WO2022124864A1 - Anticorps anti-tigit et son utilisation - Google Patents

Anticorps anti-tigit et son utilisation 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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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
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    • 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.

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Abstract

La présente invention concerne : un anticorps anti-TIGIT (domaine à immunoglobuline de lymphocytes T et ITIM) ou un fragment de liaison à l'antigène de celui-ci ; un acide nucléique codant pour celui-ci ; un vecteur d'expression recombinant contenant l'acide nucléique ; une cellule hôte qui est transinfectée par le vecteur d'expression recombinant ; un procédé de préparation de l'anticorps ou du fragment de liaison à l'antigène de celui-ci ; un anticorps bispécifique ou multi-spécifique contenant l'anticorps ou le fragment de liaison à l'antigène de celui-ci ; un anticorps bispécifique ou multi-spécifique activant la cellule immunitaire contenant au moins l'un parmi un scFv de l'anticorps et un scFv de l'anticorps se liant à l'antigène activant la cellule immunitaire ; un conjugué anticorps-médicament (ADC) dans lequel l'anticorps ou le fragment de liaison à l'antigène de celui-ci est lié à un médicament ; un récepteur antigénique chimérique (CAR) contenant le scFv de l'anticorps anti-TIGIT en tant que site de liaison à l'antigène d'un domaine extracellulaire ; une cellule immunitaire dans laquelle le récepteur antigénique chimérique a été introduit ; une composition pour une polythérapie comprenant la cellule immunitaire ; une composition pour une polythérapie comprenant l'anticorps ou le fragment de liaison à l'antigène de celui-ci ; une composition pour le traitement du cancer ou de maladies infectieuses ; et une méthode de traitement du cancer ou de maladies infectieuses.
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CN109852588A (zh) * 2018-12-24 2019-06-07 中国水产科学研究院珠江水产研究所 一种抗罗非鱼免疫球蛋白IgM的单克隆抗体及其细胞株和应用

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KR20180119653A (ko) * 2016-03-04 2018-11-02 제이엔 바이오사이언시즈 엘엘씨 Tigit에 대한 항체
KR20190103996A (ko) * 2018-02-28 2019-09-05 주식회사유한양행 신규 항-tigit 항체 및 그의 용도
KR20190133078A (ko) * 2014-08-19 2019-11-29 머크 샤프 앤드 돔 코포레이션 항-tigit 항체
WO2020020281A1 (fr) * 2018-07-25 2020-01-30 信达生物制药(苏州)有限公司 Anticorps anti-tigit et ses utilisations
US10766957B2 (en) * 2015-08-14 2020-09-08 Merck Sharp & Dohme Corp Anti-TIGIT antibodies

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KR20190133078A (ko) * 2014-08-19 2019-11-29 머크 샤프 앤드 돔 코포레이션 항-tigit 항체
US10766957B2 (en) * 2015-08-14 2020-09-08 Merck Sharp & Dohme Corp Anti-TIGIT antibodies
KR20180119653A (ko) * 2016-03-04 2018-11-02 제이엔 바이오사이언시즈 엘엘씨 Tigit에 대한 항체
KR20190103996A (ko) * 2018-02-28 2019-09-05 주식회사유한양행 신규 항-tigit 항체 및 그의 용도
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Publication number Priority date Publication date Assignee Title
CN109852588A (zh) * 2018-12-24 2019-06-07 中国水产科学研究院珠江水产研究所 一种抗罗非鱼免疫球蛋白IgM的单克隆抗体及其细胞株和应用
CN109852588B (zh) * 2018-12-24 2023-03-07 中国水产科学研究院珠江水产研究所 一种抗罗非鱼免疫球蛋白IgM的单克隆抗体及其细胞株和应用

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