WO2021225159A1 - Ｇｉｔｒ結合性分子 - Google Patents

Ｇｉｔｒ結合性分子 Download PDF

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WO2021225159A1
WO2021225159A1 PCT/JP2021/017479 JP2021017479W WO2021225159A1 WO 2021225159 A1 WO2021225159 A1 WO 2021225159A1 JP 2021017479 W JP2021017479 W JP 2021017479W WO 2021225159 A1 WO2021225159 A1 WO 2021225159A1
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amino acid
seq
acid sequence
heavy chain
gitr
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French (fr)
Japanese (ja)
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洋 珠玖
泰 赤堀
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Mie University NUC
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Mie University NUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to GITR-binding molecules and the like.
  • GITR glucocorticoid-induced TNFR-related protein
  • TNFRSF18 a membrane protein expressed in peripheral blood T cells and bone marrow cells
  • GITRL GITR ligand
  • stimulation from GITRL transmits co-stimulation to T cells, which in turn transmits activation signals via NFkB signals, and T cells are resistant to suppression of activation by Tregs.
  • a stimulation system that targets GITR and a drug distribution system that targets GITR.
  • An object of the present invention is to provide a GITR-binding molecule.
  • the present inventor has a heavy chain variable region containing heavy chain CDRs 1 to 3 containing a specific amino acid sequence, and / or a light chain containing light chain CDRs 1 to 3 containing a specific amino acid sequence. It was found that excellent binding to GITR can be exhibited by including a chain variable region.
  • the present inventor has completed the present invention as a result of further research based on this finding. That is, the present invention includes the following aspects.
  • Variable heavy chain including heavy chain CDR1 containing the amino acid sequence shown by SEQ ID NO: 8, heavy chain CDR2 containing the amino acid sequence shown by SEQ ID NO: 9, and heavy chain CDR3 containing the amino acid sequence shown by SEQ ID NO: 10.
  • Chain variable region (C) Heavy chain variable including heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 40, heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 41, and heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 42.
  • Item 2 The GITR-binding molecule according to Item 1, which has the feature (A), the feature (B), or the feature (C).
  • Item 3 The GITR-binding molecule according to Item 1 or 2, which has the above-mentioned characteristic (A).
  • Item 4. The GITR-binding molecule according to any one of Items 1 to 3, which contains an antibody structure.
  • Item 5 At least selected from the group consisting of immunoglobulin structure, Fab structure, F (ab') 2 structure, minibody structure, scFv-Fc structure, Fv structure, scFv structure, deerbody structure, triabody structure, and tetrabody structure.
  • Item 4. The GITR-binding molecule according to any one of Items 1 to 4, which comprises one type of structure.
  • Item 6 A polynucleotide encoding the GITR-binding molecule according to any one of Items 1 to 5.
  • Item 7 A cell containing the polynucleotide according to item 6.
  • Item 8 A medicament containing at least one selected from the group consisting of the GITR-binding molecule according to any one of Items 1 to 5 and the polynucleotide according to Item 6.
  • Item 9 An adjuvant containing at least one selected from the group consisting of the GITR-binding molecule according to any one of Items 1 to 5 and the polynucleotide according to Item 6.
  • Item 10 A reagent containing at least one selected from the group consisting of the GITR-binding molecule according to any one of Items 1 to 5 and the polynucleotide according to Item 6.
  • GITR-binding molecule can be provided.
  • FIG. 1 shows a schematic diagram of the antibody screen method (GITR forced expression cell screen) of Test Example 1-4
  • (B) is a schematic diagram of the antibody screen method (screen for immobilized GITR protein) of Test Example 1-5.
  • the figure is shown.
  • the antibody phage library is reacted with GITR forced expression 293T cells, and the polyclonal antibody phage bound to the cells is sorted by centrifugation using an organic solvent.
  • biotin-bound GITR-Fc binds to neutravidin while maintaining its three-dimensional structure, and phage antibodies that bind to this can be selected. By diluting the antigen concentration, clones with strong binding are selected.
  • the FACS result of Isotype control in the examination of cell surface expression GITR recognition ability (Test Example 2-2) is shown.
  • the vertical axis shows the number of cell counts, and the horizontal axis shows the fluorescence intensity.
  • the FACS result of antibody clone A in the examination of cell surface expression GITR recognition ability (Test Example 2-2) is shown.
  • the vertical axis shows the number of cell counts, and the horizontal axis shows the fluorescence intensity.
  • the FACS result of antibody clone B in the examination of cell surface expression GITR recognition ability (Test Example 2-2) is shown.
  • the vertical axis shows the number of cell counts, and the horizontal axis shows the fluorescence intensity.
  • the FACS result of antibody clone C in the examination of cell surface expression GITR recognition ability (Test Example 2-2) is shown.
  • the vertical axis shows the number of cell counts, and the horizontal axis shows the fluorescence intensity.
  • the FACS result of antibody clone D in the examination of cell surface expression GITR recognition ability (Test Example 2-2) is shown.
  • the vertical axis shows the number of cell counts, and the horizontal axis shows the fluorescence intensity.
  • the FACS result of Isotype control (mouse IgG2a) in the examination of GITR recognition on naturally activated human T cells (Test Example 3-1) is shown.
  • the FACS result of Isotype control (HERCEPTIN) in the examination of GITR recognition on naturally activated human T cells (Test Example 3-1) is shown.
  • the FACS results of a commercially available antibody in the examination of GITR recognition on naturally activated human T cells (Test Example 3-1) are shown.
  • the FACS result of antibody clone B in the examination of GITR recognition on naturally activated human T cells (Test Example 3-1) is shown.
  • the FACS results in the examination of GITR recognition on natural CAR T cells (Test Example 3-2) are shown.
  • Identity of amino acid sequences refers to the degree of coincidence of amino acid sequences with each other among two or more comparable amino acid sequences. Therefore, the higher the match between two amino acid sequences, the higher the identity or similarity of those sequences.
  • the level of amino acid sequence identity is determined, for example, using FASTA, a sequence analysis tool, using default parameters.
  • FASTA a sequence analysis tool
  • the algorithm BLAST by Karlin and Altschul Karlin and Altschul (KarlinS, Altschul SF. “Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes” Proc Natl Acad Sci USA.
  • a program called BLASTX based on such a BLAST algorithm has been developed. Specific methods for these analysis methods are known, and the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/) can be referred to.
  • NCBI National Center for Biotechnology Information
  • the "identity" of the base sequence is also defined according to the above.
  • conservative substitution means that an amino acid residue is replaced with an amino acid residue having a similar side chain.
  • substitution between amino acid residues having a basic side chain such as lysine, arginine, and histidine is a conservative substitution.
  • amino acid residues with acidic side chains such as aspartic acid and glutamic acid
  • amino acid residues with non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine
  • Amino acid residues with non-polar side chains such as proline, phenylalanine, methionine and tryptophan
  • amino acid residues with beta-branched side chains such as threonine, valine and isoleucine
  • aromatic side chains such as tyrosine, phenylalanine, tryptophan and histidine Substitution between amino acid residues is also a conservative substitution.
  • CDR is an abbreviation of C omplementarity D etermining R egion, also called complementarity determining regions.
  • the CDR is a region existing in the variable region of immunoglobulin, and is a region deeply involved in the specific binding of the antibody to the antigen.
  • the "light chain CDR” means a CDR existing in the light chain variable region of immunoglobulin
  • the “heavy chain CDR” means a CDR existing in the heavy chain variable region of immunoglobulin.
  • the "variable region” means an region including CDR1 to CDR3 (hereinafter, simply referred to as "CDRs1-3").
  • CDRs1-3 CDR1 to CDR3
  • the arrangement order of these CDRs1-3 is not particularly limited, but preferably, the frames are continuous or described later in the order of CDR1, CDR2, and CDR3 in the direction from the N-terminal side to the C-terminal side, or vice versa. It means a region arranged via another amino acid sequence called a work region (FR).
  • the "heavy chain variable region” is a region in which the above-mentioned heavy chain CDRs1-3 is arranged, and the “light chain variable region” is a region in which the above-mentioned light chain CDRs1-3 is arranged.
  • FR framework areas
  • the region between the N-terminal of the variable region and CDR1 is FR1
  • the region between CDR1 and CDR2 is FR2
  • the region between CDR2 and CDR3 is FR3
  • the region between CDR3 and the C-terminal of the variable region is Defined as FR4 respectively.
  • GITR-binding molecule having any of the following characteristics (A) to (D) (in the present specification, it may be referred to as "GITR-binding molecule of the present invention”. There is.) This will be described below.
  • the GITR-binding molecule of the present invention is particularly limited as long as it has any of the following characteristics (A) to (D) and has binding property (preferably specifically binding) to GITR. Not limited.
  • Feature (A) includes heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 8, heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 9, and heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 10.
  • Feature (B) includes heavy chain CDR1 containing the amino acid sequence set forth in SEQ ID NO: 24, heavy chain CDR2 containing the amino acid sequence set forth in SEQ ID NO: 25, and heavy chain CDR3 containing the amino acid sequence set forth in SEQ ID NO: 26.
  • Feature (C) includes heavy chain CDR1 containing the amino acid sequence set forth in SEQ ID NO: 40, heavy chain CDR2 containing the amino acid sequence set forth in SEQ ID NO: 41, and heavy chain CDR3 containing the amino acid sequence set forth in SEQ ID NO: 42.
  • Feature (D) includes heavy chain CDR1 containing the amino acid sequence set forth in SEQ ID NO: 56, heavy chain CDR2 containing the amino acid sequence set forth in SEQ ID NO: 57, and heavy chain CDR3 containing the amino acid sequence set forth in SEQ ID NO: 58.
  • the GITR binding molecule of the present invention preferably has a feature (A), a feature (B), or a feature (C), and more preferably has a feature (A) or a feature (B). It has, and more preferably has the feature (A).
  • the GITR-binding molecule of the present invention preferably contains both the heavy chain variable region and the light chain variable region in each of the characteristics (A) to (D).
  • the heavy chain variable region is preferably 90% or more of the amino acid sequence shown in SEQ ID NO: 3 or the amino acid sequence shown in SEQ ID NO: 3 ( It is a heavy chain variable region containing an amino acid sequence having an identity of preferably 95% or more, preferably 98% or more, preferably 99% or more).
  • the light chain variable region is preferably 90% or more (preferably 95% or more, preferably 98% or more, preferably 99%) of the amino acid sequence shown in SEQ ID NO: 4 or the amino acid sequence shown in SEQ ID NO: 4. It is a light chain variable region containing an amino acid sequence having the same identity as above). If there is an amino acid mutation from SEQ ID NO: 3 or 4, the mutation is preferably an amino acid substitution, more preferably a conservative substitution of an amino acid.
  • the heavy chain variable region is preferably 90% or more of the amino acid sequence shown in SEQ ID NO: 19 or the amino acid sequence shown in SEQ ID NO: 19 ( It is a heavy chain variable region containing an amino acid sequence having an identity of preferably 95% or more, preferably 98% or more, preferably 99% or more).
  • the light chain variable region is preferably 90% or more (preferably 95% or more, preferably 98% or more, preferably 99%) of the amino acid sequence shown by SEQ ID NO: 20 or the amino acid sequence shown by SEQ ID NO: 20. It is a light chain variable region containing an amino acid sequence having the same identity as above). If there is an amino acid mutation from SEQ ID NO: 19 or 20, the mutation is preferably an amino acid substitution, more preferably a conservative substitution of an amino acid.
  • the heavy chain variable region is preferably 90% or more of the amino acid sequence shown by SEQ ID NO: 35 or the amino acid sequence shown by SEQ ID NO: 35 ( It is a heavy chain variable region containing an amino acid sequence having an identity of preferably 95% or more, preferably 98% or more, preferably 99% or more).
  • the light chain variable region is preferably 90% or more (preferably 95% or more, preferably 98% or more, preferably 99%) of the amino acid sequence shown in SEQ ID NO: 36 or the amino acid sequence shown in SEQ ID NO: 36. It is a light chain variable region containing an amino acid sequence having the same identity as above). If there is an amino acid mutation from SEQ ID NO: 35 or 36, the mutation is preferably an amino acid substitution, more preferably a conservative substitution of an amino acid.
  • the heavy chain variable region is preferably 90% or more of the amino acid sequence shown by SEQ ID NO: 51 or the amino acid sequence shown by SEQ ID NO: 51 ( It is a heavy chain variable region containing an amino acid sequence having an identity of preferably 95% or more, preferably 98% or more, preferably 99% or more).
  • the light chain variable region is preferably 90% or more (preferably 95% or more, preferably 98% or more, preferably 99%) of the amino acid sequence shown by SEQ ID NO: 52 or the amino acid sequence shown by SEQ ID NO: 52. It is a light chain variable region containing an amino acid sequence having the same identity as above). If there is an amino acid mutation from SEQ ID NO: 51 or 52, the mutation is preferably an amino acid substitution, more preferably a conservative substitution of an amino acid.
  • the structure of the GITR-binding molecule of the present invention is not particularly limited.
  • the GITR-binding molecule of the present invention preferably contains an antibody structure.
  • the structure (tag, signal sequence, etc. described later) may be added.
  • the antibody structure may include a constant region of immunoglobulin or may not include a constant region of immunoglobulin.
  • the constant region When the constant region is included, the heavy chain constant region (CH1, CH2, and CH3) and the light chain constant region (CL) may be all included, and any one or more of these may be included. May include a combination of.
  • antibody structures include immunoglobulin structure, Fab structure, F (ab') 2 structure, minibody structure, scFv-Fc structure, Fv structure, scFv structure, diabody structure, and triabody structure.
  • (Triabody) structure, tetrabody structure and the like can be mentioned.
  • Immunoglobulin has a structure in which two structures consisting of one heavy chain having a heavy chain variable region and a heavy chain constant region and one light chain having a light chain variable region and a light chain constant region are combined.
  • Fab includes a heavy chain fragment containing CH1 in a heavy chain variable region and a heavy chain constant region, and a light chain containing a light chain variable region and a light chain constant region (CL), and includes a heavy chain variable region and a light chain. It has a structure in which the chain variable region is associated by the non-covalent intermolecular interaction described above or is bound by a disulfide bond.
  • CH1 and CL may be disulfide-bonded with each other by the thiol groups of the cysteine residues present in each.
  • F (ab') 2 has two pairs of the above Fabs, and CH1 is a structure formed by disulfide bonds between thiol groups of cysteine residues contained therein.
  • the minibody is a structure in which two fragments in which CH3 is bound to the heavy chain variable region constituting the following scFv are associated with each other by non-covalent intermolecular interaction.
  • scFv-Fc With scFv-Fc, the following two antibody fragments containing scFv, CH2, and CH3 are associated with each other by non-covalent intermolecular interaction between CH3s as in the case of the above minibody, and the cysteine residue contained in each CH3. It is a structure in which thiol groups of groups are disulfide-bonded to each other.
  • Fv is also called the smallest structural unit of an antibody, and is a structure in which a heavy chain variable region and a light chain variable region are associated by a non-covalent intermolecular interaction.
  • the thiol groups of cysteine residues existing in the heavy chain variable region and the light chain variable region may be disulfide-bonded to each other.
  • scFv is a structure in which the C-terminal of the heavy chain variable region and the N-terminal of the light chain variable region are linked by a linker, or the N-terminal of the heavy chain variable region and the C-terminal of the light chain variable region are linked by a linker. It is a structure and is also called a single chain antibody.
  • the above scFv forms a dimer, a trimer, and a tetramer, respectively, and like Fv, non-covalent intermolecular interactions between variable regions, etc.
  • the structure is structurally stable.
  • the GITR-binding molecule of the present invention is immunoglobulin
  • its class is not particularly limited. Examples of the class include IgA, IgD, IgE, IgG, IgM, and the like, and further include subclasses thereof.
  • the origin of the antibody structure is not particularly limited.
  • the antibody structure can be, for example, a human-derived antibody, a mouse-derived antibody, a rat-derived antibody, a rabbit-derived antibody, a monkey-derived antibody, a chimpanzee-derived antibody, or the like.
  • the antibody structure includes chimeric antibodies (for example, antibodies in which the amino acid sequence of the constant region of an antibody derived from a non-human organism (such as mouse) is replaced with the amino acid sequence of the constant region of a human-derived antibody), a humanized antibody, and a completely human antibody. It may be a chemical antibody or the like.
  • the GITR-binding molecule of the present invention is preferably a monoclonal molecule (that is, a molecular population having a single amino acid sequence and structure).
  • the molecular weight of the GITR-binding molecule of the present invention is not particularly limited, but the lower limit is, for example, 20,000, 50,000, 100,000, or 120,000, and the upper limit is, for example, 1,000,000, 500,000, or 200,000.
  • the GITR-binding molecule of the present invention may be a molecule composed of one kind of polypeptide or a molecule composed of a complex of two or more kinds of polypeptides. Further, the GITR-binding molecule of the present invention may be a molecule composed of a polypeptide or a complex thereof, or another substance (for example, a fluorescent substance, a radioactive substance, an inorganic particle, etc.) may be added to the polypeptide or the complex thereof. May be connected.
  • the GITR-binding molecule of the present invention may be chemically modified.
  • R in the ester is, for example, a C 1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl; for example, a C 3-8 cycloalkyl group such as cyclopentyl, cyclohexyl; for example, phenyl.
  • C 6-12 aryl groups such as ⁇ -naphthyl; for example, phenyl-C 1-2 alkyl groups such as benzyl, phenethyl; C 7- such as ⁇ -naphthyl-C 1-2 alkyl groups such as ⁇ -naphthylmethyl.
  • Aralkyl group; Pivaloyloxymethyl group etc. are used.
  • the polypeptide constituting the GITR-binding molecule of the present invention may have a carboxyl group (or carboxylate) other than the C-terminal amidated or esterified. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.
  • the polypeptide constituting the GITR binding molecules of the present invention the amino group of the amino acid residues of the N-terminal protecting group (e.g., formyl group, such as C 1-6 alkanoyl such as acetyl group C 1-6 Those protected with an acyl group (such as an acyl group), those in which the N-terminal glutamine residue that can be cleaved and produced in vivo is pyroglutamine-oxidized, and substituents on the side chain of amino acids in the molecule (for example, -OH,- SH, amino group, imidazole group, indol group, guanidino group, etc.) are protected by suitable protective groups (eg, C 1-6 acyl group such as C 1-6 alkanoyl group such as formyl group, acetyl group, etc.) What is included is also included.
  • suitable protective groups eg, C 1-6 acyl group such as C 1-6 alkanoyl group such as formyl
  • the GITR-binding molecule of the present invention may be one to which a protein or peptide such as a known protein tag or signal sequence is added.
  • a protein or peptide such as a known protein tag or signal sequence
  • the protein tag include biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag, fluorescent protein tag and the like.
  • the GITR-binding molecule of the present invention may be in the form of a pharmaceutically acceptable salt with an acid or base.
  • the salt is not particularly limited as long as it is a pharmaceutically acceptable salt, and either an acidic salt or a basic salt can be adopted.
  • acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; acetate, propionate, tartrate, fumarate, maleate, and apple.
  • Organic acid salts such as acid salts, citrates, methane sulfonates and paratoluene sulfonates; amino acid salts such as asparaginates and glutamates can be mentioned.
  • basic salts include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt.
  • the GITR-binding molecule of the present invention may be in the form of a solvate.
  • the solvent is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include water, ethanol, glycerol, acetic acid and the like.
  • the GITR-binding molecule of the present invention comprises, for example, culturing a host transformed with a polynucleotide encoding the GITR-binding molecule of the present invention and recovering a fraction containing the GITR-binding molecule of the present invention. It can be manufactured by the method.
  • the polynucleotide encoding the GITR-binding molecule of the present invention is not particularly limited as long as it contains the GITR-binding molecule of the present invention in an expressible state, and other than the coding sequence of the GITR-binding molecule of the present invention, other It may contain an array.
  • Other sequences include a secretory signal peptide coding sequence, a promoter sequence, an enhancer sequence, a repressor sequence, an insulator sequence, a replication base point, and a drug resistance gene coding sequence, which are arranged adjacent to the GITR-binding molecular coding sequence of the present invention. And so on.
  • the polynucleotide encoding the GITR-binding molecule of the present invention may be a linear polynucleotide or a cyclic polynucleotide (vector or the like).
  • polynucleotide examples include (I) a base sequence encoding at least one selected from the group consisting of heavy chains, heavy chain variable regions, and heavy chain CDRs1-3 of the GITR-binding molecule of the present invention.
  • Polynucleotide, (II) A polynucleotide containing a nucleotide sequence encoding at least one selected from the group consisting of the light chain, the light chain variable region, and the light chain CDRs1-3 of the GITR-binding molecule of the present invention, (III).
  • a polynucleotide containing a base sequence encoding at least one selected from the group consisting of the heavy chain, the heavy chain variable region, and the heavy chain CDRs1-3 of the GITR-binding molecule of the present invention, and the GITR-binding property of the present invention examples thereof include a polynucleotide containing a base sequence encoding at least one selected from the group consisting of the light chain, the light chain variable region, and the light chain CDRs 1-3 of the molecule.
  • the host is not particularly limited, and examples thereof include insect cells, eukaryotic cells, and mammalian cells. Among them, from the viewpoint of more efficiently expressing the antibody, mammalian cells such as HEK cells, CHO cells, NS0 cells, SP2 / O cells, and P3U1 cells are preferable.
  • mammalian cells such as HEK cells, CHO cells, NS0 cells, SP2 / O cells, and P3U1 cells are preferable.
  • the method of transformation, culturing, and recovery is not particularly limited, and a known method in antibody production can be adopted. After recovery, the GITR-binding molecule of the present invention may be purified, if necessary. Purification can be performed by known methods in antibody production, such as chromatography, dialysis and the like.
  • polynucleotides of the invention that encode the GITR-binding molecules of the invention. This will be described below.
  • the polynucleotide of the present invention may contain other sequences in addition to the coding sequence of the GITR-binding molecule of the present invention.
  • the polynucleotide of the present invention preferably contains the GITR-binding molecule of the present invention in an expressible state. Examples of other sequences include a promoter sequence, an enhancer sequence, a repressor sequence, an insulator sequence, a replication base point, a reporter protein (for example, a fluorescent protein, etc.) coding sequence, a drug resistance gene coding sequence, and the like.
  • the polynucleotide of the present invention may be a linear polynucleotide or a cyclic polynucleotide (vector or the like).
  • the vector can be a plasmid vector, or a viral vector (eg, adenovirus, or retrovirus).
  • the vector can be, for example, a cloning vector or an expression vector.
  • the expression vector include a vector for prokaryotic cells such as Escherichia coli and actinomycetes, and a vector for eukaryotic cells such as yeast cells, insect cells, and mammalian cells.
  • the polynucleotide of the present invention includes not only DNA and RNA, but also those to which known chemical modifications are applied, as illustrated below.
  • PS phosphorothioate
  • methylphosphonate methylphosphonate
  • phosphorodithionate to prevent degradation by hydrolytic enzymes such as nucleases.
  • the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide is changed to -OR (R is, for example, CH3 (2'-O-Me), CH 2 CH 2 OCH 3 (2'-O-MOE), CH.
  • the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl.
  • examples thereof include those in which the phosphoric acid moiety and the hydroxyl moiety are modified with biotin, an amino group, a lower alkylamine group, an acetyl group and the like, but the present invention is not limited thereto.
  • polynucleotide includes not only natural nucleic acids but also any of BNA (Bridged Nucleic Acid), LNA (Locked Nucleic Acid), PNA (Peptide Nucleic Acid) and the like.
  • the present invention relates, in one aspect, to cells containing the polynucleotides of the invention (sometimes referred to herein as "cells of the invention”). This will be described below.
  • the cell from which the cell of the present invention is derived is not particularly limited.
  • the derived cells include cells that can be used for protein expression (for example, insect cells, eukaryotic cells, mammalian cells, etc.). Can be mentioned.
  • Examples of cells include Escherichia coli K12 and other Escherichia coli, Bacillus subtilis MI114 and other Bacillus bacteria, Saccharomyces cerevisiae AH22 and other yeast, Spodoptera frugiperda-derived Sf cell lineage, and Trichoplusia ni-derived High Five cell lineage, and olfactory nerve cells.
  • Examples include animal cells such as insect cells and COS7 cells.
  • the animal cells are preferably cultured cells derived from mammals, specifically, COS7 cells, CHO cells, HEK293 cells, HEK293FT cells, Hela cells, PC12 cells, N1E-115 cells, SH-SY5Y cells and the like. Be done.
  • the present invention in one aspect thereof, comprises a composition containing at least one selected from the group consisting of the GITR-binding molecule of the present invention and the polynucleotide of the present invention (in the present specification, "the present invention”. It may be referred to as "composition").
  • the GITR-binding molecule of the present invention can bind to GITR expressed in cells. Therefore, at least one selected from the group consisting of the GITR-binding molecule of the present invention and the polynucleotide of the present invention can be used as an active ingredient of, for example, a drug, an adjuvant, a reagent or the like.
  • use as an adjuvant molecule that stimulates the T cell response that is, use as an agonist molecule that stimulates activated T cells
  • use as an agonist molecule that stimulates activated T cells in addition to the method of administration as a protein molecule, CAR-introduced T cells, Use as a molecule that stimulates them by co-expressing them in TCR-introduced T cells; Use as a DNA vaccine; Use as an reagent that delivers molecules to activated T cells; Since it is obtained, its use in the treatment of autoimmune diseases can be mentioned.
  • the antibody molecule having agonist activity can be selected by a reporter assay.
  • a reporter assay For example, it can be selected by the following method.
  • Jurkat E6J cells are modified into reporter cells that express CFP in an NF-kB-dependent manner, and GITR is forcibly expressed in these reporter cells. Confirm that the reporter works by the agonist molecule by GITRL administration experiment.
  • the antibody is modified into a membrane-bound type and introduced into reporter cells with a retrovirus.
  • the cells into which the agonist antibody has been introduced stimulate GITR in autocline and express CFP, so they can be selected with a cell sorter. From this, scFv is recovered and transformed into a minibody antibody to examine the agonist activity.
  • the content of the active ingredient in the composition of the present invention depends on the type of use, for example, if the use is pharmaceutical, the type of target disease, the target therapeutic effect, the administration method, the treatment period, and the patient. It can be appropriately set in consideration of the age of the patient, the weight of the patient, and the like.
  • the content of the active ingredient in the composition of the present invention can be about 0.0001 parts by weight to 100 parts by weight, assuming that the entire composition of the present invention is 100 parts by weight.
  • composition of the present invention is not particularly limited as long as the desired effect can be obtained.
  • oral administration and parenteral administration for example, intravenous injection, intramuscular injection, subcutaneous administration, rectal administration
  • Percutaneous administration, local administration can be administered to mammals including humans.
  • the preferred dosage form is parenteral administration, more preferably intravenous injection.
  • Dosage forms for oral administration and parenteral administration and methods for producing the same are well known to those skilled in the art, and the active ingredient can be produced according to a conventional method by mixing it with a pharmaceutically acceptable carrier or the like. can.
  • Formulations for parenteral administration include injectable preparations (eg, drip injections, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), external preparations (eg ointments, paps, lotions). Agents), suppository inhalants, eye agents, eye ointments, nasal drops, ear drops, liposomes and the like.
  • an injectable preparation is prepared by dissolving an antibody or a cell in distilled water for injection, and if necessary, a lysis aid, a buffer, a pH adjuster, an isotonic agent, a soothing agent, a preservative, and a preservative. Stabilizers and the like can be added.
  • the pharmaceutical can also be a lyophilized preparation for preparation of errands.
  • the carrier used for the formulation of the composition of the present invention includes a substrate, an excipient, a binder, a disintegrant, a lubricant, a colorant, a flavoring and odorant commonly used in the art, and if necessary, a stabilizer.
  • Agents, emulsifiers, absorption enhancers, surfactants, pH regulators, preservatives, antioxidants, bulking agents, wetting agents, surface activators, dispersants, buffers, preservatives, solubilizers, pain relief Agents and the like can be used.
  • the amount of the composition of the present invention to be used depends on the type of use, for example, if the use is pharmaceutical, the route of administration, the type of disease, the degree of symptoms, the age, sex, weight of the patient, and the seriousness of the disease. Based on various factors such as degree, pharmacokinetic and toxicological characteristics, availability of drug delivery system, and whether it is administered as part of a combination of other drugs, by a clinician Can be decided.
  • the dose of the drug can be, for example, about 1 ⁇ g / kg (body weight) to 10 g / kg (body weight) per day.
  • the administration schedule of the drug can also be determined in consideration of the same factors as the dose. For example, the above daily dose can be administered once a day to January.
  • Test example 1 As an antibody for the purpose of obtaining an anti-GITR antibody, it is necessary to recognize the natural form of GITR expressed on the cell surface and strongly bind to it. In addition, a means for obtaining at least a plurality of antibodies was required. This time, we have developed a screen method using an organic solvent and developed a method to obtain the desired antibody by combining it with a solid-phase antigen screen. Specifically, it was carried out as follows.
  • Test Example 1-1 Preparation of human antibody library AIMS6 With the consent of the patient, mRNA was prepared from human surgical materials (tonsil, bone marrow, peripheral blood, umbilical cord blood), and based on this, VH and VL were prepared using specific primers. Amplified. It was recombined with pTZ19R as a fusion gene (scFv-cp3) of M13 phage with cp3 to prepare a plasmid library. Escherichia coli into which this gene was introduced was amplified and infected with helper phage KO7 to prepare an antibody library AIMS6 expressing scFv-cp3 on M13 phage.
  • Test Example 1-2 Preparation of GITR-Fc A gene fragment with an Avi-tag attached to the C-terminal is prepared by fusing the extracellular domain of the human GITR gene with the Fc portion of human IgG1, and this is co-expressed with secretory biotin ligase. Incorporate into the designed pCAGGS vector, introduce into 293T cells, cultivate in GIT medium containing biotin 2 microgram / ml, collect the culture supernatant, and purify with a protein-G column to obtain biotinylated GITR-Fc protein. Obtained.
  • Test Example 1-3 Preparation of GITR forced expression 293T cells
  • the human GITR gene was introduced into pCDNA3.1neo, introduced into 293T cells, and cultured in the presence of G418 to obtain GITR forced expression 293T cells.
  • the cells were introduced into mouse cells CMS5a and cultured to obtain GITR forced expression CMS5a cells.
  • Test Example 1-4 Antibody screen against GITR forced expression cells using the human antibody library AIMS A schematic diagram of the antibody screen method of this test example is shown in FIG. 1 (A).
  • Glucose was 2% and ampicillin was 200 microgram / ml. After culturing in the containing 2xYT medium, the glucose concentration was lowered to 0.05%, and then the helper phage KO7 was infected and cultured to obtain a phage solution. The second and third organic solvent screens were layered on the organic solvent and centrifuged four times, and the antibody population with strong binding was selected. At this point, polyclone antibodies that bind to a large number of cell surface antigens, including GITR, have been obtained. In order to narrow down the target GITR recognition antibody, the following screen using biotinylated GITR-Fc was performed.
  • Test Example 1-5 Antibody screen with immobilized human GITR protein A schematic diagram of the antibody screen method of this test example is shown in FIG. 1 (B).
  • Test example 2 Antibody evaluation 1 Test Example 2-1. ELISA evaluation of antibody clones Antibody clone Escherichia coli was cultured in 0.5 mM IPTG, 200 microgram / ml ampicillin 2xYT medium to obtain a culture supernatant containing scFv-cp3. Next, this culture supernatant was reacted with a maxisorp plate on which 0.5 microgram of GITR-Fc was immobilized, and then a rabbit anti-cp3 antibody and an HRP-labeled anti-rabbit IgG antibody were reacted to develop color with TMB. From the ELISA results, four antibody clones (antibody clones A, B, C, and D) having excellent binding to human GITR were selected.
  • the amino acid sequence of each region of each antibody clone and the base sequence encoding the same are shown below.
  • the CDR sequence was estimated by IMGT.
  • Heavy chain variable region nucleotide sequence CAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTACGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACGCGGCCGTGTATTACTGTGCGAGAGTACCCCTTAGTGGCTACCTGTACTACTTTGACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGA (SEQ ID NO: 49) Light chain variable region nucleotide sequence: GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGCCCAG
  • Test Example 2-2 Examination of cell surface expression GITR recognition ability of antibody clone hGITR forced expression CMS5a cells were reacted with the supernatant of the antibody clone, then the rabbit anti-cp3 antibody was reacted and washed, and then the anti-rabbit IgG-Alexa488 was reacted and washed. Then, it was measured by FACScalibur. The results are shown in FIGS. 2 to 6. Antibody clones A, B, C, or D were found to recognize hGITR forced expression CMS5a cells.
  • Test Example 2-3 Preparation of minibody type antibody
  • a fusion protein of scFv with human IgG1 Fc was prepared, recombined with pCAGGS, introduced into 293T cells, cultured, and the supernatant was collected. This was purified with protein G sepharose to obtain a minibody type antibody.
  • Test Example 2-4 Affinity evaluation of minibody type antibody (Fig. 3) BIAcore 3000 was used to evaluate the binding activity of the minibody of antibody clones A, B, C, and D.
  • the method is as follows.
  • the human GITR protein was bound to the sensor chip CM5 by the amine coupling method, and the minibody protein diluted with HBS-EP buffer was allowed to flow at a constant rate for 60 seconds to measure the binding reaction. Next, only the HBS-EP buffer was flowed and the dissociation reaction was measured. These values were calculated to calculate the dissociation constant KD.
  • Table 1 The results are shown in Table 1.
  • Test example 3 Antibody evaluation 2 Test example 3-1. GITR recognition on natural activated human T cells Human PBMC was stimulated with OKT3 and retronectin, and the expression of GITR on activated T cells as of day 6 was stained for CD8 cells and CD4 cells, and analyzed by FACSCANT. Was done. For commercially available monoclonal antibodies, Biolegend's APC-labeled clone 621 was used. The antibody of antibody clone B was reacted with the antibody and then stained with APC-labeled anti-human IgG antibody, and at the same time, stained with anti-CD8 and anti-CD4.
  • Test Example 3-2 Naturally Activated GITR Recognition on CAR T Cells Fresh PBMCs are not activated and expression of activation markers CD25 and GITR is absent. On the other hand, when activated by OKT3 and retronectin and then the CAR gene is introduced using a retrovirus, the activated T cells can observe the expression of CD25 and GITR. Fresh PBMC isolated by the Ficoll method and T cells at the time of day 13 when the CAR gene was introduced by activating it were stained with anti-CD25 antibody, commercially available monoclone GITR antibody, and antibody clone B antibody, and then stained with FACSCANT. Measurements were made.

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* Cited by examiner, † Cited by third party
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JP2007515942A (ja) * 2003-05-23 2007-06-21 ワイス Ｇｉｔｒリガンド及びｇｉｔｒリガンド関連分子及び抗体及びその使用
JP2017526615A (ja) * 2014-05-28 2017-09-14 アジェナス インコーポレイテッド 抗ｇｉｔｒ抗体及びその使用法
JP2017531427A (ja) * 2014-10-03 2017-10-26 デイナ ファーバー キャンサー インスティチュート，インコーポレイテッド グルココルチコイド誘導腫瘍壊死因子受容体（ｇｉｔｒ）抗体およびその使用法
WO2018213297A1 (en) * 2017-05-16 2018-11-22 Bristol-Myers Squibb Company Treatment of cancer with anti-gitr agonist antibodies
JP2018535665A (ja) * 2015-10-07 2018-12-06 エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト 共刺激ｔｎｆ受容体に対する四価の二重特異性抗体発明の分野

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007515942A (ja) * 2003-05-23 2007-06-21 ワイス Ｇｉｔｒリガンド及びｇｉｔｒリガンド関連分子及び抗体及びその使用
JP2017526615A (ja) * 2014-05-28 2017-09-14 アジェナス インコーポレイテッド 抗ｇｉｔｒ抗体及びその使用法
JP2017531427A (ja) * 2014-10-03 2017-10-26 デイナ ファーバー キャンサー インスティチュート，インコーポレイテッド グルココルチコイド誘導腫瘍壊死因子受容体（ｇｉｔｒ）抗体およびその使用法
JP2018535665A (ja) * 2015-10-07 2018-12-06 エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト 共刺激ｔｎｆ受容体に対する四価の二重特異性抗体発明の分野
WO2018213297A1 (en) * 2017-05-16 2018-11-22 Bristol-Myers Squibb Company Treatment of cancer with anti-gitr agonist antibodies

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