 와해시키는 방법이 있다. 또한, 세포에 내재된 글루코스 및 아미노산 전달체, 인슐린 또는 트랜스페린의 수용체 매개 트랜스사이토시스와 같은 전달 시스템의 사용 또는 당단백질의 활성 유출 전달(efflux transporter)을 차단하는 것이 포함될 수 있다.The cancer may be, for example, glioblastoma, lung cancer, bladder cancer, oral cancer, head and neck squamous cell cancer, gallbladder cancer or cervical cancer. In particular, in the case of glioblastoma, the antibody or antigen-binding fragment thereof is a barrier formed by a tight junction in the capillary endothelial cell membrane of the brain that exists between the brain and spine and the surrounding circulatory system, the blood-brain barrier (blood-brain barrier). barrier) needs to be passed. It can be used in conjunction with a transporter to cross the BBB. In order to deliver a drug through the BBB, for example, there is a method of disrupting the osmotic pressure of the BBB using a method such as bradkinin or HIFU (Hign density fucues ultrasound). It may also include the use of delivery systems such as receptor-mediated transcytosis of glucose and amino acid transporters, insulin or transferrin, or blocking the active efflux transporter of glycoproteins in cells.
본 발명의 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토오스, 덱스트로오스, 수크로오스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산칼슘, 미세결정성 셀룰로오스, 폴리비닐피롤리돈, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. Pharmaceutically acceptable carriers included in the composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate. , microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components.
본 발명의 약제학적 조성물은 경구 또는 비경구로 투여할 수 있고, 비경구 투여인 경우에는 정맥내 주입, 피하 주입, 근육 주입, 복강 주입, 내피 투여, 국소 투여, 비내 투여, 폐내 투여 및 직장 내 투여 등으로 투여할 수 있다.The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration and rectal administration etc. can be administered.
경구 투여시, 단백질 또는 펩타이드는 소화가 되기 때문에 경구용 조성물은 활성 약제를 코팅하거나 위에서의 분해로부터 보호되도록 제형화 되어야 한다. 또한, 약제학적 조성물은 활성 물질이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다.Since the protein or peptide is digested upon oral administration, oral compositions should be formulated to coat the active agent or to protect it from degradation in the stomach. In addition, the pharmaceutical composition may be administered by any device capable of transporting the active agent to a target cell.
본 발명에 따른 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사는 소망하는 치료 또는 예방에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다. 예를 들어, 본 발명의 약제학적 조성물의 1일 투여량은 0.0001-100 ㎎/㎏이다. 본 명세서에서 용어 "약제학적 유효량"은 암 또는 감염질환의 예방 또는 치료하는 데 충분한 양을 의미한다.A suitable dosage of the composition according to the present invention varies depending on factors such as formulation method, administration mode, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity of the patient, usually Thus, a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention. For example, the daily dosage of the pharmaceutical composition of the present invention is 0.0001-100 mg/kg. As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to prevent or treat cancer or infectious diseases.
본 발명의 약제학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화 함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이 때 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 산제, 좌제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. or it may be prepared by incorporation into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in oil or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.
병용치료combination therapy
본 발명은 면역세포를 포함하는 병용 치료용 조성물에 관한 것이다. The present invention relates to a composition for combination therapy comprising immune cells.
하나의 실시예에서, 상기 조성물은 화학요법제를 더 포함할 수 있고, 상기 화합요법제는 마이탄시노이드, 오리스타틴 (MMAE, MMAF 포함), 아미노프테린, 악티노마이신, 블레오마이신, 탈리소마이신, 캄프토쎄신, N8-아세틸 스퍼미딘, 1-(2 클로로에틸)-1,2-다이메틸 술포닐 하이드라자이드, 에스퍼라마이신, 에토포사이드, 6-머캅토퓨린, 돌라스타틴, 트리코테센, 칼리케아미신, 탁솔(taxol), 탁산, 파클리탁셀(paclitaxel), 도세탁셀(docetaxel), 메토트렉세이트, 빈크리스틴, 빈블라스틴, 독소루비신, 멜팔란, 미토마이신 A, 미토마이신 C, 클로람부실, 듀오카마이신, L-아스파라기나제(L-asparaginase), 머캡토퓨린(mercaptopurine), 티오구아닌(thioguanine), 하이드록시우레아(hydroxyurea), 시타라빈(cytarabine), 사이클로포스파미드(cyclophosphamide), 이포스파미드(ifosfamide), 니트로소우레아(nitrosourea), 시스플라틴(cisplatin), 카보플라틴(carboplatin), 미토마이신(mitomycin), 다카바진(dacarbazine), 프로카바진(procarbazine), 토포테칸(topotecan), 질소 머스터드(nitrogen mustard), 사이톡산(cytoxan), 에토포시드(etoposide),5-플루오로우라실(5-fluorouracil), CNU(bischloroethylnitrosourea), 이리노테칸(irinotecan), 캄포토테신(camptothecin), 블레오마이신(bleomycin), 이다루비신(idarubicin), 다우노루비신(daunorubicin), 닥티노마이신(dactinomycin), 플리카마이신(plicamycin), 미톡산트론(mitoxantrone), 아스파라기나제(asparaginase), 비노렐빈(vinorelbine), 클로로람부실(chlorambucil), 멜파란(melphalan), 카르무스틴(carmustine), 로무스틴(lomustine), 부설판(busuLfan), 트레오설판(treosulfan), 데카바진(decarbazine), 에토포시드(etoposide), 테니포시드(teniposide), 토포테칸(topotecan), 9-아미노캠프토테신(9-aminocamptothecin), 크리스나톨(crisnatol), 미토마이신 C(mitomycin C), 트리메트렉세이트(trimetrexate), 마이코페놀산(mycophenolic acid), 티아조퓨린(tiazofurin), 리바비린(ribavirin), EICAR(5-ethynyl-1-beta-Dribofuranosylimidazole-4-carboxamide), 하이드록시우레아(hydroxyurea), 데프록사민(deferoxamine), 플룩수리딘(floxuridine), 독시플루리딘(doxifluridine), 랄티트렉세드(raltitrexed), 시타라빈(cytarabine(ara C)), 시토신 아라비노시드(cytosine arabinoside), 플루다라빈(fludarabine), 타목시펜(tamoxifen), 라록시펜(raloxifene), 메게스트롤(megestrol), 고세렐린(goserelin), 류프롤리드 아세 테이트(leuprolide acetate), 플루타미드(flutamide), 바이칼루타마이드(bicalutamide), EB1089, CB1093, KH1060, 베르테포르핀(verteporfin), 프탈로시아닌(phthalocyanine), 광감작제 Pe4(photosensitizer Pe4), 데메톡시-하이포크레린 A(demethoxy-hypocrellin A), 인터페론-α(Interferon-α), 인터페론-γ(Interferon-γ), 종양 괴사 인자(tumor necrosis factor), 겜사이타빈(Gemcitabine), 벨케이드(velcade), 레발미드(revamid), 탈라미드(thalamid), 로바스타틴(lovastatin), 1-메틸-4-페닐피리디늄 이온(1-methyl-4-phenylpyridiniumion), 스타우로스포린(staurosporine), 악티노마이신 D(actinomycin D), 닥티노마이신(dactinomycin), 블레오마이신 A2(bleomycin A2), 블레오마이신 B2(bleomycinB2), 페플로마이신(peplomycin), 에피루비신(epirubicin), 피라루비신(pirarubicin), 조루비신(zorubicin), 마이토산트론(mitoxantrone), 베라파밀(verapamil), 탑시가르긴(thapsigargin), 핵산 분해 효소 및 세균이나 동식물 유래의 독소로 구성된 군에서 선택된 하나 이상일 수 있다. In one embodiment, the composition may further include a chemotherapeutic agent, wherein the chemotherapeutic agent is maytansinoid, auristatin (including MMAE, MMAF), aminopterin, actinomycin, bleomycin, thali Somycin, camptothecin, N8-acetyl spermidine, 1-(2 chloroethyl)-1,2-dimethyl sulfonyl hydrazide, esperamicin, etoposide, 6-mercaptopurine, dolastatin, tricho Tecene, calicheamicin, taxol, taxane, paclitaxel, docetaxel, methotrexate, vincristine, vinblastine, doxorubicin, melphalan, mitomycin A, mitomycin C, chlorambucil, duo Carmycin, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifospha amide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustard (nitrogen mustard), cytoxan (cytoxan), etoposide (etoposide), 5-fluorouracil (5-fluorouracil), CNU (bischloroethylnitrosourea), irinotecan (irinotecan), camptothecin (camptothecin), bleomycin ( bleomycin), idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinorelbine , chlorambucil, melphalan, carmustine (carmu) stine), lomustine, busuLfan, treosulfan, decarbazine, etoposide, teniposide, topotecan, 9-amino Camptothecin (9-aminocamptothecin), cristol (crisnatol), mitomycin C (mitomycin C), trimetrexate, mycophenolic acid, thiazofurin, ribavirin , EICAR (5-ethynyl-1-beta-Dribofuranosylimidazole-4-carboxamide), hydroxyurea, deferoxamine, floxuridine, doxifluridine, raltitrexed (raltitrexed), cytarabine (ara C), cytosine arabinoside, fludarabine, tamoxifen, raloxifene, megestrol, goose Relin, leuprolide acetate, flutamide, bicalutamide, EB1089, CB1093, KH1060, verteporfin, phthalocyanine, photosensitizer Pe4 (photosensitizer Pe4), demethoxy-hypocrellin A, interferon-α, interferon-γ, tumor necrosis factor, gemcitabine (Gemcitabine), velcade (velcade), revalmid (revamid), thalamid (thalamid), lovastatin (lovastatin), 1-methyl-4-phenylpi Lidinium ion (1-methyl-4-phenylpyridiniumion), staurosporine, actinomycin D (actinomycin D), dactinomycin, bleomycin A2 (bleomycin A2), bleomycin B2 (bleomycin B2) , peplomycin, epirubicin, pirarubicin, zorubicin, mitoxantrone, verapamil, thapsigargin, nuclease And it may be at least one selected from the group consisting of toxins derived from bacteria or animals and plants.
하나의 실시예에서, 상기 조성물은 FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137(4-1BB), VISTA, CD258(LIGHT), MARCO, CD134(OX40), CD28, CD278(ICOS), CD27, CD154(CD40L), CD357(GITR), CD30, DR3, CD226(DNAM1), CD96, CD200, CD200R, 트랜스페린 수용체 (Transferrin receptor), c-Met, EGFR, HER2, KDR, PDGFRa 및 NRP1으로 구성된 군에서 선택된 하나 이상을 타겟하는 항체 또는 이의 항원 결합단편일 수 있다. In one embodiment, the composition comprises FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137(4-1BB), VISTA, CD258(LIGHT), MARCO, CD134(OX40) ), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2 , KDR, PDGFRa, and may be an antibody or antigen-binding fragment thereof targeting one or more selected from the group consisting of NRP1.
본 발명은 항체 또는 이의 항원 결합단편과 다음으로 구성된 군에서 선택된 하나 이상을 포함하는 병용 치료용 조성물에 관한 것이다.The present invention relates to a composition for combination therapy comprising an antibody or antigen-binding fragment thereof and at least one selected from the group consisting of the following.
(i) 면역세포; (i) immune cells;
(ii) 면역관문 억제제에 대한 항체에 대한 scFv를 세포외도메인으로 포함하는 항원 수용체(CAR)를 포함하는 면역세포; 및(ii) immune cells comprising an antigen receptor (CAR) comprising an scFv for an antibody against an immune checkpoint inhibitor as an extracellular domain; and
(iii) 면역관문억제제 (Immune checkpoint inhibitor).(iii) Immune checkpoint inhibitors.
또한, 본 발명은 상기 이중특이적 또는 다중특이적 항체 및 다음으로 구성된 군에서 선택된 하나 이상을 포함하는 병용 치료용 조성물에 관한 것이다:The present invention also relates to a composition for combination therapy comprising the bispecific or multispecific antibody and at least one selected from the group consisting of:
(i) 면역세포; (i) immune cells;
(ii) 면역관문 억제제에 대한 항체에 대한 scFv 단편을 세포외도메인으로 포함하는 항원 수용체(CAR)를 포함하는 면역세포; 및(ii) immune cells comprising an antigen receptor (CAR) comprising an scFv fragment for an antibody against an immune checkpoint inhibitor as an extracellular domain; and
(iii) 면역관문억제제 (Immune checkpoint inhibitor).(iii) Immune checkpoint inhibitors.
상기 면역세포는 면역 치료 예를 들어 면역을 유도하여 목적하는 암 치료 효과를 유발할 수 있는 것으로, 예를 들어, T 세포, NK 세포, 사이토카인 유도 살해세포(Cytokine Induced Killer cell, CIK), 활성화 세포독성 T 림프구(Cytotoxic T Lymphocyte, CTL), 마크로파지, 종양 조직 내 침투 T 세포 (Tumor-Infiltrating Lymphocytes, TIL) 및 수지상세포로 구성된 군에서 선택될 수 있으나, 이에 제한되는 것은 아니다.The immune cells are capable of inducing a desired cancer therapeutic effect by inducing immune treatment, for example, immunity, for example, T cells, NK cells, cytokine-induced killer cells (CIK), activated cells. Cytotoxic T lymphocytes (CTL), macrophages, tumor tissue infiltrating T cells (Tumor-Infiltrating Lymphocytes, TIL) and may be selected from the group consisting of dendritic cells, but is not limited thereto.
상기 면역관문 억제제에 대한 항체는 TIGIT 이외의 면역관문 억제제를 표적을 타겟하는 항체로, 예를 들어, 상기 면역관문 억제제에 대한 항체는 FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137(4-1BB), VISTA, CD258(LIGHT), MARCO, CD134(OX40), CD28, CD278(ICOS), CD27, CD154(CD40L), CD357(GITR), CD30, DR3, CD226(DNAM1), CD96, CD200, CD200R, 트랜스페린 수용체 (Transferrin receptor), c-Met, EGFR, HER2, KDR, PDGFRa 및 NRP1에 결합하는 항체 또는 이의 항원 결합단편일 수 있으나, 이에 제한되는 것은 아니다.The antibody against the checkpoint inhibitor is an antibody that targets an immune checkpoint inhibitor other than TIGIT, for example, the antibody against the checkpoint inhibitor is FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), MARCO, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, It may be an antibody or antigen-binding fragment thereof that binds to CD226 (DNAM1), CD96, CD200, CD200R, transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa and NRP1, but is not limited thereto.
상기 면역관문억제제는 항원 제시 세포 (APC, antigen presenting cell)와 면역세포 예를 들어 T세포가 만나는 부위로 T세포 억제 신호를 차단하여, T 세포 활성화를 유도할 수 있는 제제를 의미한다. 상기 면역관문억제제는 예를 들어, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137(4-1BB), CD258(LIGHT), MARCO, CD134(OX40), CD28, CD278(ICOS), CD27, CD154(CD40L), CD357(GITR), CD30, DR3, CD226(DNAM1), CD96, CD200 또는 CD200R를 타겟으로 하는 약물일 수 있으나, 이에 제한되는 것은 아니다.The immune checkpoint inhibitor refers to an agent capable of inducing T cell activation by blocking the T cell inhibitory signal at a site where antigen presenting cells (APCs) and immune cells, for example, T cells meet. The immune checkpoint inhibitor is, for example, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), CD258 (LIGHT), MARCO, CD134 (OX40), CD28, It may be a drug targeting CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200 or CD200R, but is not limited thereto.
상기 병용 투여 대상 제1성분 및 제2성분 각각은 동시에 투여될 수 있다. 또한, 상기 병용 투여 대상 제1성분 및 제2성분 각각은 일정 시간 간격을 두고 별도로 투여할 수도 있다. 상기 병용 투여 대상 중 제1성분 투여 전 또는 후 제2성분이 별도로 투여될 수 있다. Each of the first component and the second component to be administered in combination may be administered simultaneously. In addition, each of the first component and the second component to be administered in combination may be administered separately at a predetermined time interval. The second component may be separately administered before or after administration of the first component among the subject of the combined administration.
동반진단(Companion diagnostics, CDx)Companion diagnostics (CDx)
동반진단은 특정 약물치료에 대한 환자의 반응성을 예측하기 위한 분자 진단 기법의 일종으로, 약물의 적용에 있어, 적용 대상 환자의 선별 기준을 제시하고 이에 부합하는 진단, 예후 및 예측 바이오마커의 특이성 및 민감성을 고려하여, 환자에 적합한 약물을 처리할 수 있도록 하는 기준을 제시할 수 있다.Companion diagnostics is a type of molecular diagnostic technique for predicting the patient's responsiveness to a specific drug treatment. Considering the sensitivity, it is possible to present a standard that allows the treatment of a drug suitable for the patient.
항암제의 경우 반응을 보이는 환자에게서는 높은 효과를 보이지만 반응하는 환자비율이 낮기 때문에 효과를 보일 환자를 선별해내는 것이 중요하며, 동반진단을 통해 항암제에 반응하는 환자를 선별하고, 환자의 치료 효율을 증대시킬 수 있다. In the case of anticancer drugs, it is highly effective in patients who respond, but because the proportion of patients who respond is low, it is important to select patients who will be effective. can do it
상기 샘플은 환자의 조직 또는 체액으로부터 수득되는 생물학적 피검체를 의미한다. 조직 샘플의 공급원은 동결된 및/또는 보존된 기관, 조직 샘플, 생검 또는 흡인물에서 유래되는 것과 같은 고형 조직; 혈액 또는 임의의 혈액 구성성분(예, 혈청, 혈장); 골수 또는 임의의 골수 구성성분; 소변, 뇌척수액, 전혈액, 혈장 및 혈청과 같은 체액일 수 있다. 샘플은 비-세포 분획(예, 소변, 혈장, 혈청 또는 다른 비세포 체액)을 포함할 수 있다. 샘플은 체액 예를 들어, 혈액(예, 전혈액)을 포함할 수 있다. 구체적으로, 상기 샘플은 환자로부터 수득되는 전혈액 샘플, 전 골수 샘플, 전 말초혈액 샘플 또는 전 종양 샘플일 수 있다. 상기 "전" 샘플은 해당 샘플로부터 제거되거나 분리된 성분(예, 세포)이 실질적으로 없는 샘플이다. 상기 샘플, 예를 들어 혈액 샘플은 사용되기 전에 희석(예컨대, 생리적 융화성 완충액 또는 배지에 의해)될 수 있다. 다른 구체예들에서, "전" 샘플, 예컨대 전 조직 샘플 또는 전 종양 샘플은 기원 조직 유래의 미세환경을 실질적으로 유지하고, 예를 들어 종양 또는 면역 미세환경의 구조를 실질적으로 유지할 수 있다. 구체적으로, 샘플, 예컨대 종양 샘플은 더 작은 조각으로 가공(예, 마쇄, 다짐, 블렌딩, 분쇄 등)되고 희석(예컨대, 생리적 융화성 완충액 또는 배지에 의해)될 수 있다.The sample refers to a biological subject obtained from a tissue or body fluid of a patient. Sources of tissue samples include solid tissue, such as those derived from frozen and/or preserved organs, tissue samples, biopsies, or aspirates; blood or any blood component (eg, serum, plasma); bone marrow or any component of bone marrow; It can be body fluids such as urine, cerebrospinal fluid, whole blood, plasma and serum. A sample may comprise a non-cellular fraction (eg, urine, plasma, serum or other non-cellular body fluid). A sample may include a bodily fluid, such as blood (eg, whole blood). Specifically, the sample may be a whole blood sample, a whole bone marrow sample, a whole peripheral blood sample, or a whole tumor sample obtained from a patient. The "pre" sample is a sample that is substantially free of components (eg, cells) that have been removed or isolated from the sample. The sample, eg, a blood sample, may be diluted (eg, with a physiologically compatible buffer or medium) prior to use. In other embodiments, a “pre” sample, such as a whole tissue sample or a whole tumor sample, substantially retains the microenvironment from the tissue of origin, eg, may substantially retain the structure of the tumor or immune microenvironment. Specifically, a sample, such as a tumor sample, can be processed (eg, ground, minced, blended, milled, etc.) into smaller pieces and diluted (eg, with a physiologically compatible buffer or medium).
상기 유전자의 발현 확인은 해당 유전자의 존재 여부와 발현 정도를 확인하는 과정으로, 중합효소연쇄반응(PCR)에 의해 수행될 수 있고, 경우에 따라서 전사 중합효소반응(RT-PCR), 경쟁적 역전사 중합효소반응(Competitive RT-PCR), 실시간 역전사 중합효소반응(Real-time RT-PCR), RNase 보호 분석법(RPA; RNase protection assay), 노던 블럿팅(Northern blotting), DNA 칩을 사용하여 수행될 수 있다. Confirmation of the expression of the gene is a process of confirming the presence and level of expression of the gene, and may be performed by polymerase chain reaction (PCR), and in some cases transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerization Competitive RT-PCR, Real-time RT-PCR, RNase protection assay (RPA), Northern blotting, can be performed using a DNA chip. have.
중합효소연쇄반응(PCR)은 프라이머를 이용하여 유전자 증폭 반응을 통해 수행될 수 있다. 멀티플렉스 PCR, 실-시간(real-time) PCR, 분별 디스플레이 PCR(differential display PCR: DD-PCR), cDNA 말단의 신속 증폭(rapid amplification of cDNA ends: RACE), 인버스 중합효소 연쇄반응(inverse polymerase chain reaction: IPCR), 벡토레트(vectorette) PCR 및 TAIL-PCR(thermal asymmetric interlaced PCR) 등이 수행될 수 있다. Polymerase chain reaction (PCR) can be performed through a gene amplification reaction using a primer. Multiplex PCR, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), inverse polymerase chain reaction chain reaction: IPCR), vectorette PCR, and TAIL-PCR (thermal asymmetric interlaced PCR) may be performed.
유전자 코딩에 의한 단백질 발현 여부를 검출할 수도 있다. 상기 유전자로부터 발현된 단백질의 존재 여부와 발현 정도를 확인하는 과정이다. 상기 단백질에 대하여 특이적으로 결합하는 항체, 올리고펩타이드, 리간드, PNA(Peptide nucleic acid) 또는 앱타머(aptamer) 등을 이용하여 단백질의 양을 확인할 수 있다. It is also possible to detect whether a protein is expressed by gene coding. It is a process of confirming the presence and expression level of the protein expressed from the gene. The amount of the protein can be confirmed using an antibody, oligopeptide, ligand, PNA (peptide nucleic acid) or aptamer that specifically binds to the protein.
이를 위한 분석 방법으로는 웨스턴 블랏, 엘라이자(enzyme linked immunosorbent assay, ELISA), 방사선면역분석(RIA: Radioimmunoassay), 방사 면역확산법(radioimmunodiffusion), 오우크테로니(Ouchterlony) 면역 확산법, 로케트(rocket) 면역전기영동, 조직면역염색, 면역침전 분석법(Immunoprecipitation Assay), 보체 고정 분석법(Complement Fixation Assay), 유세포분석(Fluorescence Activated Cell Sorter, FACS), 단백질 칩(protein chip)을 이용한 방법 등이 포함될 수 있다.Analysis methods for this include Western blot, ELISA (enzyme linked immunosorbent assay, ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immune diffusion method, and rocket Immunoelectrophoresis, tissue immunostaining, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), a method using a protein chip, etc. may be included. .
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.
실시예 1. 후보 클론의 항원결합력 분석Example 1. Analysis of antigen binding affinity of candidate clones
재조합 인간 TIGIT-His 단백질로 마우스를 immunize하고 그 spleen과 SP2/0 myeloid cells과 fusion하여 hybridoma clones을 생성하였다. 후보 항체는 hybridoma 배양액을 사용하여 재조합 인간 TIGIT-His 단백질에 결합력을 가지는 clones을 ELISA 방법에 의해 1차 선별하였고, 인간 PBMC를 분리하여 anti-hCD3/28로 activation을 시킨 후 다시 hybridoma 배양액을 사용하여 T cells에서 결합력을 가지는 clones을 flow cytometry 방법에 의해 2차 선별하였다. flow cytometry 결과는 도 1에 나타내었다.Mice were immunized with recombinant human TIGIT-His protein, and hybridoma clones were generated by fusion with the spleen and SP2/0 myeloid cells. For candidate antibodies, clones having binding affinity to recombinant human TIGIT-His protein were first selected by ELISA using a hybridoma culture medium. Clones having binding affinity from T cells were secondarily selected by flow cytometry. The flow cytometry results are shown in FIG. 1 .
도 1에 나타낸 바와 같이, clone 58E, 62F, 63F 및 75G가 재조합 인간 TIGIT-His 단백질에 대한 항원결합력이 우수한 것을 확인하였다.As shown in FIG. 1 , it was confirmed that clones 58E, 62F, 63F and 75G had excellent antigen-binding ability to recombinant human TIGIT-His protein.
후보항체가 TIGIT과 그 counterpart인 CD155와의 결합을 inhibition할 수 있는 효능을 조사하였다. 구체적으로, 효능 실험은 TIGIT/CD155 Blockade Bioassay (Promega) kit을 사용하였으며 인간 TIGIT과 CD226을 발현하고 luciferase reporter를 가지는 Jurkat Effector cells과 CD155를 발현하는 CHO-K1 aAPC를 co-culture하였을 때, 후보항체 처리 시 항체가 TIGIT과 결합하여 CD155와의 결합을 block하고 CD155가 CD226과 결합함으로써 activation되어진 luciferase signal을 측정할 수 있는 방법을 사용하였다. 선별된 후보항체(clone 19D, 58E, 62F, 63F 및 75G)의 처리는 hybridoma 배양액으로부터 소량 정제하여 사용하였다. 후보항체의 억제 효능을 확인한 결과는 도 2에 나타내었다.The efficacy of the candidate antibody to inhibit the binding of TIGIT to its counterpart CD155 was investigated. Specifically, the efficacy test was performed using the TIGIT/CD155 Blockade Bioassay (Promega) kit. When Jurkat Effector cells expressing human TIGIT and CD226 and having a luciferase reporter and CHO-K1 aAPC expressing CD155 were co-cultured, candidate antibodies During treatment, the antibody binds to TIGIT to block the binding to CD155, and a method capable of measuring the activated luciferase signal by binding of CD155 to CD226 was used. For the treatment of the selected candidate antibodies (clone 19D, 58E, 62F, 63F and 75G), a small amount was purified from the hybridoma culture medium and used. The results of confirming the inhibitory efficacy of the candidate antibody are shown in FIG. 2 .
도 2에 나타낸 바와 같이, clone 19D, 58C, 62F 및 63F가 TIGIT과 결합하여 CD155와의 결합을 차단하는 효능이 우수한 것을 확인하였다.As shown in FIG. 2 , it was confirmed that clones 19D, 58C, 62F and 63F had excellent efficacy in blocking binding to CD155 by binding to TIGIT.
상기 결과로부터 최종 clone 19D, 58C, 58E, 62F, 63F 및 75G을 선별하였고 각 클론의 서열분석을 하였다. From the above results, final clones 19D, 58C, 58E, 62F, 63F and 75G were selected and each clone was sequenced.
실시예 2. 선별된 개별클론의 서열분석Example 2. Sequencing of selected individual clones
선별된 클론의 VH와 VL 및 CDR region 시퀀스 분석 결과는 표 1에 나타내었다.Table 1 shows the results of sequence analysis of the VH, VL, and CDR regions of the selected clones.
선별된 클론의 아미노산 서열Amino acid sequence of selected clones
클론clone 구분division 서열order 서열번호SEQ ID NO: 서열order 서열번호SEQ ID NO:
19D19D VHVH NYVMHNYVMH 1 One EVQLQQSGPELVKPGASVKMSCKASGYTFSNYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDRSSSTAYMELSSLTSE DSAVYYCARWRVNYYFDYWGQGTTLTVSS EVQLQQSGPELVKPGASVKMSCKASGYTFS NYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDRSSSTAYMELSSLTSEDSAVYYCARWRVNYYFDYWGQGTTLTVSS 77
YNDGTKYNEKFKG YNDGTKYNEKFKG 22
RWRVNYYFDY RWRVNYYFDY 33
VL VL RASSSVNYMHRASSSVNYMH 4 4 QIVLSQSPAILSASPGEKVTMTCRASSSVNYMHWYQQKPGSSPKAWISVTSNLASGVPARFSGSGSGTSYSLTISRVE AEDAATYYCQQWSSNPPWTFGGGTKLEIK QIVLSQSPAILSASPGEKVTMTC RASSSVNYMHWYQQKPGSSPKAWISVTSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPPWTFGGGTKLEIK 88
VTSNLAS VTSNLAS 55
QQWSSNPPWT QQWSSNPPWT 66
58C 58C VHVH GYIMNGYIMN 9 9 EVQLQQSGPELVKPGASMKISCKASGYSFTGYIMNWVKQSHGKNLEWIGLINPYNGGSSYNQKFKGKATLTVDKSSSTAYMELLSLTSED SAVYYCARFLRGYYAVDYWGQGTSVTVSS EVQLQQSGPELVKPGASMKISCKASGYSFT GYIMNWVKQSHGKNLEWIGLINPYNGGSSYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARFLRGYYAVDYWGQGTSVTVSS 1515
LINPYNGGSSYNQKFKG LINPYNGGSSYNQKFKG 1010
RFLRGYYAVDY RFLRGYYAVDY 1111
VL VL RSSQSLVNSYGHTYLSRSSQSLVNSYGHTYLS 1212 DVVVTQTPLSLPVSFGDQVSISCRSSQSLVNSYGHTYLSWYLHKPGQSPQLLIYGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPWTFGGGTKLEIK DVVVTQTPLSLPVSFGDQVSISC RSSQSLVNSYGHTYLSWYLHKPGQSPQLLIYGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPWTFGGGTKLEIK 1616
GISNRFS GISNRFS 1313
LQGTHQPWT LQGTHQPWT 1414
58E 58E VHVH SYVMHSYVMH 1717 EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINLNNDGTKYNEKFKGKATLSSDKSSSTAYMELSSLTSEDSAVYYCARWRVYYYAMDYWGQGTSVTVSS EVQLQQSGPELVKPGASVKMSCKASGYTFT SYVMHWVKQKPGQGLEWIGYINLNNDGTKYNEKFKGKATLSSDKSSSTAYMELSSLTSEDSAVYYCARWRVYYYAMDYWGQGTSVTVSS 2323
YINLNNDGTKYNEKFKG YINLNNDGTKYNEKFKG 1818
WRVYYYAMDY WRVYYYAMDY 1919
VL VL SASSSVNYMHSASSSVNYMH 2020 EIVLTQSPAITAASLGQKVTITCSASSSVNYMHWYQQKSGTSPRPWIYEISKLASGVPARFSGSGSGTSYSLTISSMEAEDAAIYYCQLWNYPLITFGAGTKLELK EIVLTQSPAITAASLGQKVTITC SASSSVNYMHWYQQKSGTSPRPWIYEISKLASGVPARFSGSGSGTSYSLTISSMEAEDAAIYYCQLWNYPLITFGAGTKLELK 2424
EISKLAS EISKLAS 2121
QLWNYPLIT QLWNYPLIT 2222
62F 62F VHVH EYTMHEYTMH 2525 EVQLQQSGPELVKPGASVKISCKTSGFTFTEYTMHWVKQSHGKSLEWIGGFNPNSGGSSYNQKFKGKATLTVDKSSSTAFMELRSLTSEDSAAYYCARSSYYRFDLDYWGQGTTLTVSS EVQLQQSGPELVKPGASVKISCKTSGFTFT EYTMHWVKQSHGKSLEWIGGFNPNSGGSSYNQKFKGKATLTVDKSSSTAFMELRSLTSEDSAAYYCARSSYYRFDLDYWGQGTTLTVSS 3131
GFNPNSGGSSYNQKFKG GFNPNSGGSSYNQKFKG 2626
SSYYRFDLDY SSYYRFDLDY 2727
VLVL KASQDVKTAVAKASQDVKTAVA 28 28 DIVMTQSHKFMSTSVGDRVSITCKASQDVKTAVAWYQQKPGQSPQLLIYSASFRYTGVPDRFTGSGSGADFTFTISSV QAEDLAAYYCQQHYTTPWTFGGGTKLEIK DIVMTQSHKFMSTSVGDRVSITC KASQDVKTAVAWYQQKPGQSPQLLIYSASFRYTGVPDRFTGSGSGADFTFTISSVQAEDLAAYYCQQHYTTPWTFGGGTKLEIK 3232
SASFRYT SASFRYT 2929
QQHYTTPWT QQHYTTPWT 3030
63F 63F VHVH NYWMNNYWMN 3333 EVQLEEFGGGLVQPGGSMKLSCVASGSTFSNYWMNWVRQSPEKGLEWVAEIRLKFNNYATHYVESVIGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTMVRRNWYFDVWGAGTTVTVSS EVQLEEFGGGLVQPGGSMKLSCVASGSTFS NYWMNWVRQSPEKGLEWVAEIRLKFNNYATHYVESVIGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTMVRRNWYFDVWGAGTTVTVSS 3939
EIRLKFNNYATHYVESVIG EIRLKFNNYATHYVESVIG 3434
VRRNWYFDV VRRNWYFDV 3535
VL1 VL1 SASSSVSSSYLYSASSSVSSSYLY 3636 QIVLTQSPAIMSAFPGEKVTLTCSASSSVSSSYLYWYQQKPRSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQWSSYPYTFGGGTKLEIK QIVLTQSPAIMSAFPGEKVTLTC SASSSVSSSYLYWYQQKPRSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQWSSYPYTFGGGTKLEIK 4040
STSNLAS STSNLAS 3737
HQWSSYPYT HQWSSYPYT 3838
VL2VL2 KSSQSLLDSDRKTYLNKSSQSLLDSDRKTYLN 4141 DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDRKTYLNWLLQRPGQSPKRLIYLVSKLDSVVPDRFTGCGSGTDFTLKISRVEAEDLGVYYRWQGTHFPHTFGGGTKLEIK DVVMTQTPLTLSVTIGQPASISC KSSQSLLDSDRKTYLNWLLQRPGQSPKRLIYLVSKLDSVVPDRFTGCGSGTDFTLKISRVEAEDLGVYYRWQGTHFPHTFGGGTKLEIK 44

44
LVSKLDS LVSKLDS 4242
WQGTHFPHT WQGTHFPHT 4343
75G 75G VHVH NYWMNNYWMN 3333 EVNLEESGGGLVQPGGSMKLSCVASGVTFSNYWMNWVRQSPEKGLEWVAEIRLKFNDYATHYVESVKGRFTISRDDSESSVYLQMNNLRAEDTGIYYCTMVRRNWYFDVWGAGTTVTVSS EVNLEESGGGLVQPGGSMKLSCVASGVTFS NYWMNWVRQSPEKGLEWVAEIRLKFNDYATHYVESVKGRFTISRDDSESSVYLQMNNLRAEDTGIYYCTMVRRNWYFDVWGAGTTVTVSS 4848
EIRLKFNDYATHYVESVKG EIRLKFNDYATHYVESVKG 4545
VRRNWYFDV VRRNWYFDV 3535
VLVL SASSSVSSGYLYSASSSVSSGYLY 46 46 QIVLTQSPAIMSASPGEKVTLTCSASSSVSSGYLYWYQKKPGSSPKLWIYSTSDLASGVPARFSGSGSGTFYSLTISNM EAEDAASYFCHQWSSYPYTFGGGTKLEIK QIVLTQSPAIMSASPGEKVTLTC SASSSVSSGYLYWYQKKPGSSPKLWIYSTSDLASGVPARFSGSGSGTFYSLTISNMEAEDAASYFCHQWSSYPYTFGGGTKLEIK 4949
STSDLAS STSDLAS 4747
HQWSSYPYTHQWSSYPYT 3838
선별된 클론의 핵산 서열Nucleic acid sequence of selected clones
클론clone 구분division 서열order 서열번호SEQ ID NO:
19D19D VHVH gaagtccag
ctgcagcaga gcggccctga gctggtaaag cccggtgcct cagtcaaaat gagctgcaaa
gcttctggat acacgttcag taactatgtt atgcactggg tgaaacaaaa gccagggcag
ggccttgagt ggatcggcta tattaacccc tataatgatg gaactaagta caatgagaaa
ttcaagggga aggccaccct gaccagtgac aggtcctcca gcacagccta catggaactc
agctcgctga cctctgagga ctctgccgtg tattactgtg caagatggcg ggtgaactac
tactttgatt actggggcca ggggaccact ctgacagtgt cctcagaagtccag
ctgcagcaga gcggccctga gctggtaaag cccggtgcct cagtcaaaat gagctgcaaa
gcttctggat acacgttcag taactatgtt atgcactggg tgaaacaaaa gccagggcag
ggccttgagt ggatcggcta tattaacccc tataatgatg gaactaagta caatgagaaa
ttcaagggga aggccaccct gaccagtgac aggtcctcca gcacagccta catggaactc
agctcgctga cctctgagga ctctgccgtg tattactgtg caagatggcg ggtgaactac
tactttgatt actggggcca ggggaccact ctgacagtgt cctca 		5858
VLVL caaattgtgc tctcccagtc tccagcaatc ctgagcgctt ctccagggga aaaggtaacg
atgacttgta gggccagctc atcagttaat tacatgcact ggtatcagca aaagcctggc
agcagtccca aagcttggat ttctgtgaca tccaacctgg cctctggagt ccccgcgcgg
ttcagtggct ctggatccgg gaccagttac tccttgacca taagcagagt ggaggctgaa
gacgcagcca cttattactg ccagcagtgg tcaagcaatc ctccttggac atttggtgga
ggcaccaagc ttgagatcaa acaaattgtgc tctcccagtc tccagcaatc ctgagcgctt ctccagggga aaaggtaacg
atgacttgta gggccagctc atcagttaat tacatgcact ggtatcagca aaagcctggc
agcagtccca aagcttggat ttctgtgaca tccaacctgg cctctggagt ccccgcgcgg
ttcagtggct ctggatccgg gaccagttac tccttgacca taagcagagt ggaggctgaa
gacgcagcca cttattactg ccagcagtgg tcaagcaatc ctccttggac atttggtgga
ggcaccaagc ttgagatcaa a 		5959
58C58C VHVH gaagtgcaa
ctgcagcagt ctgggccaga acttgtgaag cccggggctt caatgaagat cagttgcaag
gcaagcggtt atagcttcac cggctacatc atgaactggg tgaaacagag ccacggtaaa
aacttggagt ggatcggact cattaatcct tataatggcg gatcctccta taaccagaag
ttcaagggca aagccactct cacagtagac aagtcttcaa gcacagccta catggagctg
ctgtccctga cctctgagga cagtgcagtt tactactgtg cccggtttct gcgggggtac
tatgctgtgg attactgggg ccagggaact tcggtcaccg tcagctccgaagtgcaa
ctgcagcagt ctgggccaga acttgtgaag cccggggctt caatgaagat cagttgcaag
gcaagcggtt atagcttcac cggctacatc atgaactggg tgaaacagag ccacggtaaa
aacttggagt ggatcggact cattaatcct tataatggcg gatcctccta taaccagaag
ttcaagggca aagccactct cacagtagac aagtcttcaa gcacagccta catggagctg
ctgtccctga cctctgagga cagtgcagtt tactactgtg cccggtttct gcgggggtac
tatgctgtgg attactgggg ccagggaact tcggtcaccg tcagctcc 		6060
VLVL gacgtggtgg tgactcagac tcccctctcc ctgcccgtca gctttggtga tcaggtttcc
atatcttgca ggagctcgca aagtcttgtc aattcctatg ggcacaccta tttgtcttgg
tacctgcaca aacctgggca gtcaccacag ctgctcatct acggaattag caacagattc
agcggggtgc cagaccggtt cagtggctct ggttcaggga cggacttcac cctgaagatc
agcacaatca agcctgagga tctaggaatg tactattgtt tacaaggcac acatcagccg
tggacatttg gcggaggcac caagctggaa attaaagacgtggtgg tgactcagac tcccctctcc ctgcccgtca gctttggtga tcaggtttcc
atatcttgca ggagctcgca aagtcttgtc aattcctatg ggcacaccta tttgtcttgg
tacctgcaca aacctgggca gtcaccacag ctgctcatct acggaattag caacagattc
agcggggtgc cagaccggtt cagtggctct ggttcaggga cggacttcac cctgaagatc
agcacaatca agcctgagga tctaggaatg tactattgtt tacaaggcac acatcagccg
tggacatttg gcggaggcac caagctggaa attaaa 		6161
58E58E VHVH gaggtccag
cttcagcagt cgggccccga gctggtgaag cctggcgctt ccgtgaagat gtcctgcaag
gcatctggat acacattcac ctcatacgtt atgcactggg tgaaacagaa gccagggcag
ggcctcgaat ggatcgggta cattaatctg aacaacgacg gtactaagta taatgagaaa
tttaaaggca aggccaccct gagttcagac aagagctcca gcactgcgta catggagctg
agcagtctga catctgaaga ttctgccgta tactattgtg ccaggtggag ggtgtattac
tatgctatgg attactgggg gcaaggaacg agcgtcaccg tgtcctccgaggtccag
cttcagcagt cgggccccga gctggtgaag cctggcgctt ccgtgaagat gtcctgcaag
gcatctggat acacattcac ctcatacgtt atgcactggg tgaaacagaa gccagggcag
ggcctcgaat ggatcgggta cattaatctg aacaacgacg gtactaagta taatgagaaa
tttaaaggca aggccaccct gagttcagac aagagctcca gcactgcgta catggagctg
agcagtctga catctgaaga ttctgccgta tactattgtg ccaggtggag ggtgtattac
tatgctatgg attactgggg gcaaggaacg agcgtcaccg tgtcctcc 		6262
VLVL gaaattgtgc tcactcagtc ccctgctatc acagctgcgt ctctgggcca aaaagtcacg
atcacctgtt cggccagctc atcagtaaat tacatgcact ggtaccagca gaagagcggg
acctctccca gaccctggat ctatgaaatc tccaaactgg catctggcgt cccagcccgc
ttcagtggga gtggcagcgg aacctcctat tccctcacaa tcagctcaat ggaggcagag
gatgctgcca tatactactg ccagctgtgg aactatcctc ttatcacatt cggtgccgga
accaagctgg agttgaaggaaattgtgc tcactcagtc ccctgctatc acagctgcgt ctctgggcca aaaagtcacg
atcacctgtt cggccagctc atcagtaaat tacatgcact ggtaccagca gaagagcggg
acctctccca gaccctggat ctatgaaatc tccaaactgg catctggcgt cccagcccgc
ttcagtggga gtggcagcgg aacctcctat tccctcacaa tcagctcaat ggaggcagag
gatgctgcca tatactactg ccagctgtgg aactatcctc ttatcacatt cggtgccgga
accaagctgg agttgaag 		6363
62F62F VHVH gaggttcaa
ctgcagcaga gtggccctga actggtgaaa ccaggtgcta gtgtgaagat ctcctgcaaa
acttccggat tcacgtttac tgaatatacc atgcactggg tgaagcagtc gcatggaaaa
tctcttgagt ggattggcgg gtttaacccc aacagcgggg gatctagcta caatcagaag
ttcaagggca aagccacctt gaccgtcgac aagtccagct ccacagcctt catggagctc
cggtcactga caagcgagga ttctgcagcc tactattgtg ctagatcctc ctattacagg
tttgacctgg attactgggg ccaggggacc acactcactg tgagctcagaggttcaa
ctgcagcaga gtggccctga actggtgaaa ccaggtgcta gtgtgaagat ctcctgcaaa
acttccggat tcacgtttac tgaatatacc atgcactggg tgaagcagtc gcatggaaaa
tctcttgagt ggattggcgg gtttaacccc aacagcgggg gatctagcta caatcagaag
ttcaagggca aagccacctt gaccgtcgac aagtccagct ccacagcctt catggagctc
cggtcactga caagcgagga ttctgcagcc tactattgtg ctagatcctc ctattacagg
tttgacctgg attactgggg ccaggggacc acactcactg tgagctca 		6464
VLVL gacattgt gatgacccag tctcacaagt tcatgtccac atcagtgggc
gacagggtca gcatcacctg caaggctagt caggatgtga aaactgccgt tgcctggtat
caacagaaac caggacagtc tcctcaactg cttatctact ccgcatcctt tcggtacact
ggtgtccccg atcgctttac cggcagcgga tctggggcgg atttcacttt caccatcagc
agtgtgcaag ctgaagacct ggccgcatac tattgtcagc agcattacac gacaccctgg
acatttgggg ggggtaccaa gctcgagatc aaagacattgt gatgacccag tctcacaagt tcatgtccac atcagtgggc
gacagggtca gcatcacctg caaggctagt caggatgtga aaactgccgt tgcctggtat
caacagaaac caggacagtc tcctcaactg cttatctact ccgcatcctt tcggtacact
ggtgtccccg atcgctttac cggcagcgga tctggggcgg atttcacttt caccatcagc
agtgtgcaag ctgaagacct ggccgcatac tattgtcagc agcattacac gacaccctgg
acatttgggg ggggtaccaa gctcgagatc aaa 		6565
63F63F VHVH gaggtgcagctgg aggagtttgg aggcggcttg gtgcagcctg ggggttctat gaaactctcc
tgtgtcgcct ctgggtccac tttcagtaat tactggatga actgggtccg ccagagcccc
gagaaaggac ttgagtgggt ggccgaaatt agactgaagt tcaacaacta tgcaacacat
tacgtggaat ccgtcatcgg caggttcacc atttcaagag acgatagcaa gagtagcgtc
tacctgcaaa tgaataacct gcgggctgaa gatactggta tctactattg caccatggtt
cggaggaact ggtattttga cgtctggggc gccgggacaa cggtaaccgt ttcctcagaggtgcagctgg aggagtttgg aggcggcttg gtgcagcctg ggggttctat gaaactctcc
tgtgtcgcct ctgggtccac tttcagtaat tactggatga actgggtccg ccagagcccc
gagaaaggac ttgagtgggt ggccgaaatt agactgaagt tcaacaacta tgcaacacat
tacgtggaat ccgtcatcgg caggttcacc atttcaagag acgatagcaa gagtagcgtc
tacctgcaaa tgaataacct gcgggctgaa gatactggta tctactattg caccatggtt
cggaggaact ggtattttga cgtctggggc gccgggacaa cggtaaccgt ttcctca 		6666
VL1VL1 caaattgttt tgacgcagtc tccagcaatc atgtctgcgt ttcctggcga gaaggtgacc
ctgacctgta gtgccagctc atccgtctcc tcgtcatatc tgtactggta ccagcagaaa
ccaaggagct cccccaagct ctggatttat agcacatcta acctggcatc cggcgtgcct
gctaggttca gcggcagtgg atctgggact tcttattccc ttacaatcag tagcatggag
gctgaagacg ccgccagtta cttctgccac cagtggagct cataccccta cacttttgga
ggtgggacca agctcgaaat aaaacaaattgttt tgacgcagtc tccagcaatc atgtctgcgt ttcctggcga gaaggtgacc
ctgacctgta gtgccagctc atccgtctcc tcgtcatatc tgtactggta ccagcagaaa
ccaaggagct cccccaagct ctggatttat agcacatcta acctggcatc cggcgtgcct
gctaggttca gcggcagtgg atctgggact tcttattccc ttacaatcag tagcatggag
gctgaagacg ccgccagtta cttctgccac cagtggagct cataccccta cacttttgga
ggtgggacca agctcgaaat aaaa 		6767
VL2VL2 gatgtcgtga tgacccagac acccttgact ctgagcgtta ccattggaca accagcctccatctcctgca aatcaagtca gagcctcctc gacagtgacc ggaagacata ccttaattgg
ttgctgcagc ggccaggcca gtcccctaag cgcctaatct atctggtgag caagctggac
tctgtggtcc ccgacaggtt tactgggtgt ggtagcggaa cagattttac actcaaaatc
tcaagagtgg aagctgagga tctgggagta tactatcgat ggcaggggac gcattttccg
cacacattcg gcgggggcac caagcttgag ataaaagatgtcgtga tgacccagac acccttgact ctgagcgtta ccattggaca accagcctccatctcctgca aatcaagtca gagcctcctc gacagtgacc ggaagacata ccttaattgg
ttgctgcagc ggccaggcca gtcccctaag cgcctaatct atctggtgag caagctggac
tctgtggtcc ccgacaggtt tactgggtgt ggtagcggaa cagattttac actcaaaatc
tcaagagtgg aagctgagga tctgggagta tactatcgat ggcaggggac gcattttccg
cacacattcg gcgggggcac caagcttgag ataaaa 		6868
75G75G VHVH gaa
gtgaacctgg aggagtcagg cggggggttg gtgcaacccg gaggtagcat gaaactctcc
tgtgttgcct ctggagtgac tttctccaat tattggatga attgggttcg acagtctcct
gagaaggggc tggaatgggt ggctgagatt aggctgaagt tcaatgacta tgccacccac
tacgtggaga gcgtgaaggg caggtttaca atctcaagag acgattccga aagttctgtg
tatctgcaga tgaacaatct gcgcgctgaa gataccggca tctactactg caccatggtc
cggcgcaact ggtacttcga cgtgtggggc gcaggaacaa ctgtcacggt tagcagcgaa
gtgaacctgg aggagtcagg cggggggttg gtgcaacccg gaggtagcat gaaactctcc
tgtgttgcct ctggagtgac tttctccaat tattggatga attgggttcg acagtctcct
gagaaggggc tggaatgggt ggctgagatt aggctgaagt tcaatgacta tgccacccac
tacgtggaga gcgtgaaggg caggtttaca atctcaagag acgattccga aagttctgtg
tatctgcaga tgaacaatct gcgcgctgaa gataccggca tctactactg caccatggtc
cggcgcaact ggtacttcga cgtgtggggc gcaggaacaa ctgtcacggt tagcagc 		6969
VLVL caaattgttt tgactcagtc accagccatc atgtctgcca gccctggtga gaaggtgaca
ctgacctgca gtgcatccag ctctgtgtct tcagggtacc tttattggta ccagaaaaag
cccggatcct cccccaaact ctggatttat tcgacatccg acctggctag cggcgtccct
gccaggttct ctggcagtgg cagtggcact ttttacagcc tgaccatcag caacatggag
gctgaagatg cggcatccta tttctgtcac cagtggagct catacccata cacgtttgga
ggcgggacca agctggaaat aaaacaaattgttt tgactcagtc accagccatc atgtctgcca gccctggtga gaaggtgaca
ctgacctgca gtgcatccag ctctgtgtct tcagggtacc tttattggta ccagaaaaag
cccggatcct cccccaaact ctggatttat tcgacatccg acctggctag cggcgtccct
gccaggttct ctggcagtgg cagtggcact ttttacagcc tgaccatcag caacatggag
gctgaagatg cggcatccta tttctgtcac cagtggagct catacccata cacgtttgga
ggcgggacca agctggaaat aaaa 		7070
실시예 3. 특성 분석Example 3. Characterization
(1) 결합능(1) binding ability
선별된 각 후보항체의 VH와 VL 서열은 constant region으로 human IgG1 heavy chain과 human kappa light chain을 사용하여 chimeric 항체 형태로 cloning하였다. Chimeric 후보항체는 Expi293 transient expression system을 사용하여 항체를 생산하였고 ELISA 방법에 의하여 정제된 각 chimeric 항체의 정량적 결합력을 분석하였다. ELISA는 재조합 인간 TIGIT-His 단백질을 96-well ELISA plate에 4℃에서 O/N coating하고 5% non-fat skim milk로 RT에서 1hr동안 blocking하였다. 항체는 각 500ng 또는 200ng 농도부터 1/2 serial dilution하여 RT에서 2hr 처리하고 anti-hIgG-HRP로 detection하였고, 양성대조군으로 Oncomed-313M32 항체를 생산하여 비교하였다. 클로닝을 통한 항체 생산을 비교한 결과는 도 3에 나타내었다.The VH and VL sequences of each selected candidate antibody were cloned in the form of chimeric antibodies using human IgG1 heavy chain and human kappa light chain as constant regions. The chimeric candidate antibody was produced using the Expi293 transient expression system, and the quantitative binding ability of each purified chimeric antibody was analyzed by ELISA. For ELISA, recombinant human TIGIT-His protein was O/N coated on a 96-well ELISA plate at 4° C. and blocked with 5% non-fat skim milk for 1 hr at RT. Antibodies were subjected to 1/2 serial dilution from each concentration of 500ng or 200ng, treated for 2hr at RT, and detected with anti-hIgG-HRP, and the Oncomed-313M32 antibody was produced as a positive control and compared. The results of comparing antibody production through cloning are shown in FIG. 3 .
도 3에 나타낸 바와 같이, 선별된 chimeric-후보항체 clone 19D, 58C, 58E, 62F, 63F 및 75G가 생산되었고, 항원에 결합하는 것을 확인하였다.As shown in FIG. 3 , the selected chimeric- candidate antibody clones 19D, 58C, 58E, 62F, 63F and 75G were produced, and binding to antigen was confirmed.
chimeric-후보항체 clone 19D, 58C, 58E, 62F, 63F 및 75G의 순도 및 결합력을 SEC-HPLC와 SPR로 분석하였다. HPLC 및 SPR 분석 결과는 도 4에 나타내었다.The purity and avidity of chimeric- candidate antibody clones 19D, 58C, 58E, 62F, 63F and 75G were analyzed by SEC-HPLC and SPR. HPLC and SPR analysis results are shown in FIG. 4 .
도 4에 나타낸 바와 같이, SPR 분석에서 chimeric-후보항체 clone 58E, 62F, 63F 및 75G의 결합력은 대조군인 Oncomed-313M32 항체에 비해 높았다. 보다 상세하게는, 상기 후보항체 58E는 대조군보다 약 13배, 62F는 약 30배, 63F는 약 6배, 75G는 약 30배 높았다.As shown in FIG. 4 , in the SPR analysis, the binding affinity of the chimeric- candidate antibody clones 58E, 62F, 63F and 75G was higher than that of the control, Oncomed-313M32 antibody. More specifically, the candidate antibody 58E was about 13 times higher than the control group, 62F was about 30 times, 63F was about 6 times, and 75G was about 30 times higher.
인간 PBMC를 분리하여 anti-hCD3/28로 activation을 시킨 후 정제된 chimeric-후보항체를 처리하여 각 CD4 T 세포와 CD8 T 세포에서의 결합력을 flow cytometry 방법으로 확인하였다. Flow cytometry 결과는 도 5에 나타내었다.Human PBMCs were isolated, activated with anti-hCD3/28, treated with purified chimeric-candidate antibody, and the binding force between each CD4 T cell and CD8 T cell was confirmed by flow cytometry. Flow cytometry results are shown in FIG. 5 .
도 5에 나타낸 바와 같이, chimeric-후보항체 58C, 58E, 62F, 63F 및 75G는 CD4/CD8 T 세포 모두에서 대조군(Oncomed-313M32 항체)보다 더 높은 결합력을 보였다. As shown in FIG. 5 , the chimeric- candidate antibodies 58C, 58E, 62F, 63F and 75G showed higher avidity than the control (Oncomed-313M32 antibody) in all CD4/CD8 T cells.
(2) TIGIT/CD155 Blocking Assay(2) TIGIT/CD155 Blocking Assay
실시예 1의 항원 결합력 분석 조건을 사용하여 chimeric-후보항체가 TIGIT과 그 counterpart인 CD155와의 결합을 inhibition할 수 있는 효능을 조사하였다. hybridoma로부터 선별된 chimeric-후보항체를 생산 및 정제하여 처리하였다. chimeric-후보항체의 억제 효능을 확인한 결과는 도 6에 나타내었다.Using the antigen binding affinity analysis conditions of Example 1, the efficacy of the chimeric-candidate antibody to inhibit the binding of TIGIT to its counterpart CD155 was investigated. A chimeric-candidate antibody selected from hybridoma was produced, purified, and treated. The results confirming the inhibitory efficacy of the chimeric-candidate antibody are shown in FIG. 6 .
도 6에 나타낸 바와 같이, chimeric-후보항체 clone 62F, 63F 및 75G는 ebioscience사의 function grade TIGIT 항체 및 Oncomed-313M32 항체보다 억제 효능이 우수한 것을 확인하였다.As shown in FIG. 6 , it was confirmed that the chimeric- candidate antibody clones 62F, 63F and 75G had superior inhibitory efficacy than ebioscience's function grade TIGIT antibody and Oncomed-313M32 antibody.
실시예 4. 인간화 항체 제조Example 4. Humanized Antibody Preparation
hybridoma와 chimeric-후보 항체의 특성 및 기능 분석 결과로부터 clone 19D, 62F, 63F 및 75G에 대한 인간화 항체를 제작하였다. 제작된 인간화 항체의 서열을 분석한 결과는 표 2에 나타내었다.Humanized antibodies against clones 19D, 62F, 63F and 75G were prepared from the results of analysis of the characteristics and functions of hybridoma and chimeric-candidate antibodies. The results of sequence analysis of the prepared humanized antibody are shown in Table 2.
아미노산 서열amino acid sequence
클론clone 구분division 서열order 서열번호SEQ ID NO:
19D 19D VHVH QVQLVQSGAEVKKPGASVKVSCKASGYTFSNYVMHWVRQAPGQRLEWIG YINPYNDGTKYNEKFKGRATLTSDRSASTAYMELSSLRSEDTAVYYCARWRVNYYFDYWGQGTLVTVSSQVQLVQSGAEVKKPGASVKVSCKASGYTFSNYVMHWVRQAPGQRLEWIGYINPYNDGTKYNEKFKGRATLTSDRSASTAYMELSSLRSEDTAVYYCARWRVNYYFDYWGQGTLVTVSS 5050
VLVL DIQLTQSPSSLSASVGDRVTITCRASSSVNYMHWYQQKPGKAPKAWISVTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPPWTFGQGTKVEIKDIQLTQSPSSLSASVGDRVTITCRASSSVNYMHWYQQKPGKAPKAWISVTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPPWTFGQGTKVEIK 5151
62F62F VHVH QVQLVQSGAEVKKPGASVKVSCKASGFTFTEYTMHWVRQAPGQGLEWIGGFNPNSGGSSYNQKFKGRATLTVDKSISTAYMELSRLRSDDTAVYYCARSSYYRFDLDYWGQGTTVTVSSQVQLVQSGAEVKKPGASVKVSCKASGFTFTEYTMHWVRQAPGQGLEWIGGFNPNSGGSSYNQKFKGRATLTVDKSISTAYMELSRLRSDDTAVYYCARSSYYRFDLDYWGQGTTVTVSS 5252
VLVL DIQMTQSPSSLSASVGDRVTITCKASQDVKTAVAWYQQKPGKAPKLLIYSASFRYTGVPSRFSGSGSGADFTLTISSLQPEDFATYYCQQHYTTPWTFGQGTKVEIKDIQMTQSPSSLSASVGDRVTITCKASQDVKTAVAWYQQKPGKAPKLLIYSASFRYTGVPSRFSGSGSGADFTLTISSLQPEDFATYYCQQHYTTPWTFGQGTKVEIK 5353
63F 63F VHVH EVQLVESGGGLVQPGGSLRLSCAASGSTFSNYWMNWVRQAPGKGLEWVAEI RLKFNNYATHYVESVIGRFTISRDDSKNSVYLQMNSLKTEDTAVYYCTMVRRNWYFDVWGRGTLVTVSSEVQLVESGGGLVQPGGSLRLSCAASGSTFSNYWMNWVRQAPGKGLEWVAEIRLKFNNYATHYVESVIGRFTISRDDSKNSVYLQMNSLKTEDTAVYYCTMVRRNWYFDVWGRGTLVTVSS 5454
VLVL EIVLTQSPATLSLSPGERATLSCSASSSVSSSYLYWYQQKPGLAPRLWIYSTSNLASGIPDRFSGSGSGTDYTLTISRLEPEDFAVYYCHQWSSYPYTFGQGTKLEIKEIVLTQSPATLSLSPGERATLSCSASSSVSSSYLYWYQQKPGLAPRLWIYSTSNLASGIPDRFSGSGSGTDYTLTISRLEPEDFAVYYCHQWSSYPYTFGQGTKLEIK 5555
75G 75G VHVH EVQLVESGGGLVQPGGSLRLSCAASGVTFSNYWMNWVRQAPGKGLEWVAEI RLKFNDYATHYVESVKGRFTISRDDSKNSVYLQMNSLKTEDTAVYYCTMVRRNWYFDVWGRGTLVTVSSEVQLVESGGGLVQPGGSLRLSCAASGVTFSNYWMNWVRQAPGKGLEWVAEIRLKFNDYATHYVESVKGRFTISRDDSKNSVYLQMNSLKTEDTAVYYCTMVRRNWYFDVWGRGTLVTVSS 5656
VLVL EIVLTQSPATLSLSPGERATLSCSASSSVSSGYLYWYQQKPGLAPRLWIYSTSDLASGIPDRFSGSGSGTDYTLTISRLEPEDFAVYYCHQWSSYPYTFGQGTKLEIKEIVLTQSPATLSLSPGERATLSCSASSSVSSGYLYWYQQKPGLAPRLWIYSTSDLASGIPDRFSGSGSGTDYTLTISRLEPEDFAVYYCHQWSSYPYTFGQGTKLEIK 5757
핵산 서열nucleic acid sequence
클론clone 구분division 서열order 서열번호SEQ ID NO:
19D19D VHVH caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg
agctgcaagg ccagcggcta caccttcagc aactacgtga tgcactgggt gaggcaggcc
cccggccaga ggctggagtg gatcggctac atcaacccct acaacgacgg caccaagtac
aacgagaagt tcaagggcag ggccaccctg accagcgaca ggagcgccag caccgcctac
atggagctga gcagcctgag gagcgaggac accgccgtgt actactgcgc caggtggagg
gtgaactact acttcgacta ctggggccag ggcaccctgg tgaccgtgag cagccaggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg
agctgcaagg ccagcggcta caccttcagc aactacgtga tgcactgggt gaggcaggcc
cccggccaga ggctggagtg gatcggctac atcaacccct acaacgacgg caccaagtac
aacgagaagt tcaagggcag ggccaccctg accagcgaca ggagcgccag caccgcctac
atggagctga gcagcctgag gagcgaggac accgccgtgt actactgcgc caggtggagg
gtgaactact acttcgacta ctggggccag ggcaccctgg tgaccgtgag cagc 		7171
VLVL gacatccagc tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagggtgacc
atcacctgca gggccagcag cagcgtgaac tacatgcact ggtaccagca gaagcccggc
aaggccccca aggcctggat cagcgtgacc agcaacctgg ccagcggcgt gcccagcagg
ttcagcggca gcggcagcgg caccgactac accctgacca tcagcagcct gcagcccgag
gacttcgcca cctactactg ccagcagtgg agcagcaacc ccccctggac cttcggccag
ggcaccaagg tggagatcaa ggacatccagc tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagggtgacc
atcacctgca gggccagcag cagcgtgaac tacatgcact ggtaccagca gaagcccggc
aaggccccca aggcctggat cagcgtgacc agcaacctgg ccagcggcgt gcccagcagg
ttcagcggca gcggcagcgg caccgactac accctgacca tcagcagcct gcagcccgag
gacttcgcca cctactactg ccagcagtgg agcagcaacc ccccctggac cttcggccag
ggcaccaagg tggagatcaa g 		7272
62F62F VHVH caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg
agctgcaagg ccagcggctt caccttcacc gagtacacca tgcactgggt gaggcaggcc
cccggccagg gcctggagtg gatcggcggc ttcaacccca acagcggcgg cagcagctac
aaccagaagt tcaagggcag ggccaccctg accgtggaca agagcatcag caccgcctac
atggagctga gcaggctgag gagcgacgac accgccgtgt actactgcgc caggagcagc
tactacaggt tcgacctgga ctactggggc cagggcacca ccgtgaccgt gagcagccaggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg
agctgcaagg ccagcggctt caccttcacc gagtacacca tgcactgggt gaggcaggcc
cccggccagg gcctggagtg gatcggcggc ttcaacccca acagcggcgg cagcagctac
aaccagaagt tcaagggcag ggccaccctg accgtggaca agagcatcag caccgcctac
atggagctga gcaggctgag gagcgacgac accgccgtgt actactgcgc caggagcagc
tactacaggt tcgacctgga ctactggggc cagggcacca ccgtgaccgt gagcagc 		7373
VLVL gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagggtgacc
atcacctgca aggccagcca ggacgtgaag accgccgtgg cctggtacca gcagaagccc
ggcaaggccc ccaagctgct gatctacagc gccagcttca ggtacaccgg cgtgcccagc
aggttcagcg gcagcggcag cggcgccgac ttcaccctga ccatcagcag cctgcagccc
gaggacttcg ccacctacta ctgccagcag cactacacca ccccctggac cttcggccag
ggcaccaagg tggagatcaa ggacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagggtgacc
atcacctgca aggccagcca ggacgtgaag accgccgtgg cctggtacca gcagaagccc
ggcaaggccc ccaagctgct gatctacagc gccagcttca ggtacaccgg cgtgcccagc
aggttcagcg gcagcggcag cggcgccgac ttcaccctga ccatcagcag cctgcagccc
gaggacttcg ccacctacta ctgccagcag cactacacca ccccctggac cttcggccag
ggcaccaagg tggagatcaa g 		7474
63F63F VHVH gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg
agctgcgccg ccagcggcag caccttcagc aactactgga tgaactgggt gaggcaggcc
cccggcaagg gcctggagtg ggtggccgag atcaggctga agttcaacaa ctacgccacc
cactacgtgg agagcgtgat cggcaggttc accatcagca gggacgacag caagaacagc
gtgtacctgc agatgaacag cctgaagacc gaggacaccg ccgtgtacta ctgcaccatg
gtgaggagga actggtactt cgacgtgtgg ggcaggggca ccctggtgac cgtgagcagcgaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg
agctgcgccg ccagcggcag caccttcagc aactactgga tgaactgggt gaggcaggcc
cccggcaagg gcctggagtg ggtggccgag atcaggctga agttcaacaa ctacgccacc
cactacgtgg agagcgtgat cggcaggttc accatcagca gggacgacag caagaacagc
gtgtacctgc agatgaacag cctgaagacc gaggacaccg ccgtgtacta ctgcaccatg
gtgaggagga actggtactt cgacgtgtgg ggcaggggca ccctggtgac cgtgagcagc 		7575
VLVL gagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gagggccacc
ctgagctgca gcgccagcag cagcgtgagc agcagctacc tgtactggta ccagcagaag
cccggcctgg cccccaggct gtggatctac agcaccagca acctggccag cggcatcccc
gacaggttca gcggcagcgg cagcggcacc gactacaccc tgaccatcag caggctggag
cccgaggact tcgccgtgta ctactgccac cagtggagca gctaccccta caccttcggc
cagggcacca agctggagat caaggagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gagggccacc
ctgagctgca gcgccagcag cagcgtgagc agcagctacc tgtactggta ccagcagaag
cccggcctgg cccccaggct gtggatctac agcaccagca acctggccag cggcatcccc
gacaggttca gcggcagcgg cagcggcacc gactacaccc tgaccatcag caggctggag
cccgaggact tcgccgtgta ctactgccac cagtggagca gctaccccta caccttcggc
cagggcacca agctggagat caag 		7676
75G75G VHVH gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg
agctgcgccg ccagcggcgt gaccttcagc aactactgga tgaactgggt gaggcaggcc
cccggcaagg gcctggagtg ggtggccgag atcaggctga agttcaacga ctacgccacc
cactacgtgg agagcgtgaa gggcaggttc accatcagca gggacgacag caagaacagc
gtgtacctgc agatgaacag cctgaagacc gaggacaccg ccgtgtacta ctgcaccatg
gtgaggagga actggtactt cgacgtgtgg ggcaggggca ccctggtgac cgtgagcagcgaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg
agctgcgccg ccagcggcgt gaccttcagc aactactgga tgaactgggt gaggcaggcc
cccggcaagg gcctggagtg ggtggccgag atcaggctga agttcaacga ctacgccacc
cactacgtgg agagcgtgaa gggcaggttc accatcagca gggacgacag caagaacagc
gtgtacctgc agatgaacag cctgaagacc gaggacaccg ccgtgtacta ctgcaccatg
gtgaggagga actggtactt cgacgtgtgg ggcaggggca ccctggtgac cgtgagcagc 		7777
VLVL gagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gagggccacc
ctgagctgca gcgccagcag cagcgtgagc agcggctacc tgtactggta ccagcagaag
cccggcctgg cccccaggct gtggatctac agcaccagcg acctggccag cggcatcccc
gacaggttca gcggcagcgg cagcggcacc gactacaccc tgaccatcag caggctggag
cccgaggact tcgccgtgta ctactgccac cagtggagca gctaccccta caccttcggc
cagggcacca agctggagat caaggagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gagggccacc
ctgagctgca gcgccagcag cagcgtgagc agcggctacc tgtactggta ccagcagaag
cccggcctgg cccccaggct gtggatctac agcaccagcg acctggccag cggcatcccc
gacaggttca gcggcagcgg cagcggcacc gactacaccc tgaccatcag caggctggag
cccgaggact tcgccgtgta ctactgccac cagtggagca gctaccccta caccttcggc
cagggcacca agctggagat caag 		7878
실시예 5. 특성 분석Example 5 Characterization
(1) 결합능(1) binding ability
hybridoma와 chimeric-후보 항체의 특성 및 기능 분석 결과로부터 후보항체 clone 19D, 62F, 63F 및 75G에 대한 인간화 항체 서열을 제작하고, 각 human IgG1 heavy chain과 kappa light chain으로 클로닝하였다. 각 인간화 후보항체를 생산 및 정제한 후 SDS-PAGE 염색하여 확인하였다. 또한 Expi293 transient expression system을 사용하여 항체를 생산 및 정제하였고 ELISA 방법에 의하여 정제된 각 인간화 항체의 정량적 결합력을 분석하였다. ELISA는 실시예 3-(1)과 동일한 방법으로 하였으며 항체는 각 500ng 농도부터 1/2 serial dilution하여 처리하였다. SDS-PAGE 및 ELISA 결과는 도 7에 나타내었다.Humanized antibody sequences for candidate antibody clones 19D, 62F, 63F and 75G were prepared from the hybridoma and chimeric-candidate antibody characteristics and function analysis results, and cloned into human IgG1 heavy chain and kappa light chain, respectively. After each humanized candidate antibody was produced and purified, it was confirmed by SDS-PAGE staining. In addition, antibodies were produced and purified using the Expi293 transient expression system, and quantitative binding capacity of each purified humanized antibody was analyzed by ELISA method. ELISA was performed in the same manner as in Example 3-(1), and the antibody was treated by serial dilution of 1/2 from the concentration of 500ng each. The results of SDS-PAGE and ELISA are shown in FIG. 7 .
도 7에 나타낸 바와 같이, 인간화 후보항체 clone 19D, 62F, 63F 및 75G가 생산되었고, 항원에 결합하는 것을 확인하였다.As shown in FIG. 7 , humanized candidate antibody clones 19D, 62F, 63F and 75G were produced, and binding to antigen was confirmed.
인간화 후보항체 clone 19D, 62F, 63F 및 75G의 순도와 결합력을 SEC-HPLC와 SPR로 분석하였다. HPLC 및 SPR 결과는 도 8에 나타내었다.The purity and avidity of humanized candidate antibody clones 19D, 62F, 63F and 75G were analyzed by SEC-HPLC and SPR. HPLC and SPR results are shown in FIG. 8 .
도 8에 나타낸 바와 같이, SPR 분석 결과, 인간화 후보항체의 결합력은 clone 4개 중 19D가 가장 낮았다. 인간화 후보항체 clone 62F, 63F 및 75G는 각각 결합력이 제일 낮은 clone 19D보다 33배, 13배 및 36배 높은 것을 확인하였다.As shown in FIG. 8 , as a result of SPR analysis, the binding affinity of the humanized candidate antibody was the lowest in 19D among the 4 clones. It was confirmed that the humanized candidate antibody clones 62F, 63F and 75G were 33-fold, 13-fold and 36-fold higher than clone 19D, which had the lowest binding affinity, respectively.
인간화 후보항체는 TIGIT을 발현하는 artificial effector T 세포를 사용한 flow cytometry 방법에 의해 결합력을 확인하였다. Flow cytometry 결과는 도 9에 나타내었다.The binding ability of the humanized candidate antibody was confirmed by flow cytometry using artificial effector T cells expressing TIGIT. Flow cytometry results are shown in FIG. 9 .
도 9에 나타낸 바와 같이, 인간화 후보항체 clone 19D, 62F, 63F 및 75G는 모두 ebioscience사의 flow cytometry용 항체보다 더 높은 결합 비율을 보였다.As shown in FIG. 9, the humanized candidate antibody clones 19D, 62F, 63F and 75G all showed a higher binding ratio than ebioscience's antibody for flow cytometry.
(2) TIGIT/CD155 Blocking Assay(2) TIGIT/CD155 Blocking Assay
실시예 1의 항원 결합력 분석 조건을 사용하여 인간화 후보항체가 TIGIT과 그 counterpart인 CD155와의 결합을 inhibition할 수 있는 효능을 조사하였다. 억제 효능을 확인한 결과는 도 10에 나타내었다.The efficacy of the humanized candidate antibody to inhibit the binding of TIGIT to its counterpart CD155 was investigated using the antigen binding affinity assay conditions of Example 1. The results of confirming the inhibitory efficacy are shown in FIG. 10 .
도 10에 나타낸 바와 같이, 양성대조군(ebioscience사의 function grade TIGIT 항체, Oncomed-313M32 항체)과 비교하였을 때, 인간화 후보항체 62F, 75G 및 63F의 억제 효능이 우수한 것을 확인하였다. 인간화 후보항체 19D는 억제 효능이 OMP-313M32와 유사한 수준인 것을 확인하였다.As shown in FIG. 10 , it was confirmed that the inhibitory efficacy of the humanized candidate antibodies 62F, 75G and 63F was excellent when compared to the positive control group (ebioscience's function grade TIGIT antibody, Oncomed-313M32 antibody). It was confirmed that the humanized candidate antibody 19D had a similar inhibitory efficacy to that of OMP-313M32.
실시예 6. In vivo 효능 분석Example 6. In vivo efficacy analysis
인간화 후보항체의 in vivo 효능 분석을 위하여 hTIGIT/hPD-1 double knock-in mice에 mouse colon cancer MC38에 hPD-L1이 발현하는 세포주로 종양모델을 만들어서 항암 효능을 분석하였다. 상기 종양모델에 인간화 후보항체 62F, 63F 또는 75G를 10 mg/kg 농도로 3일 간격으로 5회 투여하였고, 종양의 크기를 분석하였다. 각 인간화 후보항체 당 5마리를 한 그룹으로 구성하였고, 인간화 후보항체 및 인간화 PD-1 2B3W 항체를 병용 투여하는 그룹도 포함하였다. 상기 인간화 PD-1 2B3W 항체는 다음의 서열로 표시되는 중쇄 및 경쇄를 포함한다.To analyze the in vivo efficacy of the humanized candidate antibody, a tumor model was created using a cell line expressing hPD-L1 in mouse colon cancer MC38 in hTIGIT/hPD-1 double knock-in mice and the anticancer efficacy was analyzed. Humanized candidate antibodies 62F, 63F or 75G were administered to the tumor model 5 times at a concentration of 10 mg/kg at 3-day intervals, and the size of the tumor was analyzed. Each humanized candidate antibody consisted of a group of 5 animals, and a group in which the humanized candidate antibody and the humanized PD-1 2B3W antibody were co-administered was also included. The humanized PD-1 2B3W antibody comprises a heavy chain and a light chain represented by the following sequence.
-인간화 PD-1 2B3W 항체의 중쇄(서열번호 104): EVQLVESGGGLVQPGGSLRLSCAASGFVFSNYDMSWVRQAPGKGLEWVAYITIGGGTTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARWVYDPLYAMDYWGQGTTVTVSS-heavy chain of humanized PD-1 2B3W antibody (SEQ ID NO: 104): EVQLVESGGGLVQPGGSLRLSCAASGFVFSNYDMSWVRQAPGKGLEWVAYITIGGGTTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARWVYDPLYAMDYWGQGTTVTVSS
-인간화 PD-1 2B3W 항체의 경쇄(서열번호 105): DIVLTQTPLSLSVTPGQPASISCRASESVDNYGISFMNWFLQKPGQPPQLLIYAASNQGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEVPWTFGQGTKVEIK -Light chain of humanized PD-1 2B3W antibody (SEQ ID NO:105): DIVLTQTPLSVTPGQPASISCRASESVDNYGISFMNWFLQKPGQPPQLLIYAASNQGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEVPWTFGQGTKVEIK
인간화 PD-1 2B3W 항체는 새로운 PD-1 항체 개발을 위하여 선별된 후보 항체를 사용하였다. 인간화 후보항체 단독 또는 병용 투여에 따른 종양 크기를 분석한 결과는 도 11에 나타내었고, 각 그룹의 평균 값을 그래프화하여 도 12에 나타내었다.The humanized PD-1 2B3W antibody was used as a candidate antibody selected for the development of a new PD-1 antibody. The results of analyzing the tumor size following administration of the humanized candidate antibody alone or in combination are shown in FIG. 11 , and the average value of each group is graphed and shown in FIG. 12 .
도 11 및 12에 나타낸 바와 같이, 단독 항체 투여에서는 인간화 후보항체 62F 및 75G는 암성장이 지연되는 현상이 관찰되었다. 그러나 인간화 PD-1 2B3W 항체는 단독 투여만으로도 항암 효능이 관찰되었다. 대조군인 hIgG1 투여군의 1개체는 암 괴사에 의해 탈락되었다.As shown in FIGS. 11 and 12 , a phenomenon in which cancer growth was delayed was observed with humanized candidate antibodies 62F and 75G when administered alone. However, the humanized PD-1 2B3W antibody was observed to have anticancer efficacy only when administered alone. One subject in the hIgG1 administration group as a control group was dropped out due to cancer necrosis.
또한 인간화 후보항체 및 인간화 PD-1 2B3W 항체 병용 투여군은 단독 투여군에 비해 종양의 크기가 현저히 감소된 것을 확인하였다. 특히 인간화 후보항체 75G(anti-TIGIT) 및 인간화 PD-1 2B3W 항체(anti-PD-1) 병용 투여군은 모든 개체에서 종양이 제거된 것을 확인하였다.In addition, it was confirmed that the humanized candidate antibody and humanized PD-1 2B3W antibody combined administration group significantly reduced the size of the tumor compared to the single administration group. In particular, it was confirmed that tumors were removed from all subjects in the group administered with the humanized candidate antibody 75G (anti-TIGIT) and humanized PD-1 2B3W antibody (anti-PD-1).
각 그룹의 동물로부터 혈액을 채취하여 CD8 T 세포의 비율 변화를 분석하였다. 또한 동물을 희생시킨 후 혈액을 채취하여 CD8 T 세포의 비율을 추가 분석하였다. 분석 결과는 도 13에 나타내었다.Blood was collected from animals in each group and analyzed for changes in the percentage of CD8 T cells. In addition, after sacrificing the animals, blood was collected and the ratio of CD8 T cells was further analyzed. The analysis results are shown in FIG. 13 .
도 13에 나타낸 바와 같이, 인간화 후보항체 투여 전 (pre-bleed)과 비교하여, 3회 인간화 후보항체 투여 후 (post-bleed)에 CD8 T 세포는 모든 그룹에서 그 비율이 증가하였다. 특히, 단독 투여보다 병용 투여에서 더 높은 비율로 증가하였다.As shown in FIG. 13 , compared to before administration of the humanized candidate antibody (pre-bleed), the ratio of CD8 T cells increased in all groups after administration of the humanized candidate antibody three times (post-bleed). In particular, it increased at a higher rate in the combined administration than in the single administration.
또한 실험 종료 후 혈액을 채취하여 CD8 T 세포의 비율을 분석한 결과, 단독 투여에서는 대조군과 비교하여 CD8 T 세포 비율의 변화가 없었으며 병용 투여 그룹에서는 CD8 T 세포 비율이 증가 및 유지되고 있었다. Also, as a result of collecting blood after the end of the experiment and analyzing the CD8 T cell ratio, there was no change in the CD8 T cell ratio in the single administration compared to the control group, and the CD8 T cell ratio in the combination administration group was increased and maintained.
한편, 종양내 TIL (tumor infiltrated lymphocyte) 분석은 병용 투여 그룹의 종양이 거의 제거되어 분석할 수 없었다.On the other hand, intratumoral TIL (tumor infiltrated lymphocyte) analysis could not be performed because almost all tumors in the combined administration group were removed.
실험 종료 및 동물을 희생시킨 후 각 그룹의 동물로부터 혈액을 채취하여 간 독성 측정(즉, ALT, AST, BUN 및 T-BIL 수치 측정)하였으며, 그 결과는 도 14에 나타내었다.After the end of the experiment and animals were sacrificed, blood was collected from animals in each group to measure liver toxicity (ie, ALT, AST, BUN and T-BIL levels were measured), and the results are shown in FIG. 14 .
도 14에 나타낸 바와 같이, 인간화 후보항체 63F 단독 투여군이 타 그룹에 비해 수치가 증가하였고, 다른 인간화 후보항체 및 인간화 PD-1 2B3W 항체 병용 투여군은 각 수치가 거의 변화하지 않았다.As shown in FIG. 14 , the level increased in the group administered with the humanized candidate antibody 63F alone, compared to other groups, and the values of the group administered with the other humanized candidate antibody and the humanized PD-1 2B3W antibody in combination did not change substantially.
실시예 7. 후보항체 58E의 gene engineering 및 효능 평가Example 7. Gene engineering and efficacy evaluation of candidate antibody 58E
(1) 후보항체 58E의 gene engineering 및 서열분석(1) Gene engineering and sequencing of candidate antibody 58E
후보항체인 58E의 항체에 대한 친화도를 증가시키기 위하여, gene engineering을 수행하였다. 후보항체 T21.01, T21.08, T21.09, T21.10, T21.11, T21.12 및 T21.13를 얻었으며, 보다 구체적인 중쇄 및 경쇄 정보는 표 3에 나타내었다.In order to increase the affinity for the candidate antibody 58E, gene engineering was performed. Candidate antibodies T21.01, T21.08, T21.09, T21.10, T21.11, T21.12 and T21.13 were obtained, and more specific heavy and light chain information is shown in Table 3.
Figure PCTKR2021018795-appb-img-000001
Figure PCTKR2021018795-appb-img-000001
표 3에 나타낸 바와 같이, T21.01, T21.08 및 T21.09는 58E 항체의 인간화되지 않은 hybridoma의 중쇄를 포함한다. 또한 T21.10, T21.11, T21.12, T21.13, T21.14, T21.16 및 T21.33은 58E 인간화 항체의 중쇄를 포함한다. 경쇄와 관련하여, T21.01, T21.10 및 T21.11은 58E 항체의 인간화되지 않은 hybridoma의 경쇄를 포함하며, T21.08, T21.09, T21.12, T21.13, T21.14, T21.16 및 T21.33은 인간화된 경쇄 서열을 포함한다. 또한 T21.14, T21.16 및 T21.33은 58E 인간화 항체의 중쇄 및 인간화 T21L01 경쇄를 포함한다.As shown in Table 3, T21.01, T21.08 and T21.09 contain the heavy chain of the non-humanized hybridoma of the 58E antibody. T21.10, T21.11, T21.12, T21.13, T21.14, T21.16 and T21.33 also contain the heavy chain of the 58E humanized antibody. With respect to the light chains, T21.01, T21.10 and T21.11 include the light chains of the non-humanized hybridoma of the 58E antibody, T21.08, T21.09, T21.12, T21.13, T21.14, T21.16 and T21.33 contain humanized light chain sequences. T21.14, T21.16 and T21.33 also contain the heavy chain and humanized T21L01 light chain of the 58E humanized antibody.
각 항체의 특성을 SDS-PAGE, SEC-HPLC 및 SPR로 분석하였다. 상기 SDS-PAGE 및 SEC-HPLC 결과는 도 15에 나타내었으며, SPR 결과는 도 16에 나타내었다. The characteristics of each antibody were analyzed by SDS-PAGE, SEC-HPLC and SPR. The SDS-PAGE and SEC-HPLC results are shown in FIG. 15 , and the SPR results are shown in FIG. 16 .
도 15에 나타낸 바와 같이, gene engineering를 통해 얻은 후보항체들은 Expi293F transient expression system을 이용하여 항체를 정제 분리하였고 SDS-PAGE 방법에 의한 전기영동 후 염색하여 non-reduceing에서는 전체 항체 크기를, reducing 조건에서는 항체의 각 heavy chain과 light chain의 크기를 비교하였다. 더불어 SEC-HPLC 분석에 의하여 정제된 항체의 순도를 확인하였다. As shown in Fig. 15, candidate antibodies obtained through gene engineering were purified and separated using the Expi293F transient expression system, and were stained after electrophoresis by SDS-PAGE method to reduce the total antibody size in non-reducing and reducing conditions. The size of each heavy chain and light chain of the antibody was compared. In addition, the purity of the purified antibody was confirmed by SEC-HPLC analysis.
도 16에 나타낸 바와 같이, gene engineering를 통해 얻은 후보항체 중 T21.01, T21.09, T21.11 및 T21.13은 후보항체 58E보다 항원 친화도가 높은 것을 확인하였다.As shown in FIG. 16 , it was confirmed that among the candidate antibodies obtained through gene engineering, T21.01, T21.09, T21.11 and T21.13 had higher antigen affinity than the candidate antibody 58E.
상기 결과로부터 항원 친화도가 높다고 판단된 clone T21.01, T21.09, T21.11 및 T21.13을 선별하였다. 또한 친화도가 가장 높은 T21.11 항체의 경쇄 T21L01의 서열을 인간화하여 T21.11과 유사한 친화력을 가지는 T21.14, T21.16 및 T21.33을 선별하였고 각 인간화한 T21L01.01, T21L01.03 및 T21L01.20의 경쇄 서열을 포함한다.From the above results, clones T21.01, T21.09, T21.11 and T21.13 that were judged to have high antigen affinity were selected. In addition, by humanizing the sequence of the light chain T21L01 of the T21.11 antibody with the highest affinity, T21.14, T21.16 and T21.33 having similar affinity to T21.11 were selected, and humanized T21L01.01 and T21L01.03 respectively. and the light chain sequence of T21L01.20.
선별된 clone T21.01, T21.09, T21.11, T21.13, T21.14, T21.16 및 T21.33의 서열을 분석한 결과는 하기 표 4에 나타내었다.The results of sequence analysis of the selected clones T21.01, T21.09, T21.11, T21.13, T21.14, T21.16 and T21.33 are shown in Table 4 below.
CloneClone 구분division 서열order 서열번호SEQ ID NO: 서열order 서열번호SEQ ID NO:
T21.01T21.01 VH VH SYVMHSYVMH 1717 EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINLNNDGTKYNEKFKGKATLSSDKSSSTAYMELSSLTSEDSAVYYCARWRVYYYAMDYWGQGTSVTVSS EVQLQQSGPELVKPGASVKMSCKASGYTFT SYVMHWVKQKPGQGLEWIGYINLNNDGTKYNEKFKGKATLSSDKSSSTAYMELSSLTSEDSAVYYCARWRVYYYAMDYWGQGTSVTVSS 2323
YINLNNDGTKYNEKFKG YINLNNDGTKYNEKFKG 1818
WRVYYYAMDY WRVYYYAMDY 1919
VLVL NASSSVDYVHNASSSVDYVH 7979 EIVLTQSPAITAASLGQKVTITCNASSSVDYVHWYQQKSGTSPRPWIYEISKLASGVPARFSGSGSGTSYSLTISSMEAEDAAIYYCQLWNYPLITFGAGTKLELK EIVLTQSPAITAASLGQKVTITCNASSSVDYVHWYQQKSGTSPRPWIYEISKLASGVPARFSGSGSGTSYSLTISSMEAEDAAIYYCQLWNYPLITFGAGTKLELK 8080
EISKLAS EISKLAS 2121
QLWNYPLIT QLWNYPLIT 2222
T21.09T21.09 VH VH SYVMHSYVMH 1717 EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINLNNDGTKYNEKFKGKATLSSDKSSSTAYMELSSLTSEDSAVYYCARWRVYYYAMDYWGQGTSVTVSS EVQLQQSGPELVKPGASVKMSCKASGYTFT SYVMHWVKQKPGQGLEWIGYINLNNDGTKYNEKFKGKATLSSDKSSSTAYMELSSLTSEDSAVYYCARWRVYYYAMDYWGQGTSVTVSS 2323
YINLNNDGTKYNEKFKG YINLNNDGTKYNEKFKG 1818
WRVYYYAMDY WRVYYYAMDY 1919
VLVL NASSSVDYVHNASSSVDYVH 7979 EIVLTQSPATLSLSPGERATLSCNASSSVDYVHWYQQKPGQAPRPWIYEISKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQLWNYPLITFGQGTKVEIKEIVLTQSPATLSLSPGERATLSCNASSSVDYVHWYQQKPGQAPRPWIYEISKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQLWNYPLITFGQGTKVEIK 8181
EISKLAS EISKLAS 2121
QLWNYPLIT QLWNYPLIT 2222
T21.11T21.11 VH VH SYVMHSYVMH 1717 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYINLNNDGTKYNEKFKGRATLTSDKSASTAYMELSSLRSEDTAVYYCARWRVYYYAMDYWGQGTLVTVSSQVQLVQSGAEVKKPGASVKVSCKASGYTFT SYVMH WVRQAPGQRLEWIG YINLNNDGTKYNEKFKG RATLTSDKSASTAYMELSSLRSEDTAVYYCAR WRVYYYAMDY WGQGTLVTVSS 8282
YINLNNDGTKYNEKFKG YINLNNDGTKYNEKFKG 1818
WRVYYYAMDY WRVYYYAMDY 1919
VLVL NASSSVDYVHNASSSVDYVH 7979 EIVLTQSPAITAASLGQKVTITCNASSSVDYVHWYQQKSGTSPRPWIYEISKLASGVPARFSGSGSGTSYSLTISSMEAEDAAIYYCQLWNYPLITFGAGTKLELK EIVLTQSPAITAASLGQKVTITCNASSSVDYVHWYQQKSGTSPRPWIYEISKLASGVPARFSGSGSGTSYSLTISSMEAEDAAIYYCQLWNYPLITFGAGTKLELK 8080
EISKLAS EISKLAS 2121
QLWNYPLIT QLWNYPLIT 2222
T21.13T21.13 VH VH SYVMHSYVMH 1717 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYINLNNDGTKYNEKFKGRATLTSDKSASTAYMELSSLRSEDTAVYYCARWRVYYYAMDYWGQGTLVTVSS QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYVMH WVRQAPGQRLEWIG YINLNNDGTKYNEKFKG RATLTSDKSASTAYMELSSLRSEDTAVYYCAR WRVYYYAMDY WGQGTLVTVSS 8282
YINLNNDGTKYNEKFKG YINLNNDGTKYNEKFKG 1818
WRVYYYAMDY WRVYYYAMDY 1919
VLVL NASSSVDYVHNASSSVDYVH 7979 EIVLTQSPATLSLSPGERATLSCNASSSVDYVHWYQQKPGQAPRPWIYEISKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQLWNYPLITFGQGTKVEIKEIVLTQSPATLSLSPGERATLSCNASSSVDYVHWYQQKPGQAPRPWIYEISKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQLWNYPLITFGQGTKVEIK 8181
EISKLAS EISKLAS 2121
QLWNYPLIT QLWNYPLIT 2222
T21.14T21.14 VH VH SYVMHSYVMH 1717 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYINLNNDGTKYNEKFKGRATLTSDKSASTAYMELSSLRSEDTAVYYCARWRVYYYAMDYWGQGTLVTVSSQVQLVQSGAEVKKPGASVKVSCKASGYTFT SYVMH WVRQAPGQRLEWIG YINLNNDGTKYNEKFKG RATLTSDKSASTAYMELSSLRSEDTAVYYCAR WRVYYYAMDY WGQGTLVTVSS 8282
YINLNNDGTKYNEKFKG YINLNNDGTKYNEKFKG 1818
WRVYYYAMDY WRVYYYAMDY 1919
VLVL NASSSVDYVHNASSSVDYVH 7979 EIVLTQSPAITAASLGQKVTITCNASSSVDYVHWYQQKPGQAPRPWIYEISKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQLWNYPLITFGQGTKVEIK EIVLTQSPAITAASLGQKVTITCNASSSVDYVHWYQQKPGQAPRPWIYEISKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQLWNYPLITFGQGTKVEIK 102102
EISKLAS EISKLAS 2121
QLWNYPLIT QLWNYPLIT 2222
T21.16T21.16 VH VH SYVMHSYVMH 1717 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYINLNNDGTKYNEKFKGRATLTSDKSASTAYMELSSLRSEDTAVYYCARWRVYYYAMDYWGQGTLVTVSSQVQLVQSGAEVKKPGASVKVSCKASGYTFT SYVMH WVRQAPGQRLEWIG YINLNNDGTKYNEKFKG RATLTSDKSASTAYMELSSLRSEDTAVYYCAR WRVYYYAMDY WGQGTLVTVSS 8282
YINLNNDGTKYNEKFKG YINLNNDGTKYNEKFKG 1818
WRVYYYAMDY WRVYYYAMDY 1919
VLVL NASSSVDYVHNASSSVDYVH 7979 EIVLTQSPATLSLSPGERATLSCNASSSVDYVHWYQQKPGQAPRPWIYEISKLASGVPARFSGSGSGTSYSLTISSMEAEDAAIYYCQLWNYPLITFGQGTKVEIK EIVLTQSPATLSLSPGERATLSCNASSSVDYVHWYQQKPGQAPRPWIYEISKLASGVPARFSGSGSGTSYSLTISSMEAEDAAIYYCQLWNYPLITFGQGTKVEIK 101101
EISKLAS EISKLAS 2121
QLWNYPLIT QLWNYPLIT 2222
T21.33T21.33 VH VH SYVMHSYVMH 1717 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYINLNNDGTKYNEKFKGRATLTSDKSASTAYMELSSLRSEDTAVYYCARWRVYYYAMDYWGQGTLVTVSSQVQLVQSGAEVKKPGASVKVSCKASGYTFT SYVMH WVRQAPGQRLEWIG YINLNNDGTKYNEKFKG RATLTSDKSASTAYMELSSLRSEDTAVYYCAR WRVYYYAMDY WGQGTLVTVSS 8282
YINLNNDGTKYNEKFKG YINLNNDGTKYNEKFKG 1818
WRVYYYAMDY WRVYYYAMDY 1919
VLVL NASSSVDYVHNASSSVDYVH 7979 EIVLTQSPATLSLSPGERATLSCNASSSVDYVHWYQQKPGQAPRPWIYEISKLASGIPARFSGSGSGTSYTLTISSLEPEDFAVYYCQLWNYPLITFGQGTKVEIK EIVLTQSPATLSLSPGERATLSCNASSSVDYVHWYQQKPGQAPRPWIYEISKLASGIPARFSGSGSGTSYTLTISSLEPEDFAVYYCQLWNYPLITFGQGTKVEIK 103103
EISKLAS EISKLAS 2121
QLWNYPLIT QLWNYPLIT 2222
(2) TIGIT/CD155 cell based Blocking Assay(2) TIGIT/CD155 cell based Blocking Assay
상기 표 4의 친화도가 개선된 후보항체가 TIGIT과 그 counterpart인 CD155와의 결합을 억제할 수 있는지 확인하였다. 실험은 TIGIT/CD155 Blockade Bioassay (Promega) kit을 사용하였다. 구체적으로, 인간 TIGIT과 CD226을 발현하고 luciferase reporter를 가지는 Jurkat Effector cells과 CD155를 발현하는 CHO-K1 aAPC를 co-culture하였다. 그 후 친화도가 개선된 후보항체 처리 시 항체가 TIGIT과 결합하여 CD155와의 결합을 block하고 CD155가 CD226과 결합함으로써 activation된 luciferase signal을 측정하였다. 각 후보항체 별 측정 결과는 도 17a에 나타내었고, 도 17a에 기반으로 그래프를 작성하여 도 17b에 나타내었다.It was confirmed whether the candidate antibody with improved affinity in Table 4 could inhibit the binding of TIGIT to its counterpart CD155. For the experiment, TIGIT/CD155 Blockade Bioassay (Promega) kit was used. Specifically, Jurkat Effector cells expressing human TIGIT and CD226 and having a luciferase reporter and CHO-K1 aAPC expressing CD155 were co-cultured. After that, when the affinity-improved candidate antibody was treated, the antibody bound to TIGIT to block the binding to CD155, and the luciferase signal activated by binding of CD155 to CD226 was measured. Measurement results for each candidate antibody are shown in FIG. 17A, and a graph was prepared based on FIG. 17A and shown in FIG. 17B.
도 17a 및 b에 나타낸 바와 같이, 후보항체 58E 및 친화도가 개선된 후보항체는 시판 항체 Oncomed-313M32보다 억제 효능이 우수한 것을 확인하였다. 특히, 친화도가 개선된 후보항체 중 T21.11 clone은 후보항체 58E보다 억제 효능이 더 높았다.As shown in FIGS. 17A and 17B , it was confirmed that the candidate antibody 58E and the candidate antibody with improved affinity had superior inhibitory efficacy than the commercially available antibody Oncomed-313M32. In particular, among the candidate antibodies with improved affinity, the T21.11 clone had a higher inhibitory effect than the candidate antibody 58E.
실시예 8. 인간화 후보항체 62F(hu62F)의 affinity engineering 및 효능 평가Example 8. Affinity engineering and efficacy evaluation of humanized candidate antibody 62F (hu62F)
(1) Anti-TIGIT hu62F scFv 항체를 주형으로 하는 CDR 라이브러리 제작(1) Construction of a CDR library using the Anti-TIGIT hu62F scFv antibody as a template
(i) CDR 라이브러리 제작을 위한 primer 디자인(i) Primer design for CDR library construction
상기 표 2의 인간화 후보항체 62F(hu62F)의 서열을 분석하여 6종의 CDR을 정의하였으며, 그 결과는 도 18에 나타내었다. 무작위 서열을 도입할 CDR 아미노산을 선정하였다. 선정된 CDR 아미노산은 표 5와 같다. Six kinds of CDRs were defined by analyzing the sequence of the humanized candidate antibody 62F (hu62F) in Table 2, and the results are shown in FIG. 18 . CDR amino acids into which random sequences were to be introduced were selected. The selected CDR amino acids are shown in Table 5.
Figure PCTKR2021018795-appb-img-000002
Figure PCTKR2021018795-appb-img-000002
표 5의 아미노산을 표적으로 하여 randomization시키는 primer를 디자인하였고, 라이브러리를 구현하기 위한 primer 22종을 IDT technology (www.IDTDNA.com)에서 제작하였다. 제작된 프라이머는 도 19와 같다.Primers for randomization targeting the amino acids in Table 5 were designed, and 22 types of primers to implement the library were produced by IDT technology (www.IDTDNA.com). The prepared primer is shown in FIG. 19 .
(ii) hu62F (VH-VL) template DNA 제작(ii) hu62F (VH-VL) template DNA construction
Library를 만들기 위하여, hu62F scFv를 pYD5 벡터에 클로닝하였다. pYD5 플라스미드에 VH(G4S)3-VL scFv 형태로 제작하였다. 제작된 플라스미드 pYD5의 개열지도는 도 20과 같으며, IDT technology에 의뢰하여 합성하였다. 각 scFv의 c-terminal EcoR1 site 뒤에 V5-tag를 접합하였고, Nhe1, EcoR1 restriction enzyme으로 자른 pYD5 vector의 양 말단과 homologous한 sequence를 합성 유전자의 양 말단에 합성하였다. 합성이 완료된 insert gene과 linearized vector를 In-Fusion HD Cloning Kit(Clontech)을 사용하여 클로닝하고, 시퀀싱 프라이머는 T7을 사용하여 확인하였다. 클로닝이 완료된 플라스미드를 Stellar Competent Cells(Clontech)에 transfection한 후 ZymoPUREII Plasmid Midiprep kit(Zymo research, Cat# D4201)로 midi-prep하여 DNA를 15 ug 이상 수득하였다.To make the library, hu62F scFv was cloned into pYD5 vector. It was constructed in the form of VH(G4S)3-VL scFv on pYD5 plasmid. The cleavage map of the produced plasmid pYD5 is shown in FIG. 20, and it was synthesized by requesting IDT technology. V5-tag was spliced after the c-terminal EcoR1 site of each scFv, and sequences homologous to both ends of the pYD5 vector cut with Nhe1 and EcoR1 restriction enzymes were synthesized at both ends of the synthetic gene. The synthesized insert gene and linearized vector were cloned using the In-Fusion HD Cloning Kit (Clontech), and the sequencing primer was confirmed using T7. After transfection of the cloned plasmid into Stellar Competent Cells (Clontech), midi-prep with ZymoPUREII Plasmid Midiprep kit (Zymo research, Cat# D4201) to obtain more than 15 ug of DNA.
(iii) Random library 유전자 제작(iii) Random library gene production
10개의 CDR region의 random library를 만들기 위하여, random primer를 포함한 2쌍의 primer로 fragment를 2개를 PCR reaction으로 증폭시켰다. PCR 산물을 gel-extraction한 후 2개의 fragment를 overlap extension PCR로 1개의 CDR region에 random sequence가 있는 library를 제작하였다. 각 CDR region에 대한 PCR을 통해 library를 완성하였다. 상기 CDR region에 대한 PCR(1st PCR)은 도 21의 모식도 및 실험 조건으로 수행하였다. 구체적으로, 1st PCR은 hu62F pYD5를 template로 하여 PCR reaction volume 50ul 당 100ng 넣어주고, dNTP(invitrogen), 2uM forward 및 reverse primer, 5X Phusion HF buffer(Thermo scientific), 1.5ul DMSO, 2unit Phusion high fidelity polymerse(Thermo scientific, cat#F530L)을 각각 넣고 나머지 volume은 distilled water로 채웠다. PCR reaction은 위의 표와 같은 방법으로 진행하였고, 1.5%의 agarose gel을 만들어 전기영동으로 증폭된 DNA를 확인하였다 각 fragment는 DNA cleanup kit (Geneaid, cat#DFM025)로 extraction한 후 UV spectrophotometer (Thermo, Model: Multiskan Go)로 농도를 측정하였다 CDR region에 대한 PCR 결과는 도 22에 나타내었다.To create a random library of 10 CDR regions, two fragments were amplified by PCR reaction with two pairs of primers including random primers. After gel-extraction of the PCR product, a library with a random sequence in one CDR region was prepared by overlap extension PCR with two fragments. The library was completed through PCR for each CDR region. PCR (1 st PCR) for the CDR region was performed according to the schematic diagram of FIG. 21 and experimental conditions. Specifically, 1st PCR uses hu62F pYD5 as a template and puts 100ng per 50ul of PCR reaction volume, dNTP (invitrogen), 2uM forward and reverse primer, 5X Phusion HF buffer (Thermo scientific), 1.5ul DMSO, 2unit Phusion high fidelity polymerse (Thermo scientific, cat#F530L) was added, and the remaining volume was filled with distilled water. The PCR reaction was carried out in the same manner as in the table above, and the DNA amplified by electrophoresis was confirmed by making a 1.5% agarose gel. Each fragment was extracted with a DNA cleanup kit (Geneaid, cat#DFM025) and then UV spectrophotometer (Thermo , Model: Multiskan Go) was used to measure the concentration. PCR results for the CDR region are shown in FIG. 22 .
1st PCR에서 얻은 fragment 1 및 2를 이용하여 2nd PCR을 수행하였다. 구체적으로, 1st PCR에서 얻은 fragment 1 및 2를 molar ratio가 1 : 1이 되도록 총 400ng의 DNA를 넣고, 10mM each dNTP(invtrogen), 5X Phusion HF buffer(Thermo scientific), 2unit Phusion high fidelity polymerase(Thermo scientific, cat#F530L)을 각각 넣고 total volume 50ul가 되도록 나머지 volume은 distilled water(Invitrogen, cat#10977)로 채웠다. 2nd PCR의 모식도 및 반응 조건은 도 23과 같다. 2nd PCR 결과는 도 24에 나타내었다. 2nd PCR was performed using fragments 1 and 2 obtained in 1st PCR. Specifically, a total of 400 ng of DNA was added to fragments 1 and 2 obtained in 1 st PCR so that the molar ratio is 1:1, 10mM each dNTP (invtrogen), 5X Phusion HF buffer (Thermo scientific), 2unit Phusion high fidelity polymerase ( Thermo scientific, cat#F530L) was added, and the remaining volume was filled with distilled water (Invitrogen, cat#10977) to a total volume of 50ul. A schematic diagram and reaction conditions of 2nd PCR are shown in FIG. 23 . 2nd PCR results are shown in FIG. 24 .
1.5% agarose gel을 만들어 전기영동으로 증폭된 DNA를 확인하였다 2nd PCR 이후 각 PCR product는 DNA cleanup kit (Geneaid, cat#DFM025)로 extraction한 후 spectrophotometer(Thermo, Model: Multiskan Go)로 농도를 측정하였다.After the 2nd PCR, each PCR product was extracted with a DNA cleanup kit (Geneaid, cat#DFM025) and the concentration was measured with a spectrophotometer (Thermo, Model: Multiskan Go). did
2nd PCR을 통해 얻은 각 insert DNA는 5ug 이상 되도록 준비하였다. Yeast transformation에 사용하기 위해 pYD5 vector를 Nhe1(NEB, cat# R3131L)과 EcoR1(NEB, cat# R3101S)으로 cut하여 DNA cleanup kit (Geneaid, cat#DFM025)으로 추출하였다. Transformation에 사용될 vector와 insert를 library 당 각각 1 및 5ug씩 준비한 후 Pellet Paint®Co Precipitant (Novagen, Cat#690493)를 사용하여 최종 volume이 각각 1 및 5ul 이하가 되도록 농축하였다.Each insert DNA obtained through 2nd PCR was prepared to be 5ug or more. To use for yeast transformation, pYD5 vector was cut with Nhe1 (NEB, cat# R3131L) and EcoR1 (NEB, cat# R3101S) and extracted with a DNA cleanup kit (Geneaid, cat#DFM025). After preparing 1 and 5 μg of vector and insert to be used for transformation per library, Pellet Paint®Co Precipitant (Novagen, Cat#690493) was used to concentrate the final volume to 1 and 5ul or less, respectively.
(iv) Yeast transformation(iv) Yeast transformation
Yeast transformation은 도 25와 같이 진행하였다. 구체적으로, Yeast competent cell은 EBY100 yeast strain을 YPD plate에 streaking하여 30℃에서 2일 동안 배양한 후 yeast colony 1개를 picking하여 5ml YPD media에서 30℃250rpm으로 overnight 배양하였다. 배양한 yeast cell을 50ml scale로 계대배양하여 약 6시간 동안 동일한 조건에서 O.D 측정값이 약 1.0 내지 1.5가 될 때까지 키운 후, 0.5ml의 sterilized Tris DTT solution(0.39g 1,4 dithiothreitol in 1ml 1M Tris pH8.0 buffer)을 넣어주고 15분 동안 incubation하였다. 배양이 끝난 yeast cell은 centrifuge 후 cold E buffer(1.2 g Tris base, 92.4g sucrose, 1M MgCl2 in distilled water)로 washing하고 total volume이 300ul가 되도록 E buffer로 resuspension하여 competent cell을 만들었다. 준비된 competent cell 50ul와 농축된 insert DNA 5ug, vector 1ug을 electro cuvette에 넣고 0.54kV 및 25uF 조건으로 electroporation을 하였다. 그 후 YPD media 1ml을 즉시 넣었고, 30℃250rpm에서 1시간 동안 incubation하였다.Yeast transformation was performed as shown in FIG. 25 . Specifically, yeast competent cells were streaked with EBY100 yeast strain on a YPD plate and cultured at 30°C for 2 days, then one yeast colony was picked and cultured overnight at 30°C and 250rpm in 5ml YPD media. The cultured yeast cells were subcultured on a 50ml scale and grown under the same conditions for about 6 hours until the measured OD value was about 1.0 to 1.5, and then 0.5ml of sterilized Tris DTT solution (0.39 g 1,4 dithiothreitol in 1ml 1M). Tris pH8.0 buffer) was added and incubated for 15 minutes. After culturing, the yeast cells were centrifuged, washed with cold E buffer (1.2 g Tris base, 92.4 g sucrose, 1M MgCl 2 in distilled water), and resuspensioned with E buffer to make a total volume of 300 μl to prepare competent cells. 50ul of the prepared competent cells, 5ug of concentrated insert DNA, and 1ug of vector were placed in an electro cuvette and electroporated at 0.54kV and 25uF conditions. After that, 1ml of YPD media was immediately added, and incubated at 30°C and 250rpm for 1 hour.
(v) Diversity 측정 및 라이브러리 배양(v) Diversity measurement and library culture
Transformation 후 YPD에서 배양한 yeast cell을 100ug/ml kanamycin 항생제를 넣은 10ml SDCAA media(20g glucose, 14.7g sodium citrate, 4.3g citric acid monohydrate, 6.7g yeast nitrogen base, 5g bacto casamino acid in 1L distilled water)로 resuspension 후 1/100 SDCAA media로 희석하여 104 cell이 되도록 100ul를 SDCAA::Km plate(100ug/ml kanamycin, 20g glucose, 14.7g sodium citrate, 4.3g citric acid monohydrate, 6.7g yeast nitrogen base, 5g bacto casamino acid, 16g bacto agar) in 1L distilled water에 spreading하였다. 그리고 1/10씩 serial dilution하여 각각 105, 106, 107 cell이 되도록 spreading 후 diversity를 측정하였다. Diversity 측정 결과는 표 6에 나타내었다.After transformation, yeast cells cultured in YPD were treated with 10 ml SDCAA media (20 g glucose, 14.7 g sodium citrate, 4.3 g citric acid monohydrate, 6.7 g yeast nitrogen base, 5 g bacto casamino acid in 1 L distilled water) containing 100 μg/ml kanamycin antibiotic. After resuspension, dilute with 1/100 SDCAA media to make 10 4 cells, and 100ul of SDCAA::Km plate (100ug/ml kanamycin, 20g glucose, 14.7g sodium citrate, 4.3g citric acid monohydrate, 6.7g yeast nitrogen base, 5g bacto) casamino acid, 16g bacto agar) in 1L distilled water. Then, by serial dilution by 1/10, diversity was measured after spreading to become 10 5 , 10 6 , and 10 7 cells, respectively. Diversity measurement results are shown in Table 6.
Figure PCTKR2021018795-appb-img-000003
Figure PCTKR2021018795-appb-img-000003
Transformed yeast cell을 SDCAA media에 resuspension하여 initial O.D가 0.1이 되도록 100ml SDCAA media에서 24시간 동안 배양하였다 동일한 방법으로 계대배양 한 후에 initial O.D가 1이 되도록 100ug/ml kanamycin 항생제를 넣은 100ml SGCAA media(20g galactose, 14.7g sodium citrate, 4.3g citric acid monohydrate, 6.7g yeast nitrogen base, 5g bacto casamino acid in 1L distilled water) 로 resuspension 후 30 ℃, 250rpm으로 16 내지 20시간 동안 induction하였다.Transformed yeast cells were resuspensed in SDCAA media and cultured in 100ml SDCAA media for 24 hours so that the initial O.D was 0.1. After subculture in the same way, 100ml SGCAA media (20g galactose) containing 100ug/ml kanamycin antibiotic so that the initial O.D is 1. , 14.7 g sodium citrate, 4.3 g citric acid monohydrate, 6.7 g yeast nitrogen base, 5 g bacto casamino acid in 1 L distilled water), followed by induction at 30 °C and 250 rpm for 16 to 20 hours.
(2) 라이브러리의 enrichment(2) enrichment of the library
항원 결합력을 향상시키기 위하여, CDR region에 mutation이 들어가지 않고 wild type의 비중이 높은 L3A 라이브러리를 제외한 9개의 라이브러리를 FACS sorting을 통해 yeast cell 표면에 발현된 scFv를 staining하였다. 구체적으로, 1차 staining은 항원 결합력을 확인할 수 있는 0.5~1nM biotinylated TIGIT his와 scFv의 발현을 확인할 수 있는 1:500 anti V5 antibody(Invitrogen, cat#R960 25)를 FACS buffer 0.1% BSA in pH7.4 PBS buffer)로 희석하여 room temperature에서 30분간 rotational mixer에서 incubation하였다. 1차 staining이 끝난 후 FACS buffer로 washing한 후 1:100 비율로 Streptavidin, R Phycoerythrin Conjugate (SAPE) (Invitrogen, cat#S866)와 Anti Mouse IgG (H+L) FITC antibody (Invitrogen/Cat No.11-4011-85)를 FACS buffer에 희석하여 빛이 차단된 4℃에서 20분 동안 incubation하였다. 2차 staining이 끝난 cell은 FACS buffer로 2번 washing 후 FACS buffer로 resuspension하여 분석 sample을 준비하였다 각각의 라이브러리는 wild type인 hu62F의 결합력보다 높은 부분을 0.1~1% 사이에서 gating하여, 0.5~1nM의 항원 농도 조건 하에서 초 당 20,000~30,000개의 속도로 sorting하였다. FACS 결과는 도 26a 내지 d에 나타내었다.In order to improve antigen-binding capacity, nine libraries except for the L3A library, which did not contain mutations in the CDR region and had a high proportion of wild type, were stained with scFv expressed on the yeast cell surface through FACS sorting. Specifically, for the first staining, 0.5~1nM biotinylated TIGIT his, which can confirm antigen binding, and 1:500 anti V5 antibody (Invitrogen, cat#R960 25), which can confirm the expression of scFv, were used in FACS buffer 0.1% BSA in pH7. 4 PBS buffer) and incubated in a rotational mixer at room temperature for 30 minutes. After primary staining, washing with FACS buffer and 1:100 ratio of Streptavidin, R Phycoerythrin Conjugate (SAPE) (Invitrogen, cat#S866) and Anti Mouse IgG (H+L) FITC antibody (Invitrogen/Cat No.11) -4011-85) was diluted in FACS buffer and incubated for 20 minutes at 4°C where light was blocked. Cells after secondary staining were washed twice with FACS buffer and then resuspensioned with FACS buffer to prepare analysis samples. Each library was gating between 0.1 and 1% of the binding strength of wild type hu62F, 0.5 to 1 nM. Sorting was performed at a rate of 20,000 to 30,000 per second under the antigen concentration condition of FACS results are shown in Figures 26a to d.
도 26a 내지 d에 나타낸 바와 같이, 항원 결합력을 향상시키기 위하여 각각의 라이브러리와 TIGIT-항원을 staining하여 유세포 분석하여 hu62F보다 TIGIT-항원 결합력이 증가된 세포들을 분리하였다.As shown in FIGS. 26A to 26D , each library and TIGIT-antigen were stained and flow cytometric analysis to improve antigen binding, and cells with increased TIGIT-antigen binding than hu62F were isolated.
(3) Affinity 향상 후보 개별 클론의 항원결합력 분석(3) Affinity improvement candidate antigen-binding strength analysis of individual clones
마지막 sorting round 후 sorted cell을 일부 SDCAA::Km plate에서 배양하였다 자란 colony를 picking하여 SDCAA media에서 키운 후 SGCAA media로 induction하여 상기 실시예 (2)와 같은 방법으로 FACS analysis를 진행하였다. FACS 결과는 도 27a 내지 f에 나타내었다.After the last sorting round, the sorted cells were cultured on some SDCAA::Km plate. The grown colonies were picked, grown in SDCAA media, and then inducted into SGCAA media, and FACS analysis was performed in the same manner as in Example (2). FACS results are shown in Figures 27a to f.
도 27a 내지 f에 나타낸 바와 같이, 라이브러리로부터 분리한 hu62F보다 TIGIT-항원 결합력이 증가된 세포들은 단일 클론으로 분리한 후 각 Yeast 표면에 발현시켜서 TIGIT-항원과의 결합력을 flow cytometry 분석하였다. As shown in FIGS. 27A to 27F , the cells with increased TIGIT-antigen avidity compared to hu62F isolated from the library were separated into single clones and expressed on the surface of each yeast, and the binding ability with TIGIT-antigen was analyzed by flow cytometry.
(4) 선별된 개별클론의 서열분석(4) Sequence analysis of selected individual clones
SDCAA::Km plate에서 colony를 picking하여 SDCAA::Km media로 키운 yeast cell을 ZymoPrep Yeast Plasmid MiniPrep II Kit(zymo research, Cat#D2004 50) 로 DNA prep을 하였다. Extraction된 DNA는 E.coli Stellar™Competent Cells에 transformation한 후 sequencing을 진행하였다. 선별된 개별클론의 VH와 아미노산 서열 및 염기서열을 분석 결과는 각각 표 7 및 8에 나타내었다.Yeast cells grown with SDCAA::Km media by picking colonies from SDCAA::Km plate were DNA prep with ZymoPrep Yeast Plasmid MiniPrep II Kit (zymo research, Cat#D2004 50). The extracted DNA was transformed into E. coli Stellar™ Competent Cells and then sequencing was performed. The results of analysis of the VH and amino acid sequences and nucleotide sequences of the selected individual clones are shown in Tables 7 and 8, respectively.
CloneClone 구분division 아미노산 서열amino acid sequence 서열번호SEQ ID NO:
T02H01T02H01 VHVH QVQLVQSGAEVKKPGASVKVSCKASGFTFTGRTIHWVRQAPGQGLEWIGGFNPNSGGSSYNQKFKGRATLTVDKSISTAYMELSRLRSDDTAVYYCARSSYYRFDLDYWGQG TTVTVSSQVQLVQSGAEVKKPGASVKVSCKASGFTFTGRTIHWVRQAPGQGLEWIGGFNPNSGGSSYNQKFKGRATLTVDKSISTAYMELSRLRSDDTAVYYCARSSYYRFDLDYWGQG TTVTVSS 8383
T02H02T02H02 VHVH QVQLVQSGAEVKKPGASVKVSCKASGFTFTGRTMHWVRQAPGQGLEWIGGFNPNSGGSSYNQKFKGRATLTVDKSISTAYMELSRLRSDDTAVYYCARSSYYRFDLDYWGQGTTVTVSSQVQLVQSGAEVKKPGASVKVSCKASGFTFTGRTMHWVRQAPGQGLEWIGGFNPNSGGSSYNQKFKGRATLTVDKSISTAYMELSRLRSDDTAVYYCARSSYYRFDLDYWGQGTTVTVSS 8484
T02H03T02H03 VHVH QVQLVQSGAEVKKPGASVKVSCKASGFTFTGRSIHWVRQAPGQGLEWIGGFNPNSGGSSYNQKFKGRATLTVDKSISTAYMELSRLRSDDTAVYYCARSSYYRFDLDYWGQGTTVTVSSQVQLVQSGAEVKKPGASVKVSCKASGFTFTGRSIHWVRQAPGQGLEWIGGFNPNSGGSSYNQKFKGRATLTVDKSISTAYMELSRLRSDDTAVYYCARSSYYRFDLDYWGQGTTVTVSS 8585
T02H04T02H04 VHVH QVQLVQSGAEVKKPGASVKVSCKASGFTFTGRSMHWVRQAPGQGLEWIGGFNPNSGGSSYNQKFKGRATLTVDKSISTAYMELSRLRSDDTAVYYCARSSYYRFDLDYWGQGTTVTVSSQVQLVQSGAEVKKPGASVKVSCKASGFTFTGRSMHWVRQAPGQGLEWIGGFNPNSGGSSYNQKFKGRATLTVDKSISTAYMELSRLRSDDTAVYYCARSSYYRFDLDYWGQGTTVTVSS 8686
T02H05T02H05 VHVH QVQLVQSGAEVKKPGASVKVSCKASGFTFTEYTMHWVRQAPGQGLEWIGGINAKFGRQLIHQKFKGRATLTVDKSISTAYMELSRLRSDDTAVYYCARSSYYRFDLDYWGQGTTVTVSSQVQLVQSGAEVKKPGASVKVSCKASGFTFTEYTMHWVRQAPGQGLEWIGGINAKFGRQLIHQKFKGRATLTVDKSISTAYMELSRLRSDDTAVYYCARSSYYRFDLDYWGQGTTVTVSS 8787
T02H06T02H06 VHVH QVQLVQSGAEVKKPGASVKVSCKASGFTFTEYTMHWVRQAPGQGLEWIGGIRAKTGGSSYNQKFKGRATLTVDKSISTAYMELSRLRSDDTAVYYCARSSYYRFDLDYWGQGTTVTVSSQVQLVQSGAEVKKPGASVKVSCKASGFTFTEYTMHWVRQAPGQGLEWIGGIRAKTGGSSYNQKFKGRATLTVDKSISTAYMELSRLRSDDTAVYYCARSSYYRFDLDYWGQGTTVTVSS 8888
T02H07T02H07 VHVH QVQLVQSGAEVKKPGASVKVSCKASGFTFTEYTMHWVRQAPGQGLEWIGGFNPNSGRSAYNQKFKGRATLTVDKSISTAYMELSRLRSDDTAVYYCARSSYYRFDLDYWGQGTTVTVSSQVQLVQSGAEVKKPGASVKVSCKASGFTFTEYTMHWVRQAPGQGLEWIGGFNPNSGRSAYNQKFKGRATLTVDKSISTAYMELSRLRSDDTAVYYCARSSYYRFDLDYWGQGTTVTVSS 8989
T02L01T02L01 VLVL DIQMTQSPSSLSASVGDRVTITCKASQDVKTAVAWYQQKPGKAPKLLIYSASWRYSGVPSRFSGSGSGADFTLTISSLQPEDFATYYCQQHYTTPWTFGQGTKVEIKDIQMTQSPSSLSASVGDRVTITCKASQDVKTAVAWYQQKPGKAPKLLIYSASWRYSGVPSRFSGSGSGADFTLTISSLQPEDFATYYCQQHYTTPWTFGQGTKVEIK 9090
T02L02T02L02 VLVL DIQMTQSPSSLSASVGDRVTITCKASQDVKTAVAWYQQKPGKAPKLLIYSASFRYTGVPSRFSGSGSGADFTLTISSLQPEDFATYYCQQHYLTPWTFGQGTKVEIKDIQMTQSPSSLSASVGDRVTITCKASQDVKTAVAWYQQKPGKAPKLLIYSASFRYTGVPSRFSGSGSGADFTLTISSLQPEDFATYYCQQHYLTPWTFGQGTKVEIK 9191
CloneClone 구분division 염기서열base sequence 서열번호SEQ ID NO:
T02H01T02H01 VHVH CAGGTGCAGCTGGTCCAATCAGGGGCAGAAGTAAAGAAACCCGGCGCATCCGTTAAAGTCTCATGCAAAGCCTCCGGATTCACATTCACTGGACGTACTATTCACTGGGTACGTCAGGCGCCCGGACAGGGATTGGAGTGGATTGGCGGATTTAATCCTAACTCTGGCGGCAGCTCATACAATCAGAAATTTAAGGGAAGGGCCACGTTGACCGTAGACAAAAGTATTAGCACGGCCTACATGGAGCTTTCAAGATTACGTTCCGACGATACGGCGGTCTACTACTGCGCGAGGTCCAGCTATTATAGGTTTGACCTAGATTACTGGGGGCAGGGCACAACCGTAACAGTTTCTAGCCAGGTGCAGCTGGTCCAATCAGGGGCAGAAGTAAAGAAACCCGGCGCATCCGTTAAAGTCTCATGCAAAGCCTCCGGATTCACATTCACTGGACGTACTATTCACTGGGTACGTCAGGCGCCCGGACAGGGATTGGAGTGGATTGGCGGATTTAATCCTAACTCTGGCGGCAGCTCATACAATCAGAAATTTAAGGGAAGGGCCACGTTGACCGTAGACAAAAGTATTAGCACGGCCTACATGGAGCTTTCAAGATTACGTTCCGACGATACGGCGGTCTACTACTGCGCGAGGTCCAGCTATTATAGGTTTGACCTAGATTACTGGGGGCAGGGCACAACCGTAACAGTTTCTAGC 9292
T02H02T02H02 VHVH CAGGTGCAGCTGGTCCAATCAGGGGCAGAAGTAAAGAAACCCGGCGCATCCGTTAAAGTCTCATGCAAAGCCTCCGGATTCACATTCACTGGACGTACTATGCACTGGGTACGTCAGGCGCCCGGACAGGGATTGGAGTGGATT GGCGGATTTAATCCTAACTCTGGCGGCAGCTCATACAATCAGAAATTTAAGGGAAGGGCCACGTTGACCGTA GACAAAAGTATTAGCACGGCCTACATGGAGCTTTCAAGATTACGTTCCGACGATACGGCGGTCTACTACTGC GCGAGGTCCAGCTATTATAGGTTTGACCTAGATTACTGGGGGCAGGGCACAACCGTAACAGTTTCTAGCCAGGTGCAGCTGGTCCAATCAGGGGCAGAAGTAAAGAAACCCGGCGCATCCGTTAAAGTCTCATGCAAAGCCTCCGGATTCACATTCACTGGACGTACTATGCACTGGGTACGTCAGGCGCCCGGACAGGGATTGGAGTGGATT GGCGGATTTAATCCTAACTCTGGCGGCAGCTCATACAATCAGAAATTTAAGGGAAGGGCCACGTTGACCGTA GACAAAAGTATTAGCACGGCCTACATGGAGCTTTCAAGATTACGTTCCGACGATACGGCGGTCTACTACTGC GCGAGGTCCAGCTATTATAGGTTTGACCTAGATTACTGGGGGCAGGGCACAACCGTAACAGTTTCTAGC 9393
T02H03T02H03 VHVH CAGGTGCAGCTGGTCCAATCAGGGGCAGAAGTAAAGAAACCCGGCGCATCCGTTAAAGTCTCATGCAAAGCCTCCGGATTCACATTCACTGGACGTAGTATTCACTGGGTACGTCAGGCGCCCGGACAGGGATTGGAGTGGATT GGCGGATTTAATCCTAACTCTGGCGGCAGCTCATACAATCAGAAATTTAAGGGAAGGGCCACGTTGACCGTA GACAAAAGTATTAGCACGGCCTACATGGAGCTTTCAAGATTACGTTCCGACGATACGGCGGTCTACTACTGC GCGAGGTCCAGCTATTATAGGTTTGACCTAGATTACTGGGGGCAGGGCACAACCGTAACAGTTTCTAGCCAGGTGCAGCTGGTCCAATCAGGGGCAGAAGTAAAGAAACCCGGCGCATCCGTTAAAGTCTCATGCAAAGCCTCCGGATTCACATTCACTGGACGTAGTATTCACTGGGTACGTCAGGCGCCCGGACAGGGATTGGAGTGGATT GGCGGATTTAATCCTAACTCTGGCGGCAGCTCATACAATCAGAAATTTAAGGGAAGGGCCACGTTGACCGTA GACAAAAGTATTAGCACGGCCTACATGGAGCTTTCAAGATTACGTTCCGACGATACGGCGGTCTACTACTGC GCGAGGTCCAGCTATTATAGGTTTGACCTAGATTACTGGGGGCAGGGCACAACCGTAACAGTTTCTAGC 9494
T02H04T02H04 VHVH CAGGTGCAGCTGGTCCAATCAGGGGCAGAAGTAAAGAAACCCGGCGCATCCGTTAAAGTCTCATGCAAAGCCTCCGGATTCACATTCACTGGACGTAGTATGCACTGGGTACGTCAGGCGCCCGGACAGGGATTGGAGTGGATT GGCGGATTTAATCCTAACTCTGGCGGCAGCTCATACAATCAGAAATTTAAGGGAAGGGCCACGTTGACCGTA GACAAAAGTATTAGCACGGCCTACATGGAGCTTTCAAGATTACGTTCCGACGATACGGCGGTCTACTACTGC GCGAGGTCCAGCTATTATAGGTTTGACCTAGATTACTGGGGGCAGGGCACAACCGTAACAGTTTCTAGCCAGGTGCAGCTGGTCCAATCAGGGGCAGAAGTAAAGAAACCCGGCGCATCCGTTAAAGTCTCATGCAAAGCCTCCGGATTCACATTCACTGGACGTAGTATGCACTGGGTACGTCAGGCGCCCGGACAGGGATTGGAGTGGATT GGCGGATTTAATCCTAACTCTGGCGGCAGCTCATACAATCAGAAATTTAAGGGAAGGGCCACGTTGACCGTA GACAAAAGTATTAGCACGGCCTACATGGAGCTTTCAAGATTACGTTCCGACGATACGGCGGTCTACTACTGC GCGAGGTCCAGCTATTATAGGTTTGACCTAGATTACTGGGGGCAGGGCACAACCGTAACAGTTTCTAGC 9595
T02H05T02H05 VHVH CAGGTGCAGCTGGTCCAATCAGGGGCAGAAGTAAAGAAACCCGGCGCATCCGTTAAAGTCTCATGCAAAGCCTCCGGATTCACATTCACTGAATATACTATGCACTGGGTACGTCAGGCGCCCGGACAGGGATTGGAGTGGATT GGCGGTATTAATGCCAAATTCGGGCGGCAGCTCATACATCAGAAATTTAAGGGAAGGGCCACGTTGACCGTA GACAAAAGTATTAGCACGGCCTACATGGAGCTTTCAAGATTACGTTCCGACGATACGGCGGTCTACTACTGC GCGAGGTCCAGCTATTATAGGTTTGACCTAGATTACTGGGGGCAGGGCACAACCGTAACAGTTTCTAGCCAGGTGCAGCTGGTCCAATCAGGGGCAGAAGTAAAGAAACCCGGCGCATCCGTTAAAGTCTCATGCAAAGCCTCCGGATTCACATTCACTGAATATACTATGCACTGGGTACGTCAGGCGCCCGGACAGGGATTGGAGTGGATT GGCGGTATTAATGCCAAATTCGGGCGGCAGCTCATACATCAGAAATTTAAGGGAAGGGCCACGTTGACCGTA GACAAAAGTATTAGCACGGCCTACATGGAGCTTTCAAGATTACGTTCCGACGATACGGCGGTCTACTACTGC GCGAGGTCCAGCTATTATAGGTTTGACCTAGATTACTGGGGGCAGGGCACAACCGTAACAGTTTCTAGC 9696
T02H06T02H06 VHVH CAGGTGCAGCTGGTCCAATCAGGGGCAGAAGTAAAGAAACCCGGCGCATCCGTTAAAGTCTCATGCAAAGCCTCCGGATTCACATTCACTGAATATACTATGCACTGGGTACGTCAGGCGCCCGGACAGGGATTGGAGTGGATT GGCGGGATTAGGGCGAAGACCGGCGGCAGCTCATACAATCAGAAATTTAAGGGAAGGGCCACGTTGACCGTAGACAAAAGTATTAGCACGGCCTACATGGAGCTTTCAAGATTACGTTCCGACGATACGGCGGTCTACTACTGCGCGAGGTCCAGCTATTATAGGTTTGACCTAGATTACTGGGGGCAGGGCACAACCGTAACAGTTTCTAGCCAGGTGCAGCTGGTCCAATCAGGGGCAGAAGTAAAGAAACCCGGCGCATCCGTTAAAGTCTCATGCAAAGCCTCCGGATTCACATTCACTGAATATACTATGCACTGGGTACGTCAGGCGCCCGGACAGGGATTGGAGTGGATT GGCGGGATTAGGGCGAAGACCGGCGGCAGCTCATACAATCAGAAATTTAAGGGAAGGGCCACGTTGACCGTAGACAAAAGTATTAGCACGGCCTACATGGAGCTTTCAAGATTACGTTCCGACGATACGGCGGTCTACTACTGCGCGAGGTCCAGCTATTATAGGTTTGACCTAGATTACTGGGGGCAGGGCACAACCGTAACAGTTTCTAGC 9797
T02H07T02H07 VHVH CAGGTGCAGCTGGTCCAATCAGGGGCAGAAGTAAAGAAACCCGGCGCATCCGTTAAAGTCTCATGCAAAGCCTCCGGATTCACATTCACTGAATATACTATGCACTGGGTACGTCAGGCGCCCGGACAGGGATTGGAGTGGATT GGCGGATTTAATCCTAACTCTGGTCGGTCGGCCTACAACCAGAAATTTAAGGGAAGGGCCACGTTGACCGTA GACAAAAGTATTAGCACGGCCTACATGGAGCTTTCAAGATTACGTTCCGACGATACGGCGGTCTACTACTGC GCGAGGTCCAGCTATTATAGGTTTGACCTAGATTACTGGGGGCAGGGCACAACCGTAACAGTTTCTAGCCAGGTGCAGCTGGTCCAATCAGGGGCAGAAGTAAAGAAACCCGGCGCATCCGTTAAAGTCTCATGCAAAGCCTCCGGATTCACATTCACTGAATATACTATGCACTGGGTACGTCAGGCGCCCGGACAGGGATTGGAGTGGATT GGCGGATTTAATCCTAACTCTGGTCGGTCGGCCTACAACCAGAAATTTAAGGGAAGGGCCACGTTGACCGTA GACAAAAGTATTAGCACGGCCTACATGGAGCTTTCAAGATTACGTTCCGACGATACGGCGGTCTACTACTGC GCGAGGTCCAGCTATTATAGGTTTGACCTAGATTACTGGGGGCAGGGCACAACCGTAACAGTTTCTAGC 9898
T02L01T02L01 VLVL GATATCCAAATGACCCAGAGCCCAAGTAGTTTATCAGCCAGTGTGGGAGACCGTGTAACGATAACTTGTAAG GCTAGTCAAGATGTCAAGACGGCAGTTGCCTGGTACCAACAGAAACCTGGCAAAGCACCCAAACTGTTAATT TATTCCGCATCTTGGAGGTATTCTGGCGTACCTAGCAGATTCAGTGGCAGTGGTTCTGGTGCAGACTTTACC CTAACTATATCTAGTCTTCAACCAGAGGATTTTGCGACATACTATTGCCAACAACATTATACCACCCCTTGG ACCTTCGGGCAAGGGACAAAGGTCGAAATCAAGGATATCCAAATGACCCAGAGCCCAAGTAGTTTATCAGCCAGTGTGGGAGACCGTGTAACGATAACTTGTAAG GCTAGTCAAGATGTCAAGACGGCAGTTGCCTGGTACCAACAGAAACCTGGCAAAGCACCCAAACTGTTAATT TATTCCGCATCTTGGAGGTATTCTGGCGTACCTAGCAGATTCAGTGGCAGTGGTTCTGGTGCAGACTTTACC CTAACTATATCTAGTCTTCAACCAGAGGATTTTGCGACATACTATTGCCAACAACATTATACCACCCCTTGG ACCTTCGGGCAAGGGACAAAGGTCGAAATCAAG 9999
T02L02T02L02 VLVL GATATCCAAATGACCCAGAGCCCAAGTAGTTTATCAGCCAGTGTGGGAGACCGTGTAACGATAACTTGTAAG GCTAGTCAAGATGTCAAGACGGCAGTTGCCTGGTACCAACAGAAACCTGGCAAAGCACCCAAACTGTTAATT TATTCCGCATCTTTCAGGTATACTGGCGTACCTAGCAGATTCAGTGGCAGTGGTTCTGGTGCAGACTTTACC CTAACTATATCTAGTCTTCAACCAGAGGATTTTGCGACATACTATTGCCAACAACATTATCTTACCCCTTGG ACCTTCGGGCAAGGGACAAAGGTCGAAATCAAGGATATCCAAATGACCCAGAGCCCAAGTAGTTTATCAGCCAGTGTGGGAGACCGTGTAACGATAACTTGTAAG GCTAGTCAAGATGTCAAGACGGCAGTTGCCTGGTACCAACAGAAACCTGGCAAAGCACCCAAACTGTTAATT TATTCCGCATCTTTCAGGTATACTGGCGTACCTAGCAGATTCAGTGGCAGTGGTTCTGGTGCAGACTTTACC CTAACTATATCTAGTCTTCAACCAGAGGATTTTGCGACATACTATTGCCAACAACATTATCTTACCCCTTGG ACCTTCGGGCAAGGGACAAAGGTCGAAATCAAG 100100
(5) IgG 항체 클로닝(5) IgG antibody cloning
개별 클론 분석을 통해 선정된 서열 중 heavy chain은 동물세포 발현벡터인 pOptivec (Invitrogen)AAA (L234A, L235A, K322A) mutation이 도입되어 Fc의 effector function이 없는 플라스미드로, light chain은 pcDNA3.3(Invitrogen) 플라스미드 제작하고 IDT에서 합성하였다. HC은 EcoR1, Nhe1 restriction enzyme으로 자른 pOptivec(AAA) vector, LC는 EcoR1, BsiW1 restriction enzyme으로 자른 pcDNA3.3 vector의 양 말단과 homologous한 sequence를 합성 유전자 양 말단에 넣어 합성하였다. 상기 pOptivec(AAA) vector 및 pcDNA3.3 vector의 개열지도는 도 28에 나타내었다. 각 개별 클론은 hu6H8.3 backbone에 도출된 mutation site를 각각 도입하여 모두 클로닝하였다. 합성이 완료된 insert gene 과 linearized vector를 In Fusion®HD Cloning Kit(Clontech)을 사용하여 각각의 insert gene을 넣어 클로닝하고 sequencing primer는 CmV Forward, EMCV IRES reverse primer를 사용하여 확인하였다.Among the sequences selected through individual clonal analysis, the heavy chain is a plasmid without the effector function of Fc by introducing the pOptivec (Invitrogen)AAA (L234A, L235A, K322A) mutation, an animal cell expression vector, and the light chain is pcDNA3.3 (Invitrogen). ) plasmid was fabricated and synthesized by IDT. HC was synthesized by adding sequences homologous to both ends of the pOptivec (AAA) vector cut with EcoR1 and Nhe1 restriction enzymes, and pcDNA3.3 vector cut with EcoR1 and BsiW1 restriction enzymes for LC at both ends of the synthetic gene. The cleavage maps of the pOptivec (AAA) vector and pcDNA3.3 vector are shown in FIG. 28 . Each individual clone was cloned by introducing a mutation site derived from the hu6H8.3 backbone, respectively. The synthesized insert gene and linearized vector were cloned by inserting each insert gene using the In Fusion®HD Cloning Kit (Clontech), and sequencing primers were confirmed using CmV Forward and EMCV IRES reverse primers.
제작된 IgG clone의 정보는 표 9에 나타내었다.Information on the prepared IgG clone is shown in Table 9.
Figure PCTKR2021018795-appb-img-000004
Figure PCTKR2021018795-appb-img-000004
(6) 결합력 향상 항체 후보군의 생산(6) Production of aggregation-improving antibody candidates
디자인된 인간화 항체 유전자가 도입된 플라스미드를 Expi293 expression system(Invitrogen)을 이용하여 IgG 형태로 발현하였으며, 이를 AktaPure(GE healthcare)와 AktaPrime purifier(GE healthcare) 및 MabselectSURE 컬럼(GE healthcare, Cat#11-0034-95)를 이용하여 정제하였다. 정제된 항체는 Desalting 컬럼(GE healthcare, Cat#17-1408-01)를 통하여 PBS로 buffer change하였으며 Multiskan GO(Thermo)를 통하여 농도를 측정하였다. 또한 농도 측정 결과에 기초하여 수율을 계산하였다. 농도 측정 결과 및 수율을 계산한 결과는 표 10에 나타내었다.The plasmid into which the designed humanized antibody gene was introduced was expressed in the form of IgG using the Expi293 expression system (Invitrogen), which was obtained by AktaPure (GE healthcare), AktaPrime purifier (GE healthcare) and MabselectSURE column (GE healthcare, Cat#11-0034). -95) was used for purification. The purified antibody was buffer-changed with PBS through a desalting column (GE healthcare, Cat#17-1408-01), and the concentration was measured through Multiskan GO (Thermo). In addition, the yield was calculated based on the concentration measurement results. The results of measuring the concentration and calculating the yield are shown in Table 10.
Figure PCTKR2021018795-appb-img-000005
Figure PCTKR2021018795-appb-img-000005
표 10에 나타낸 바와 같이, 제작된 IgG clone 10종은 hu62F에 비해 수율이 다소 낮은 것을 확인하였다. 제작된 IgG clone 중 T02.08의 수율이 0.37로, 가장 높았다.As shown in Table 10, it was confirmed that the yield of 10 produced IgG clones was somewhat lower than that of hu62F. Among the prepared IgG clones, the yield of T02.08 was 0.37, which was the highest.
(7) 결합력 향상 항체 후보군의 특성분석(7) Characterization of candidate group for improved binding affinity
(i) SDS-PAGE를 통한 항체 분석(i) Antibody analysis by SDS-PAGE
생산된 항체(1μg)를 LDS sample buffer(Invitrogen, Cat#B0007)를 넣어준 후 reducing condition group에는 sample reducing agent(Invitrogen, Cat#B0004)를 넣어주고 70°C에서 10분 동안 열처리하여 sample을 준비하였다. SDS running buffer(Bio-rad, Cat#1610732)를 채운 Mini gel tank에 Mini PROTEIN TGX Stain Free Gel(Bio-rad, Cat#456-8096)을 설치한 후 power supply(Bio-rad)를 이용하여, 160V, 30분 동안 sample을 running하였다 Sample running이 완료된 gel은 분리 후 Chemidoc(Bio-rad)를 통해 분석하였다. SDS-PAGE 결과는 도 29에 나타내었다.After adding the produced antibody (1μg) to the LDS sample buffer (Invitrogen, Cat#B0007), add the sample reducing agent (Invitrogen, Cat#B0004) to the reducing condition group and heat-treat at 70°C for 10 minutes to prepare the sample did After installing Mini PROTEIN TGX Stain Free Gel (Bio-rad, Cat#456-8096) in the mini gel tank filled with SDS running buffer (Bio-rad, Cat#1610732), using the power supply (Bio-rad), The sample was run at 160V for 30 minutes. After the sample running was completed, the gel was separated and analyzed by Chemidoc (Bio-rad). The SDS-PAGE results are shown in FIG. 29 .
도 29에 나타낸 바와 같이, 정제된 결합력 향상 후보항체들은 SDS-PAGE 방법에 의한 전기영동 후 염색하여 non-reduceing에서는 전체 항체 크기를, reducing 조건에서는 항체의 각 heavy chain과 light chain의 크기를 비교하였고 정제된 항체의 순도를 확인하였다. As shown in Figure 29, the purified binding strength improvement candidate antibodies were stained after electrophoresis by the SDS-PAGE method to compare the total antibody size in non-reducing, and the size of each heavy chain and light chain of the antibody in reducing conditions. The purity of the purified antibody was confirmed.
(ii) Size exclusion chromatography 분석(ii) Size exclusion chromatography analysis
생산된 IgG 항체를 HPLC(Agilent Technologies, 1260 infinity II LC system) 로 size exclusion column(Tosoh, TSKgel G3000 SWXL, 7.8×300mm, Part No.0008541, Column No.023D08819D)을 사용하여 분석하였다. 이동상은 10X Phosphate Buffered Saline buffer(wellgene, Cat#LB204 02)를 3차 증류수에 1X PBS로 희석한 후 Corning 1000 mL Vacuum Filter/Storage Bottle System, 0.22μm Pore 54.5cm² PES Membrane, Sterile, 12/Case를 사용하여 filtration하였다. 분석방법은 flow rate 1ml/min, 280nm 파장대로 20min 동안 분석하였고 시료는 20ul injection하였다. Standard 시료는 Gel filtration standard(BIO RAD, Cat.#151-1901)를 사용하였다. 생산된 IgG 항체의 HPLC 결과는 도 30a 내지 c에 나타내었다.The produced IgG antibody was analyzed using a size exclusion column (Tosoh, TSKgel G3000 SWXL, 7.8×300mm, Part No.0008541, Column No.023D08819D) by HPLC (Agilent Technologies, 1260 infinity II LC system). For the mobile phase, 10X Phosphate Buffered Saline buffer (wellgene, Cat#LB204 02) was diluted with 1X PBS in tertiary distilled water, and Corning 1000 mL Vacuum Filter/Storage Bottle System, 0.22μm Pore 54.5cm² PES Membrane, Sterile, 12/Case was used for filtration. The analysis method was flow rate 1ml/min, 280nm wavelength band was analyzed for 20min, and 20ul of the sample was injected. As a standard sample, a gel filtration standard (BIO RAD, Cat. #151-1901) was used. HPLC results of the produced IgG antibody are shown in FIGS. 30a to c.
도 30a 내지 c에 나타낸 바와 같이, 정제된 결합력 향상 후보항체들은 SEC-HPLC 방법으로 분석하여 정제된 항체의 정성, 정량을 분석하였다. As shown in FIGS. 30A to 30C , the purified candidate antibodies to improve binding were analyzed by SEC-HPLC to analyze the qualitative and quantitative analysis of the purified antibody.
(iii) 결합력 향상 항체 후보군의 항원결합력 분석(iii) Analysis of the antigen-binding affinity of the candidate group for the improved binding affinity
Human capture kit(GE healthcare, Cat#BR-1008-39)를 이용하여 anti-human Fc antibody를 고정한 CM5 chip(GE Healthcare, Cat#BR-1005-30)를 준비하였다. 생산된 항체를 1ug/mL로 희석한 후, 상기 CM5 chip에 10uL/min 유속으로, 60초 동안 흘려주어 항체를 고정하였으며 TIGIT-His(Sino)를 100, 50, 25, 12.5, 6.25, 3.125nM로 association time을 150초, dissociation time을 240초 injection하였다. Biacore T200(GE healthcare) 장비를 통하여 측정된 sensorgram을 1:1 binding model로 fitting하여 결합력을 분석하였다. 생산된 항체의 항원 결합력을 분석한 결과는 표 11에 나타내었다. 또한 Biacore를 이용한 Hu62F 및 T02.10의 항원-항체 결합력을 분석한 결과는 도 31에 나타내었다.A CM5 chip (GE Healthcare, Cat#BR-1005-30) immobilized with an anti-human Fc antibody was prepared using a human capture kit (GE healthcare, Cat#BR-1008-39). After diluting the produced antibody to 1ug/mL, it was flowed to the CM5 chip at a flow rate of 10uL/min for 60 seconds to fix the antibody, and TIGIT-His(Sino) was added to 100, 50, 25, 12.5, 6.25, 3.125nM The association time was 150 seconds and the dissociation time was 240 seconds. The binding force was analyzed by fitting the sensorgram measured through the Biacore T200 (GE healthcare) equipment to a 1:1 binding model. The results of analyzing the antigen-binding ability of the produced antibodies are shown in Table 11. In addition, the results of analyzing the antigen-antibody binding capacity of Hu62F and T02.10 using Biacore are shown in FIG. 31 .
Figure PCTKR2021018795-appb-img-000006
Figure PCTKR2021018795-appb-img-000006
표 11에 나타낸 바와 같이, 10종의 후보항체 중 T02.08 및 T02.10는 Hu62F보다 항원 결합력이 높은 것을 확인하였다. 특히, T02.10 항체는 Hu62F에 비해 항원 결합력이 약 27배 높은 것을 확인하였다.As shown in Table 11, it was confirmed that among the 10 candidate antibodies, T02.08 and T02.10 had higher antigen-binding ability than Hu62F. In particular, it was confirmed that the T02.10 antibody had about 27 times higher antigen-binding ability than that of Hu62F.
도 31에 나타낸 바와 같이, T02.10은 hu62F와 비교하였을 때 항체-항원 결합력(ka)은 증가하였고, 반면에 항체-항원 해리도(kd)는 감소하여 전체적으로 hu62F보다 T02.10의 항체-항원 결합력이 증가하였다. As shown in FIG. 31 , the antibody-antigen avidity (ka) of T02.10 was increased when compared to hu62F, whereas the degree of antibody-antigen dissociation (kd) was decreased. The bonding strength was increased.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술한 바, 당 업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As the specific parts of the present invention have been described in detail above, for those of ordinary skill in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (42)

  1. 다음을 포함하는 TIGIT (T cell immunoglobulin and ITIM domain)에 특이적으로 결합하는 항체 또는 이의 항원 결합단편:An antibody or antigen-binding fragment thereof that specifically binds to TIGIT (T cell immunoglobulin and ITIM domain), comprising:
    서열번호 1, 9, 17, 25 및 33로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 중쇄 CDR1, heavy chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 9, 17, 25 and 33;
    서열번호 2, 10, 18, 26, 34 및 45로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 중쇄 CDR2, a heavy chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 10, 18, 26, 34 and 45;
    서열번호 3, 11, 19, 27 및 35로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 중쇄 CDR3, 및a heavy chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11, 19, 27 and 35, and
    서열번호 4, 12, 20, 28, 36, 41, 46 및 79로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 경쇄 CDR1, a light chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 12, 20, 28, 36, 41, 46 and 79;
    서열번호 5, 13, 21, 29, 37, 42 및 47로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 경쇄 CDR2 및a light chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37, 42 and 47;
    서열번호 6, 14, 22, 30, 38 및 43으로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 경쇄 CDR3.A light chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 14, 22, 30, 38 and 43.
  2. 제1항에 있어서, 상기 항체 또는 이의 항원 결합단편은 (i) 서열번호 1의 중쇄 CDR1, 서열번호 2의 중쇄 CDR2 및 서열번호 3의 중쇄 CDR3을 포함하는 중쇄 가변영역, 및 서열번호 4의 경쇄 CDR1, 서열번호 5의 경쇄 CDR2 및 서열번호 6의 경쇄 CDR3를 포함하는 경쇄 가변영역;According to claim 1, wherein the antibody or antigen-binding fragment thereof is (i) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 2 and the heavy chain CDR3 of SEQ ID NO: 3, and the light chain of SEQ ID NO: 4 a light chain variable region comprising CDR1, light chain CDR2 of SEQ ID NO: 5 and light chain CDR3 of SEQ ID NO: 6;
    (ii) 서열번호 9의 중쇄 CDR1, 서열번호 10의 중쇄 CDR2 및 서열번호 11의 중쇄 CDR3을 포함하는 중쇄 가변영역, 및 서열번호 12의 경쇄 CDR1, 서열번호 13의 경쇄 CDR2 및 서열번호 14의 경쇄 CDR3를 포함하는 경쇄 가변영역; (ii) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 9, the heavy chain CDR2 of SEQ ID NO: 10 and the heavy chain CDR3 of SEQ ID NO: 11, and the light chain CDR1 of SEQ ID NO: 12, the light chain CDR2 of SEQ ID NO: 13 and the light chain of SEQ ID NO: 14 a light chain variable region comprising CDR3;
    (iii) 서열번호 17의 중쇄 CDR1, 서열번호 18의 중쇄 CDR2 및 서열번호 19의 중쇄 CDR3을 포함하는 중쇄 가변영역, 및 서열번호 20의 경쇄 CDR1, 서열번호 21의 경쇄 CDR2 및 서열번호 22의 경쇄 CDR3를 포함하는 경쇄 가변영역;(iii) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 17, the heavy chain CDR2 of SEQ ID NO: 18 and the heavy chain CDR3 of SEQ ID NO: 19, and the light chain CDR1 of SEQ ID NO: 20, the light chain CDR2 of SEQ ID NO: 21 and the light chain of SEQ ID NO: 22 a light chain variable region comprising CDR3;
    (iv) 서열번호 25의 중쇄 CDR1, 서열번호 26의 중쇄 CDR2 및 서열번호 27의 중쇄 CDR3을 포함하는 중쇄 가변영역, 및 서열번호 28의 경쇄 CDR1, 서열번호 29의 경쇄 CDR2 및 서열번호 30의 경쇄 CDR3를 포함하는 경쇄 가변영역;(iv) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 25, the heavy chain CDR2 of SEQ ID NO: 26 and the heavy chain CDR3 of SEQ ID NO: 27, and the light chain CDR1 of SEQ ID NO: 28, the light chain CDR2 of SEQ ID NO: 29 and the light chain of SEQ ID NO: 30 a light chain variable region comprising CDR3;
    (v) 서열번호 33의 중쇄 CDR1, 서열번호 34의 중쇄 CDR2 및 서열번호 35의 중쇄 CDR3을 포함하는 중쇄 가변영역, 및 서열번호 36의 경쇄 CDR1, 서열번호 37의 경쇄 CDR2 및 서열번호 38의 경쇄 CDR3를 포함하는 경쇄 가변영역;(v) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 33, the heavy chain CDR2 of SEQ ID NO: 34 and the heavy chain CDR3 of SEQ ID NO: 35, and the light chain CDR1 of SEQ ID NO: 36, the light chain CDR2 of SEQ ID NO: 37 and the light chain of SEQ ID NO: 38 a light chain variable region comprising CDR3;
    (vi) 서열번호 33의 중쇄 CDR1, 서열번호 34의 중쇄 CDR2 및 서열번호 35의 중쇄 CDR3을 포함하는 중쇄 가변영역, 및 서열번호 41의 경쇄 CDR1, 서열번호 42의 경쇄 CDR2 및 서열번호 43의 경쇄 CDR3를 포함하는 경쇄 가변영역;(vi) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 33, the heavy chain CDR2 of SEQ ID NO: 34 and the heavy chain CDR3 of SEQ ID NO: 35, and the light chain CDR1 of SEQ ID NO: 41, the light chain CDR2 of SEQ ID NO: 42 and the light chain of SEQ ID NO: 43 a light chain variable region comprising CDR3;
    (vii) 서열번호 33의 중쇄 CDR1, 서열번호 45의 중쇄 CDR2 및 서열번호 35의 중쇄 CDR3을 포함하는 중쇄 가변영역, 및 서열번호 46의 경쇄 CDR1, 서열번호 47의 경쇄 CDR2 및 서열번호 38의 경쇄 CDR3를 포함하는 경쇄 가변영역; 또는(vii) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 33, the heavy chain CDR2 of SEQ ID NO: 45 and the heavy chain CDR3 of SEQ ID NO: 35, and the light chain CDR1 of SEQ ID NO: 46, the light chain CDR2 of SEQ ID NO: 47 and the light chain of SEQ ID NO: 38 a light chain variable region comprising CDR3; or
    (viii) 서열번호 17의 중쇄 CDR1, 서열번호 18의 중쇄 CDR2 및 서열번호 19의 중쇄 CDR3을 포함하는 중쇄 가변영역, 및 서열번호 79의 경쇄 CDR1, 서열번호 21의 경쇄 CDR2 및 서열번호 22의 경쇄 CDR3를 포함하는 경쇄 가변영역;을 포함하는, 항체 또는 이의 항원 결합단편.(viii) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 17, the heavy chain CDR2 of SEQ ID NO: 18 and the heavy chain CDR3 of SEQ ID NO: 19, and the light chain CDR1 of SEQ ID NO: 79, the light chain CDR2 of SEQ ID NO: 21 and the light chain of SEQ ID NO: 22 A light chain variable region comprising CDR3; comprising, an antibody or antigen-binding fragment thereof.
  3. 제1항에 있어서, 상기 항체 또는 이의 항원 결합단편은 서열번호 7, 15, 23, 31, 39, 48, 50, 52, 54, 56 및 82로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 중쇄 가변영역을 포함하는, 항체 또는 이의 항원 결합단편.According to claim 1, wherein the antibody or antigen-binding fragment thereof is a heavy chain comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 15, 23, 31, 39, 48, 50, 52, 54, 56 and 82 An antibody or antigen-binding fragment thereof comprising a variable region.
  4. 제1항에 있어서, 항체 또는 이의 항원 결합단편은 서열번호 8, 16, 24, 32, 40, 44, 49, 51, 53, 55, 57, 80, 81, 101, 102 및 103으로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 경쇄 가변영역을 포함하는, 항체 또는 이의 항원 결합단편.The method according to claim 1, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NOs: 8, 16, 24, 32, 40, 44, 49, 51, 53, 55, 57, 80, 81, 101, 102 and 103. An antibody or antigen-binding fragment thereof comprising a light chain variable region comprising one or more selected amino acid sequences.
  5. 제1항에 있어서, 상기 항원 또는 이의 항원 결합단편은,According to claim 1, wherein the antigen or antigen-binding fragment thereof,
    서열번호 7의 중쇄 가변영역 및 서열번호 8의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 7 and a light chain variable region of SEQ ID NO: 8;
    서열번호 15의 중쇄 가변영역 및 서열번호 16의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 15 and a light chain variable region of SEQ ID NO: 16;
    서열번호 23의 중쇄 가변영역 및 서열번호 24의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 23 and a light chain variable region of SEQ ID NO: 24;
    서열번호 31의 중쇄 가변영역 및 서열번호 32의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 31 and a light chain variable region of SEQ ID NO: 32;
    서열번호 39의 중쇄 가변영역 및 서열번호 40의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 39 and a light chain variable region of SEQ ID NO: 40;
    서열번호 39의 중쇄 가변영역 및 서열번호 44의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 39 and a light chain variable region of SEQ ID NO: 44;
    서열번호 48의 중쇄 가변영역 및 서열번호 49의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 48 and a light chain variable region of SEQ ID NO: 49;
    서열번호 50의 중쇄 가변영역 및 서열번호 51의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 50 and a light chain variable region of SEQ ID NO: 51;
    서열번호 52의 중쇄 가변영역 및 서열번호 53의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO: 53;
    서열번호 54의 중쇄 가변영역 및 서열번호 55의 경쇄 가변영역; a heavy chain variable region of SEQ ID NO: 54 and a light chain variable region of SEQ ID NO: 55;
    서열번호 56의 중쇄 가변영역 및 서열번호 57의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 56 and a light chain variable region of SEQ ID NO: 57;
    서열번호 23의 중쇄 가변영역 및 서열번호 80의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 23 and a light chain variable region of SEQ ID NO: 80;
    서열번호 23의 중쇄 가변영역 및 서열번호 81의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 23 and a light chain variable region of SEQ ID NO: 81;
    서열번호 82의 중쇄 가변영역 및 서열번호 80의 경쇄 가변영역; a heavy chain variable region of SEQ ID NO: 82 and a light chain variable region of SEQ ID NO: 80;
    서열번호 82의 중쇄 가변영역 및 서열번호 81의 경쇄 가변영역; a heavy chain variable region of SEQ ID NO: 82 and a light chain variable region of SEQ ID NO: 81;
    서열번호 82의 중쇄 가변영역 및 서열번호 101의 경쇄 가변영역; a heavy chain variable region of SEQ ID NO: 82 and a light chain variable region of SEQ ID NO: 101;
    서열번호 82의 중쇄 가변영역 및 서열번호 102의 경쇄 가변영역; 또는a heavy chain variable region of SEQ ID NO: 82 and a light chain variable region of SEQ ID NO: 102; or
    서열번호 82의 중쇄 가변영역 및 서열번호 103의 경쇄 가변영역;을 포함하는, 항체 또는 이의 항원 결합단편.A heavy chain variable region of SEQ ID NO: 82 and a light chain variable region of SEQ ID NO: 103; An antibody or antigen-binding fragment thereof, comprising:
  6. 제1항에 있어서, 상기 항체 또는 이의 항원 결합단편은 단쇄 Fvs (scFv), 단쇄 항체, Fab, F(ab'), 다이설파이드-결합 Fvs (sdFv)을 포함하는, 항체 또는 이의 항원 결합단편.The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a single chain Fvs (scFv), a single chain antibody, Fab, F(ab'), a disulfide-bonded Fvs (sdFv).
  7. 제6항에 있어서, 상기 scFv는 중쇄 가변영역과 경쇄 가변영역이 링커를 통해 연결된 것을 특징으로 하는, 항체 또는 이의 항원 결합단편.The antibody or antigen-binding fragment thereof according to claim 6, wherein the scFv has a heavy chain variable region and a light chain variable region connected through a linker.
  8. 서열번호 83 내지 89로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 중쇄 가변영역; 및a heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 83 to 89; and
    서열번호 90 및 91로 구성된 군에서 선택된 하나 이상의 아미노산 서열을 포함하는 경쇄 가변영역;을 포함하는, TIGIT (T cell immunoglobulin and ITIM domain)에 특이적으로 결합하는 항체 또는 이의 항원 결합단편.An antibody or antigen-binding fragment thereof that specifically binds to TIGIT (T cell immunoglobulin and ITIM domain), including a light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 90 and 91.
  9. 제8항에 있어서, 상기 항체 또는 항원 결합단편은,The method of claim 8, wherein the antibody or antigen-binding fragment,
    서열번호 83의 중쇄 가변영역 및 서열번호 53의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 83 and a light chain variable region of SEQ ID NO: 53;
    서열번호 84의 중쇄 가변영역 및 서열번호 53의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 84 and a light chain variable region of SEQ ID NO: 53;
    서열번호 85의 중쇄 가변영역 및 서열번호 53의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 85 and a light chain variable region of SEQ ID NO: 53;
    서열번호 86의 중쇄 가변영역 및 서열번호 53의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 86 and a light chain variable region of SEQ ID NO: 53;
    서열번호 52의 중쇄 가변영역 및 서열번호 90의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO: 90;
    서열번호 52의 중쇄 가변영역 및 서열번호 91의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO: 91;
    서열번호 87의 중쇄 가변영역 및 서열번호 53의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 87 and a light chain variable region of SEQ ID NO: 53;
    서열번호 88의 중쇄 가변영역 및 서열번호 53의 경쇄 가변영역;a heavy chain variable region of SEQ ID NO: 88 and a light chain variable region of SEQ ID NO: 53;
    서열번호 89의 중쇄 가변영역 및 서열번호 53의 경쇄 가변영역; 또는a heavy chain variable region of SEQ ID NO: 89 and a light chain variable region of SEQ ID NO: 53; or
    서열번호 84의 중쇄 가변영역 및 서열번호 91의 경쇄 가변영역;을 포함하는 항체 또는 항원 결합단편.An antibody or antigen-binding fragment comprising; the heavy chain variable region of SEQ ID NO: 84 and the light chain variable region of SEQ ID NO: 91.
  10. 제1항 내지 제9항 중 어느 한 항에 따른 항체 또는 이의 항원 결합단편을 코딩하는 핵산.A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.
  11. 제10항의 핵산을 포함하는 재조합 발현벡터.A recombinant expression vector comprising the nucleic acid of claim 10.
  12. 제11항의 재조합 발현벡터로 형질감염된 숙주세포.A host cell transfected with the recombinant expression vector of claim 11 .
  13. 제12항에 있어서, 상기 숙주세포는 COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO/-DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2/0, NS-0, U20S 또는 HT1080인 숙주세포.13. The method of claim 12, wherein the host cell is COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO/-DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK , BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2/0, NS-0, U20S or HT1080 host cells .
  14. 제12항의 숙주세포를 배양하여 항체를 생성하는 단계; 및 생성된 항체를 분리 및 정제하는 단계를 포함하는, TIGIT에 특이적으로 결합하는 항체 또는 이의 항원 결합단편의 제조방법.Culturing the host cell of claim 12 to generate an antibody; And a method for producing an antibody or antigen-binding fragment thereof that specifically binds to TIGIT, comprising the step of isolating and purifying the generated antibody.
  15. 제1항 내지 제9항 중 어느 한 항에 따른 항체 또는 이의 항원 결합단편을 포함하는 이중 또는 다중특이적 항체.A bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.
  16. 제15항에 있어서, 상기 항체는 FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137(4-1BB), VISTA, CD258(LIGHT), MARCO, CD134(OX40), CD28, CD278(ICOS), CD27, CD154(CD40L), CD357(GITR), CD30, DR3, CD226(DNAM1), CD96, CD200, CD200R, 트랜스페린 수용체 (Transferrin receptor), c-Met, EGFR, HER2, KDR, PDGFRa 및 NRP1으로 구성된 군에서 선택된 하나 이상에 결합하는 항체 또는 이의 항원 결합단편을 파트너로 포함하는 이중 또는 다중특이적 항체.16. The method of claim 15, wherein the antibody is FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137(4-1BB), VISTA, CD258(LIGHT), MARCO, CD134(OX40) ), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2 , KDR, PDGFRa, and a dual or multispecific antibody comprising as a partner an antibody or antigen-binding fragment thereof that binds to one or more selected from the group consisting of.
  17. 제1항 내지 제9항 중 어느 한 항에 따른 항체의 scFv 및 면역세포 활성화 항원에 결합하는 항체의 scFv를 하나 이상 포함하는, 면역세포 인게이징(immune cell engage) 이중특이적 또는 다중특이적 항체.An immune cell engage bispecific or multispecific antibody comprising an scFv of an antibody according to any one of claims 1 to 9 and at least one scFv of an antibody that binds to an immune cell activating antigen .
  18. 제17항에 있어서, 상기 면역세포 활성화 항원은 다음에 해당하는, 이중특이적 또는 다중특이적 항체:The bispecific or multispecific antibody according to claim 17, wherein the immune cell activating antigen corresponds to:
    T 세포 활성화 항원으로 CD3, TCRα, TCRβ, TCRγ,TCRξ 또는 CD226;CD3, TCRα, TCRβ, TCRγ, TCRξ or CD226 as a T cell activating antigen;
    NK세포 활성화 항원으로 NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (CD16a 또는 CD16b), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (SLAMF4 또는 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E 또는 CD160;NK cell activating antigens NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (CD16a or CD16b), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E or CD160;
    B세포 활성화 항원으로 OX40, CD40 또는 CD70;OX40, CD40 or CD70 as a B cell activating antigen;
    대식세포 활성화 항원으로 CD2 아고니스트, CD40, CD70, TCR (Toll-like Receptor) 아고니스트, CD47, STING 또는 OX40L; 또는CD2 agonist, CD40, CD70, Toll-like receptor (TCR) agonist, CD47, STING or OX40L as macrophage activating antigen; or
    수지상세포 활성화 항원은 CD2 아고니스트, OX40, OX40L, 41BB 아고니스트, TCR 아고니스트, CD47 아고니스트 또는 STING 아고니스트.The dendritic cell activating antigen is a CD2 agonist, OX40, OX40L, 41BB agonist, TCR agonist, CD47 agonist or STING agonist.
  19. 제1항 내지 제9항 중 어느 한 항에 따른 항체 또는 이의 항원 결합단편이 약물에 결합된 항체-약물 접합체 (ADC).An antibody-drug conjugate (ADC) wherein the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 is bound to a drug.
  20. 제19항에 있어서, 상기 약물은 마이탄시노이드, 오리스타틴 (MMAE, MMAF 포함), 아미노프테린, 악티노마이신, 블레오마이신, 탈리소마이신, 캄프토쎄신, N8-아세틸 스퍼미딘, 1-(2 클로로에틸)-1,2-다이메틸 술포닐 하이드라자이드, 에스퍼라마이신, 에토포사이드, 6-머캅토퓨린, 돌라스타틴, 트리코테센, 칼리케아미신, 탁솔(taxol), 탁산, 파클리탁셀(paclitaxel), 도세탁셀(docetaxel), 메토트렉세이트, 빈크리스틴, 빈블라스틴, 독소루비신, 멜팔란, 미토마이신 A, 미토마이신 C, 클로람부실, 듀오카마이신, L-아스파라기나제(L-asparaginase), 머캡토퓨린(mercaptopurine), 티오구아닌(thioguanine), 하이드록시우레아(hydroxyurea), 시타라빈(cytarabine), 사이클로포스파미드(cyclophosphamide), 이포스파미드(ifosfamide), 니트로소우레아(nitrosourea), 시스플라틴(cisplatin), 카보플라틴(carboplatin), 미토마이신(mitomycin), 다카바진(dacarbazine), 프로카바진(procarbazine), 토포테칸(topotecan), 질소 머스터드(nitrogen mustard), 사이톡산(cytoxan), 에토포시드(etoposide),5-플루오로우라실(5-fluorouracil), CNU(bischloroethylnitrosourea), 이리노테칸(irinotecan), 캄포토테신(camptothecin), 블레오마이신(bleomycin), 이다루비신(idarubicin), 다우노루비신(daunorubicin), 닥티노마이신(dactinomycin), 플리카마이신(plicamycin), 미톡산트론(mitoxantrone), 아스파라기나제(asparaginase), 비노렐빈(vinorelbine), 클로로람부실(chlorambucil), 멜파란(melphalan), 카르무스틴(carmustine), 로무스틴(lomustine), 부설판(busuLfan), 트레오설판(treosulfan), 데카바진(decarbazine), 에토포시드(etoposide), 테니포시드(teniposide), 토포테칸(topotecan), 9-아미노캠프토테신(9-aminocamptothecin), 크리스나톨(crisnatol), 미토마이신 C(mitomycin C), 트리메트렉세이트(trimetrexate), 마이코페놀산(mycophenolic acid), 티아조퓨린(tiazofurin), 리바비린(ribavirin), EICAR(5-ethynyl-1-beta-Dribofuranosylimidazole-4-carboxamide), 하이드록시우레아(hydroxyurea), 데프록사민(deferoxamine), 플룩수리딘(floxuridine), 독시플루리딘(doxifluridine), 랄티트렉세드(raltitrexed), 시타라빈(cytarabine(ara C)), 시토신 아라비노시드(cytosine arabinoside), 플루다라빈(fludarabine), 타목시펜(tamoxifen), 라록시펜(raloxifene), 메게스트롤(megestrol), 고세렐린(goserelin), 류프롤리드 아세 테이트(leuprolide acetate), 플루타미드(flutamide), 바이칼루타마이드(bicalutamide), EB1089, CB1093, KH1060, 베르테포르핀(verteporfin), 프탈로시아닌(phthalocyanine), 광감작제 Pe4(photosensitizer Pe4), 데메톡시-하이포크레린 A(demethoxy-hypocrellin A), 인터페론-α(Interferon-α), 인터페론-γ(Interferon-γ), 종양 괴사 인자(tumor necrosis factor), 겜사이타빈(Gemcitabine), 벨케이드(velcade), 레발미드(revamid), 탈라미드(thalamid), 로바스타틴(lovastatin), 1-메틸-4-페닐피리디늄 이온(1-methyl-4-phenylpyridiniumion), 스타우로스포린(staurosporine), 악티노마이신 D(actinomycin D), 닥티노마이신(dactinomycin), 블레오마이신 A2(bleomycin A2), 블레오마이신 B2(bleomycinB2), 페플로마이신(peplomycin), 에피루비신(epirubicin), 피라루비신(pirarubicin), 조루비신(zorubicin), 마이토산트론(mitoxantrone), 베라파밀(verapamil), 탑시가르긴(thapsigargin), 핵산 분해 효소 및 세균이나 동식물 유래의 독소로 구성된 군에서 선택된 하나 이상인 것을 특징으로 하는 항체-약물 접합체.20. The method of claim 19, wherein the drug is maytansinoid, auristatin (including MMAE, MMAF), aminopterin, actinomycin, bleomycin, thalisomycin, camptothecin, N8-acetyl spermidine, 1- (2 chloroethyl)-1,2-dimethyl sulfonyl hydrazide, esperamicin, etoposide, 6-mercaptopurine, dolastatin, trichothecene, calicheamicin, taxol, taxane, paclitaxel ( paclitaxel), docetaxel, methotrexate, vincristine, vinblastine, doxorubicin, melphalan, mitomycin A, mitomycin C, chlorambucil, duocarmycin, L-asparaginase, mer mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin ), carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustard, cytoxan, etoposide (etoposide), 5-fluorouracil, CNU (bischloroethylnitrosourea), irinotecan, camptothecin, bleomycin, idarubicin, daunorubicin ), dactinomycin, plicamycin, mitoxantrone, asparaginase, vinorelbine, chlorambucil, melphalan, carr Mustine (carmustine), lomustine (lomustine), busulfan (b usuLfan), treosulfan, decarbazine, etoposide, teniposide, topotecan, 9-aminocamptothecin, cristol ( crisnatol), mitomycin C, trimetrexate, mycophenolic acid, tiazofurin, ribavirin, EICAR (5-ethynyl-1-beta-Dribofuranosylimidazole) -4-carboxamide), hydroxyurea, deferoxamine, floxuridine, doxifluridine, raltitrexed, cytarabine (ara C) ), cytosine arabinoside, fludarabine, tamoxifen, raloxifene, megestrol, goserelin, leuprolide acetate acetate), flutamide, bicalutamide, EB1089, CB1093, KH1060, verteporfin, phthalocyanine, photosensitizer Pe4 (photosensitizer Pe4), demethoxy-hypocrerin A (demethoxy-hypocrellin A), interferon-α (Interferon-α), interferon-γ (Interferon-γ), tumor necrosis factor, gemcitabine, velcade, levalmid ( revamid), thalamid, lovastatin, 1-methyl-4-phenylpyridinium ion (1-methyl-4-phenylpyridi) niumion), staurosporine, actinomycin D, dactinomycin, bleomycin A2, bleomycin B2, peplomycin, epiruby Group consisting of epirubicin, pirarubicin, zorubicin, mitoxantrone, verapamil, thapsigargin, nucleases and toxins derived from bacteria, animals or plants An antibody-drug conjugate, characterized in that at least one selected from
  21. 제19항에 있어서, 상기 항체 또는 이의 항원 결합단편은 약물과 링커를 통하여 결합되는 것을 특징으로 하는 항체-약물 접합체.The antibody-drug conjugate according to claim 19, wherein the antibody or antigen-binding fragment thereof is coupled to a drug through a linker.
  22. 제21항에 있어서, 상기 링커는 절단성 링커 또는 비절단성 링커인 것을 특징으로 하는 항체-약물 접합체.22. The antibody-drug conjugate of claim 21, wherein the linker is a cleavable linker or a non-cleavable linker.
  23. 제22항에 있어서, 상기 절단성 링커 또는 비절단성 링커는 산성 불안정 링커 (acid-labile linker), 이황화 링커, 펩타이드 링커, 베타-글루쿠로나이드 (beta-glucuronide) 링커, 티오에테르기 또는 말레이미도카프로일기를 포함하는 것을 특징으로 하는 항체-약물 접합체.23. The method of claim 22, wherein the cleavable linker or non-cleavable linker is an acid labile linker, a disulfide linker, a peptide linker, a beta-glucuronide linker, a thioether group, or a maleimido group. An antibody-drug conjugate comprising a caproyl group.
  24. 제21항에 있어서, 상기 링커는 항체의 이황화 결합 환원시 노출되는 시스테인 잔기 또는 항체에 결합된 태그에 존재하는 시스테인 잔기에 결합되는 것을 특징으로 하는 항체-약물 접합체.22. The antibody-drug conjugate of claim 21, wherein the linker is coupled to a cysteine residue exposed upon reduction of a disulfide bond of the antibody or a cysteine residue present in a tag bound to the antibody.
  25. 항원 결합 부위를 포함하는 세포외도메인, 트랜스 멤브레인 도메인 및 세포내 신호전달 도메인을 포함하는 키메라 항원 수용체(CAR)로, 상기 세포외도메인의 항원 결합 부위는 제1항 내지 제9항 중 어느 한 항에 따른 항체의 scFv인 것을 특징으로 하는 키메라 항원 수용체.A chimeric antigen receptor (CAR) comprising an extracellular domain comprising an antigen-binding site, a trans-membrane domain and an intracellular signaling domain, wherein the antigen-binding site of the extracellular domain is any one of claims 1 to 9 Chimeric antigen receptor, characterized in that the scFv of the antibody according to.
  26. 제25항에 따른 키메라 항원 수용체(CAR)가 도입되어 있는 면역세포.An immune cell into which the chimeric antigen receptor (CAR) according to claim 25 is introduced.
  27. 제26항에 있어서, 상기 면역세포는 T 세포, NK 세포, 사이토카인 유도 살해세포(Cytokine Induced Killer cell, CIK), 활성화 세포독성 T 림프구(Cytotoxic T Lymphocyte, CTL), 마크로파지, 종양 조직 내 침투 T 세포 (Tumor-Infiltrating Lymphocytes, TIL) 및 수지상세포로 구성된 군에서 선택된 하나 이상인 면역세포.The method of claim 26, wherein the immune cells are T cells, NK cells, cytokine-induced killer cells (CIK), activated cytotoxic T lymphocytes (CTL), macrophages, infiltrating T in tumor tissue One or more immune cells selected from the group consisting of cells (Tumor-Infiltrating Lymphocytes, TIL) and dendritic cells.
  28. 제26항에 있어서, 상기 면역세포는 면역관문 억제제에 대한 항체에 대한 scFv가 세포외도메인의 항원 결합 부위로 포함된 키메라 항원 수용체(CAR)가 추가로 도입되어 있는 면역세포.The immune cell according to claim 26, wherein the immune cell is further introduced with a chimeric antigen receptor (CAR) in which an scFv for an antibody against an immune checkpoint inhibitor is included as an antigen-binding site of an extracellular domain.
  29. 제28항에 있어서, 상기 면역관문 억제제에 대한 항체는 FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137(4-1BB), VISTA, CD258(LIGHT), MARCO, CD134(OX40), CD28, CD278(ICOS), CD27, CD154(CD40L), CD357(GITR), CD30, DR3, CD226(DNAM1), CD96, CD200, CD200R, 트랜스페린 수용체 (Transferrin receptor), c-Met, EGFR, HER2, KDR, PDGFRa 및 NRP1으로 구성된 군에서 선택된 하나 이상을 타겟하는 항체 또는 이의 항원 결합단편인 면역세포.29. The method of claim 28, wherein the antibody against the checkpoint inhibitor is FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), MARCO, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c- Met, EGFR, HER2, KDR, PDGFRa and an immune cell that is an antibody or antigen-binding fragment thereof targeting one or more selected from the group consisting of NRP1.
  30. 제26항의 면역세포를 포함하는 병용 치료용 조성물.A composition for combination therapy comprising the immune cell of claim 26 .
  31. 제30항에 있어서, 상기 조성물은 FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137(4-1BB), VISTA, CD258(LIGHT), MARCO, CD134(OX40), CD28, CD278(ICOS), CD27, CD154(CD40L), CD357(GITR), CD30, DR3, CD226(DNAM1), CD96, CD200, CD200R, 트랜스페린 수용체 (Transferrin receptor), c-Met, EGFR, HER2, KDR, PDGFRa 및 NRP1으로 구성된 군에서 선택된 하나 이상을 타겟하는 항체 또는 이의 항원 결합 단편을 더 포함하는, 항체 또는 이의 항원 결합단편인 조성물.31. The method of claim 30, wherein the composition comprises FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137(4-1BB), VISTA, CD258(LIGHT), MARCO, CD134(OX40). ), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2 , KDR, PDGFRa and a composition that further comprises an antibody or antigen-binding fragment thereof targeting one or more selected from the group consisting of, an antibody or antigen-binding fragment thereof.
  32. 제1항 내지 제9항 중 어느 한 항에 따른 항체 또는 이의 항원 결합단편과 다음으로 구성된 군에서 선택된 하나 이상을 포함하는 병용 치료용 조성물:A composition for combination therapy comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 and at least one selected from the group consisting of:
    (i) 면역세포; (i) immune cells;
    (ii) 면역관문 억제제에 대한 항체에 대한 scFv를 세포외도메인으로 포함하는 항원 수용체(CAR)를 포함하는 면역세포; 및(ii) immune cells comprising an antigen receptor (CAR) comprising an scFv for an antibody against an immune checkpoint inhibitor as an extracellular domain; and
    (iii) 면역관문억제제 (Immune checkpoint inhibitor).(iii) Immune checkpoint inhibitors.
  33. 제32항에 있어서, 상기 면역세포는 T 세포, NK 세포, 사이토카인 유도 살해세포(Cytokine Induced Killer cell, CIK), 활성화 세포독성 T 림프구(Cytotoxic T Lymphocyte, CTL), 마크로파지, 종양 조직 내 침투 T 세포 (Tumor-Infiltrating Lymphocytes, TIL) 및 수지상세포로 구성된 군에서 선택된 하나 이상인 조성물.33. The method of claim 32, wherein the immune cells are T cells, NK cells, cytokine-induced killer cells (CIK), activated cytotoxic T lymphocytes (CTL), macrophages, infiltrating T in tumor tissue At least one composition selected from the group consisting of cells (Tumor-Infiltrating Lymphocytes, TIL) and dendritic cells.
  34. 제32항에 있어서, 상기 면역관문 억제제에 대한 항체는 FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137(4-1BB), VISTA, CD258(LIGHT), MARCO, CD134(OX40), CD28, CD278(ICOS), CD27, CD154(CD40L), CD357(GITR), CD30, DR3, CD226(DNAM1), CD96, CD200, CD200R, 트랜스페린 수용체 (Transferrin receptor), c-Met, EGFR, HER2, KDR, PDGFRa 및 NRP1으로 구성된 군에서 선택된 하나 이상을 타겟하는 항체 또는 이의 항원 결합단편인 조성물.33. The method of claim 32, wherein the antibody to the checkpoint inhibitor is FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), MARCO, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c- Met, EGFR, HER2, KDR, PDGFRa and a composition that is an antibody or antigen-binding fragment thereof targeting at least one selected from the group consisting of NRP1.
  35. 제32항에 있어서, 상기 면역관문억제제는 PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137(4-1BB), CD258(LIGHT), MARCO, CD134(OX40), CD28, CD278(ICOS), CD27, CD154(CD40L), CD357(GITR), CD30, DR3, CD226(DNAM1), CD96, CD200 또는 CD200R를 타겟으로 하는 약물인 조성물. 33. The method of claim 32, wherein the checkpoint inhibitor is PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137(4-1BB), CD258(LIGHT), MARCO, CD134(OX40), A composition which is a drug targeting CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200 or CD200R.
  36. 제15항에 따른 이중특이적 또는 다중특이적 항체 및 다음으로 구성된 군에서 선택된 하나 이상을 포함하는 병용 치료용 조성물:A composition for combination therapy comprising the bispecific or multispecific antibody according to claim 15 and at least one selected from the group consisting of:
    (i) 면역세포; (i) immune cells;
    (ii) 면역관문 억제제에 대한 항체에 대한 scFv 단편을 세포외도메인으로 포함하는 항원 수용체(CAR)를 포함하는 면역세포; 및(ii) immune cells comprising an antigen receptor (CAR) comprising an scFv fragment for an antibody against an immune checkpoint inhibitor as an extracellular domain; and
    (iii) 면역관문억제제 (Immune checkpoint inhibitor).(iii) Immune checkpoint inhibitors.
  37. 제36항에 있어서, 상기 면역관문 억제제에 대한 항체는 FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137(4-1BB), VISTA, CD258(LIGHT), MARCO, CD134(OX40), CD28, CD278(ICOS), CD27, CD154(CD40L), CD357(GITR), CD30, DR3, CD226(DNAM1), CD96, CD200, CD200R, 트랜스페린 수용체 (Transferrin receptor), c-Met, EGFR, HER2, KDR, PDGFRa 및 NRP1으로 구성된 군에서 선택된 하나 이상을 타겟하는 항체 또는 이의 항원 결합 단편인 조성물.37. The method of claim 36, wherein the antibody to the checkpoint inhibitor is FGFR3, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), MARCO, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c- Met, EGFR, HER2, KDR, PDGFRa, and a composition that is an antibody or antigen-binding fragment thereof targeting at least one selected from the group consisting of NRP1.
  38. 제36항에 있어서, 상기 면역관문억제제는 PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137(4-1BB), CD258(LIGHT), MARCO, CD134(OX40), CD28, CD278(ICOS), CD27, CD154(CD40L), CD357(GITR), CD30, DR3, CD226(DNAM1), CD96, CD200 또는 CD200R를 타겟으로 하는 약물인 조성물.37. The method of claim 36, wherein the checkpoint inhibitor is PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137(4-1BB), CD258(LIGHT), MARCO, CD134(OX40), A composition which is a drug targeting CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200 or CD200R.
  39. 제1항 내지 제9항 중 어느 한 항에 따른 항체 또는 이의 항원 결합단편;The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9;
    상기 항체 또는 이의 항원 결합단편을 포함하는 이중 또는 다중특이적 항체;a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof;
    상기 항체 또는 이의 항원 결합단편을 포함하는 항체-약물 접합체;an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof;
    상기 항체 또는 이의 항원 결합단편을 포함하는 키메라 항원 수용체; 또는 a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof; or
    상기 키메라 항원 수용체를 포함하는 면역세포;를 포함하는 암 예방 또는 치료용 조성물.A composition for preventing or treating cancer, comprising: immune cells comprising the chimeric antigen receptor.
  40. 제1항 내지 제9항 중 어느 한 항에 따른 항체 또는 이의 항원 결합단편;The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9;
    상기 항체 또는 이의 항원 결합단편을 포함하는 이중 또는 다중특이적 항체;a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof;
    상기 항체 또는 이의 항원 결합단편을 포함하는 항체-약물 접합체;an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof;
    상기 항체 또는 이의 항원 결합단편을 포함하는 키메라 항원 수용체; 또는a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof; or
    상기 키메라 항원 수용체를 포함하는 면역세포;를 포함하는 감염질환 예방 또는 치료용 조성물.Immune cells comprising the chimeric antigen receptor; a composition for preventing or treating infectious diseases, including.
  41. 제1항 내지 제9항 중 어느 한 항에 따른 항체 또는 이의 항원 결합단편;The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9;
    상기 항체 또는 이의 항원 결합단편을 포함하는 이중 또는 다중특이적 항체;a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof;
    상기 항체 또는 이의 항원 결합단편을 포함하는 항체-약물 접합체;an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof;
    상기 항체 또는 이의 항원 결합단편을 포함하는 키메라 항원 수용체; 또는 a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof; or
    상기 키메라 항원 수용체를 포함하는 면역세포;를 이를 필요로 하는 개체에 투여하는 단계를 포함하는, 암 예방 또는 치료 방법.A method for preventing or treating cancer, comprising administering an immune cell comprising the chimeric antigen receptor to an individual in need thereof.
  42. 제1항 내지 제9항 중 어느 한 항에 따른 항체 또는 이의 항원 결합단편;The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9;
    상기 항체 또는 이의 항원 결합단편을 포함하는 이중 또는 다중특이적 항체;a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof;
    상기 항체 또는 이의 항원 결합단편을 포함하는 항체-약물 접합체;an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof;
    상기 항체 또는 이의 항원 결합단편을 포함하는 키메라 항원 수용체; 또는 a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof; or
    상기 키메라 항원 수용체를 포함하는 면역세포;를 이를 필요로 하는 개체에 투여하는 단계를 포함하는, 감염질환 예방 또는 치료 방법.An immune cell comprising the chimeric antigen receptor; a method for preventing or treating an infectious disease comprising administering to an individual in need thereof.
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