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WO2025147169A1 - Anticorps multispécifique pour dégradation lysosomale - Google Patents

Anticorps multispécifique pour dégradation lysosomale Download PDF

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Publication number
WO2025147169A1
WO2025147169A1 PCT/KR2025/000209 KR2025000209W WO2025147169A1 WO 2025147169 A1 WO2025147169 A1 WO 2025147169A1 KR 2025000209 W KR2025000209 W KR 2025000209W WO 2025147169 A1 WO2025147169 A1 WO 2025147169A1
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Prior art keywords
region
multispecific antibody
lysosomal
target protein
binding region
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Inventor
Jung-Eun Kim
Hyekang KIM
Soo-Bin PARK
Jungsoon YOON
Dong-Sik Kim
Sung Jun Lee
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GREEN CROSS Corp
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GREEN CROSS Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/53—Hinge
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—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
    • 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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the present invention relates to a multispecific antibody for the degradation of a target protein using lysosomes in the body and a method for producing the same.
  • bi- or multi-specific antibodies capable of simultaneously binding to two or more different antigens have also been developed.
  • Such antibodies capable of targeting multiple proteins like this have high potential for use in the treatment of diseases caused by the complex action of multiple proteins, and are expected to be used in various forms for the development of therapeutic agents (KR 10-2023-0163305 A).
  • Targeted protein degradation technology is a technology that directly degrades and eliminates disease-causing proteins, and can be expected to have a high therapeutic effect since the proteins that cause diseases are fundamentally eliminated.
  • PROTAC Proteolysis-Targeting Chimera
  • LYTAC Lisosome Targeting Chimera
  • AUTAC Autophagy-targeting Chimera
  • LYTAC is a degradation system in which LYTAC binds to both target protein and the M6P receptors on the cell membrane to form endosomes, and the endosomes mature to lysosomes, thereby target degrading proteins existing in the cell membrane and outside the cell, and has a form in which antibodies and M6P chains are connected.
  • LYTAC technology uses antibodies and has the advantage of high selectivity for target proteins, but has the problem that the synthetic process of linking sugar chains to antibodies is complex and takes a long time.
  • An object of the present invention is to provide a multispecific antibody comprising a lysosomal transporting receptor (LTR) binding region and a target protein binding region.
  • LTR lysosomal transporting receptor
  • Another object of the present invention is to provide a polynucleotide encoding the multispecific antibody.
  • Still another object of the present invention is to provide an expression vector comprising the polynucleotide.
  • Still another object of the present invention is to provide a cell transformed with an expression vector comprising the polynucleotide.
  • Still another object of the present invention is to provide a method for producing the multispecific antibody.
  • Still another object of the present invention is to provide a use of the multispecific antibody for the degradation of a target protein.
  • the multispecific antibody according to the present invention transports target proteins to lysosomes and induces degradation thereof, and can be used to treat diseases associated with the target proteins.
  • FIG. 1 is a diagram illustrating three types of multispecific antibodies constructed according to the present invention
  • FIG. 2 is a diagram illustrating the results that multispecific antibodies of different sequences containing the M6PR binding region are prepared as A and C type and their degradation efficacy for EGFR is evaluated;
  • FIG. 3 is a diagram comparing the EGFR degradation of a multispecific antibody including an M6PR binding region in the form of scFv and a multispecific antibody including an M6PR binding region in the form of an antibody VH domain.
  • An aspect embodying the present invention is a multispecific antibody comprising a lysosomal transporting receptor binding region and a target protein binding region.
  • the multispecific antibody comprises a lysosomal transporting receptor binding region, a target protein binding region, and an Fc region.
  • the lysosomal transporting receptor binding region and the target protein binding region are linked to the Fc region on the opposite side.
  • the lysosomal transporting receptor binding region includes a heavy chain variable region (VH or VHH) of an antibody against a lysosomal transporting receptor, a fragment thereof, or an antigen-binding site thereof
  • the target protein binding region includes a heavy chain variable region, a heavy chain constant region, a light chain constant region, and a light chain variable region
  • the lysosomal transporting receptor binding region and the target protein binding region are connected to different termini of the Fc region.
  • the lysosomal transporting receptor binding region is not a single-chain variable fragment (scFv).
  • the Fc region includes a hinge region.
  • the multispecific antibody has a structure in which monomers in which a lysosomal transporting receptor binding region, an Fc region, and a target protein binding region are connected symmetrically form a dimer.
  • the multispecific antibody symmetrically forms a dimer due to a disulfide bond in the hinge region.
  • the lysosomal transporting receptor binding region includes a heavy chain variable region (VH or VHH) of an antibody against a lysosomal transporting receptor, a fragment thereof, or an antigen-binding site thereof.
  • VH or VHH heavy chain variable region
  • the lysosomal transporting receptor binding region is connected to a C-terminus of the Fc region and the target protein binding region is connected to an N-terminus of the Fc region.
  • the Fc region includes a hinge region, the lysosomal transporting receptor binding region is connected to a C-terminus of the Fc region, and the target protein binding region is connected to an N-terminus of the Fc region.
  • the lysosomal transporting receptor binding region binds to mannose 6-phosphate receptor (M6PR) or amyloid precursor-like protein 2 (APLP2).
  • M6PR mannose 6-phosphate receptor
  • APLP2 amyloid precursor-like protein 2
  • the target protein binding region binds to a target protein.
  • the target protein is epidermal growth factor receptor (EGFR).
  • EGFR epidermal growth factor receptor
  • the lysosomal transporting receptor binding region and Fc region are connected via a linker.
  • the multispecific antibody induces endocytosis and degradation by a lysosome of a target protein.
  • Another aspect embodying the present invention is a multispecific antibody comprising an antibody or an antigen-binding fragment thereof that specifically binds to mannose 6-phosphate receptor (M6PR).
  • M6PR mannose 6-phosphate receptor
  • Still another aspect embodying the present invention is an expression vector comprising the polynucleotide.
  • Still another aspect embodying the present invention is a method for producing a multispecific antibody, comprising culturing the cell to express a multispecific antibody.
  • Still another aspect embodying the present invention is a composition comprising the multispecific antibody.
  • the composition is a pharmaceutical composition.
  • Still another aspect embodying the present invention is a use of the multispecific antibody for the degradation of a target protein.
  • An aspect embodying the present invention provides a multispecific antibody comprising a lysosomal transporting receptor binding region and a target protein binding region.
  • the multispecific antibody of the present invention has a form such as Type C in FIG. 1.
  • the lysosomal transporting receptor binding region and the target protein binding region are not connected to be adjacent to each other but are connected through a separate moiety.
  • the multispecific antibody of the present invention may comprise a lysosomal transporting receptor binding region, a target protein binding region, and an Fc region
  • the lysosomal transporting receptor binding region may include a heavy chain variable region (VH or VHH) of an antibody against a lysosomal transporting receptor
  • the target protein binding region may include a heavy chain variable region, a heavy chain constant region, a light chain constant region, and a light chain variable region
  • the lysosomal transporting receptor binding region and the target protein binding region may be connected to different termini of the Fc region.
  • the lysosomal transporting receptor binding region may consist of, but is not limited to, a VH or VHH domain.
  • the lysosomal transporting receptor binding region is not a single-chain variable fragment (scFv).
  • the multispecific antibody of the present invention may comprise:
  • a lysosomal transporting receptor binding region including or consisting of a heavy chain variable region (VH or VHH) of an antibody against a lysosomal transporting receptor;
  • VH or VHH heavy chain variable region
  • a target protein binding region including or consisting of a heavy chain variable region of an antibody against a target protein, a heavy chain constant region, a light chain constant region, and a light chain variable region;
  • the lysosomal transporting receptor binding region and the target protein binding region may be connected to different termini of the Fc region.
  • the multispecific antibody of the present invention may comprise:
  • a lysosomal transporting receptor binding region including or consisting of a heavy chain variable region (VH or VHH) of an antibody against a lysosomal transporting receptor;
  • VH or VHH heavy chain variable region
  • a target protein binding region including or consisting of a heavy chain variable region of an antibody against a target protein, a heavy chain constant region, a light chain constant region, and a light chain variable region;
  • the lysosomal transporting receptor binding region and the target protein binding region may be connected to different termini of the Fc region.
  • the multispecific antibody of the present invention may comprise:
  • a lysosomal transporting receptor binding region including or consisting of a heavy chain variable region (VH or VHH) of an antibody against a lysosomal transporting receptor;
  • VH or VHH heavy chain variable region
  • a target protein binding region including or consisting of a heavy chain variable region of an antibody against a target protein, a heavy chain constant region, a light chain constant region, and a light chain variable region;
  • the lysosomal transporting receptor binding region is connected to a C-terminus of the Fc region and the target protein binding region is connected to an N-terminus of the Fc region, and
  • dimer monomers in which the lysosomal transporting receptor binding region, the Fc region, and the target protein binding region are connected may symmetrically form a dimer.
  • the dimer may be due to a disulfide bond in the hinge region, but is not limited thereto.
  • the multispecific antibody of the present invention may have a structure such as Type C in FIG. 1, but is not limited thereto.
  • the multispecific antibody provided in the present invention comprises a site that specifically binds to a lysosomal transporting receptor and a site that specifically binds to a target protein to induce degradation of the target protein through lysosomes, and is thus capable of achieving degradation of a target protein in lysosomes.
  • the multispecific antibody of the present invention comprises a lysosomal transporting receptor binding region in the form of a VH or VHH domain, and has higher target protein degradation compared to a multispecific antibody comprising a lysosomal transporting receptor binding region in the form of a single-chain variable fragment (scFv), and this provides a more effective lysosomal transporting antibody-based multispecific antibody platform.
  • Type C bi-specific antibodies have the advantage of being more easily assembled when the antibodies are produced using wild-type Fc than Type A antibodies, having 2+2 valent for each target to bind to more antigens simultaneously than Type A antibodies having 1+1 valent, and being able to bind more strongly by the avidity effect.
  • antibody in the present invention refers to a protein molecule capable of specifically recognizing an antigenic site, including an immunoglobulin or a portion thereof that immunologically has reactivity with a specific antigen.
  • the antibody of the present invention includes all polyclonal antibodies, monoclonal antibodies, whole antibodies, and antibody fragments.
  • the antibody of the present invention includes mouse, human, rabbit, and rat antibodies, and the origin is not limited.
  • the antibody of the present invention includes chimeric antibodies (for example, humanized murine antibodies), humanized antibodies, and minibodies.
  • the antibody of the present invention additionally includes single-chain antibodies having a binding function to FcRn (neonatal Fc receptor), scaffolds, derivatives of antibody constant regions, and artificial antibodies based on protein scaffolds.
  • a whole antibody has a structure having two full-length light chains (LC) and two full-length heavy chains (HC), and each light chain may be connected to a heavy chain due to a disulfide bond.
  • the whole antibody includes IgA, IgD, IgE, IgM and IgG, and IgG includes IgG1, IgG2, IgG3 and IgG4 as subtypes.
  • Such an antibody may be produced by cloning each gene into an expression vector by a conventional method, obtaining a protein encoded by the marker gene, and producing the antibody from the obtained protein by a conventional method, but is not limited thereto.
  • the multispecific antibody of the present invention may have an IgG-like structure and additionally include a target protein binding region based on the structure of IgG, but is not limited thereto.
  • fragment or "antibody fragment” means an arbitrary part of an antibody, and the antibody fragment may be scFv, dsFv, Fab, Fab', F(ab')2, Fc, Fd, sdAb, and nanobody, and combinations thereof, and the antibody fragment may include, but is not limited to, an antigen-recognizing site.
  • antigen-binding site or "antigen-binding site fragment” in the present invention means a fragment possessing an antigen binding function, and the antigen here may be a lysosomal transporting receptor or a target protein.
  • the antigen-binding site may be a fragment including a site capable of recognizing an antigenic site.
  • immunoglobulins have a heavy chain and a light chain, and each heavy chain and each light chain includes a constant region and a variable region.
  • the variable regions of the light chain and heavy chain include three variable regions called complementarity determining regions (CDRs) and four framework regions (FRs).
  • the CDRs mainly play a role in binding to the antigenic determinant of an antigen.
  • the CDRs of each chain are typically called sequentially CDR1, CDR2, and CDR3, starting from the C-terminus, and are also identified by the chain on which a particular CDR is located.
  • the complementarity determining regions are located between relatively conserved regions, called constant regions (FR).
  • Each VH and each VL consist of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy chain and light chain contain binding domains that interact with an antigen.
  • the CDRs of the heavy chain variable region may be referred to as CDR-H1, CDR-H2, and CDR-H3, the CDRs of the light chain variable region may be referred to as CDR-L1, CDR-L2, and CDR-L3, the FRs of the heavy chain variable region may be referred to as HFR1, HFR2, HFR3, and HFR4, and the FRs of the light chain variable region may be referred to as LFR1, LFR2, LFR3, and LFR4.
  • the heavy or light chain variable region may be in the form of a domain including three CDRs, but is not limited thereto.
  • the "multispecific antibody” of the present invention means an antibody having binding specificity to two or more different antigens.
  • the multispecific antibody may be in a form that does not exist in nature and is produced by genetic engineering or an arbitrary method.
  • the multispecific antibody of the present invention may include a bi-specific or higher antibody, a tri-specific or higher antibody, for example, bi-specific antibodies, tri-specific antibodies, tetra-specific antibodies, or antibodies targeting more than four targets.
  • the multispecific antibody of the present invention may simultaneously bind to a lysosomal transporting receptor and one or more, two or more, or three or more target proteins, but is not limited thereto.
  • the multispecific antibody of the present invention may be, but is not limited to, a bi-specific antibody comprising a target protein binding region that can bind to a lysosomal transporting receptor and one target protein.
  • bi-specific antibody means a protein capable of binding to two different kinds of antigens.
  • the bi-specific antibody of the present invention may bind to a target protein (EGFR or the like) and a lysosomal transporting receptor (APLP2, M6PR) that transports the target protein to lysosomes.
  • a target protein EGFR or the like
  • APLP2, M6PR lysosomal transporting receptor
  • bi-specific antibody of the present invention may be used interchangeably with the terms “dual targeting antibody”, “dual antibody” or “dual antibody protein”.
  • the bi-specific antibody of the present invention may have a lysosomal transporting receptor (for example, APLP2 or M6PR) and a target protein as antigens.
  • the form of the bi-specific antibody of the present invention is not particularly limited thereto, but includes a form constructed based on an IgG form.
  • the bi-specific antibody is named "Lportbody”.
  • the bi-specific antibody refers to a molecule of which antigen-binding sites are connected directly or via a linker or can form a heterodimer by electrostatic interaction.
  • the “valent” refers to the presence of a specified number of antigen-specific binding sites in a molecule. Therefore, the terms “monovalent”, “bivalent”, “tetravalent”, and “hexavalent” refer to the presence of one, two, four, and six binding sites, respectively, specific to antigens in a molecule.
  • the bi-specific antibody according to the present invention may be a bi-specific antibody having a domain that specifically binds to a lysosomal transporting receptor and a domain that specifically binds to a target protein, the domains that specifically bind to the respective antigens are typically VH/VL pairs or VH(H) domains, and bi-specific antibodies may be divided into monovalent or bivalent depending on the VH/VL pair or VH(H) domain that binds to each antigen.
  • the bi-specific antibody according to the present invention comprises a lysosomal transporting receptor binding region and a target protein binding region, the lysosomal transporting receptor binding region may include a VH(H) domain, and the target protein binding region may include a VH(H) domain.
  • lysosomal transporting receptor refers to a receptor that functions to transport a target substance to a lysosome and degrade the target substance, and in the present invention, plays a role in transporting a target protein to a lysosome by binding to the multispecific antibody according to the present invention.
  • the lysosomal transporting receptor according to the present invention may be recycled back to the cell surface after endocytosis or degraded together with a target protein, and may be expressed in various tissues and used as a therapeutic agent for various diseases.
  • the lysosomal transporting receptor of the present invention may be, but is not limited to, mannose 6-phosphate receptor (M6PR) or amyloid precursor-like protein 2 (APLP2).
  • lysosomal transporting receptor binding region means a site in the multispecific antibody according to the present invention that can specifically bind to a lysosomal transporting receptor, and may specifically mean a site capable of binding to M6PR and/or APLP2, but is not limited thereto.
  • the lysosomal transporting receptor binding region may include, but is not limited to, a substance capable of specifically binding to a lysosomal transporting receptor, for example, an antibody, a variable region including an antigen (lysosomal transporting receptor) binding site of the antibody or a fragment thereof, an antigen-binding site, a peptide capable of specifically binding to a lysosomal transporting receptor, a nanobody, a single domain antibody, a nucleic acid, and an aptamer.
  • a substance capable of specifically binding to a lysosomal transporting receptor for example, an antibody, a variable region including an antigen (lysosomal transporting receptor) binding site of the antibody or a fragment thereof, an antigen-binding site, a peptide capable of specifically binding to a lysosomal transporting receptor, a nanobody, a single domain antibody, a nucleic acid, and an aptamer.
  • the lysosomal transporting receptor binding region of the present invention may include a heavy chain variable region.
  • the lysosomal transporting receptor binding region of the present invention may include, but is not limited to, a heavy chain variable region (VH or VHH) of an antibody against a lysosomal transporting receptor, a fragment thereof, or an antigen-binding site thereof.
  • VH or VHH heavy chain variable region
  • the lysosomal transporting receptor binding region comprised in the multispecific antibody of the present invention may be, but is not limited to, a human-derived VH domain or an animal-derived VHH domain such as a camelid of the heavy chain variable region of an antibody against a lysosomal transporting receptor.
  • a single domain may be a heavy chain variable region having three CDRs.
  • the multispecific antibody of the present invention comprises a lysosomal transporting receptor binding region in the form of a domain and has superior target protein degradation compared to a multispecific antibody comprising a lysosomal transporting receptor binding region in the form of a single-chain variable fragment (scFv).
  • scFv single-chain variable fragment
  • the multispecific antibody of the present invention has a structure in which the lysosomal transporting receptor binding region and the target protein binding region are not adjacently connected, and has a separate moiety mediating the two binding regions. Such a moiety may be an Fc region. Therefore, in the multispecific antibody of the present invention, the target protein binding region-Fc region-lysosomal transporting receptor binding region may be connected in this order.
  • the term "Fc region” refers to a site including the heavy chain constant region 2(CH2) and/or the heavy chain constant region 3(CH3) portion, excluding the heavy and light chain variable regions of an immunoglobulin.
  • the "Fc region” may be used interchangeably with “immunoglobulin Fc region”.
  • the Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, or IgM or by a combination thereof or a hybrid thereof, but is not limited thereto.
  • the immunoglobulin Fc region is a natural immunoglobulin Fc region, or a variant in which at least one or more amino acids have undergone variation selected from the group consisting of substitution, addition, deletion, modification, or a combination thereof, and a variant may be included in the present invention without limitation as long as it exhibits biological activity equivalent to that of the natural immunoglobulin Fc.
  • Such an Fc region may be obtained from a natural form isolated from the living body of animals such as humans, cows, goats, pigs, mice, rabbits, hamsters, rats or guinea pigs, or may be a recombinant obtained from a transformed animal cell or microorganism or a derivative thereof.
  • the method for obtaining the Fc region from a natural form may be a method in which the entire immunoglobulin is isolated from the living body of a human or animal and then treated with a protease. The entire immunoglobulin is cleaved into Fab and Fc when treated with papain and is cleaved into pF'c and F(ab)2 when treated with pepsin. This may be subjected to size-exclusion chromatography or the like to separate Fc or pF'c.
  • the immunoglobulin Fc region of the present invention may be a monomer or may be a dimer formed to include a hinge region in the heavy chain constant region, but is not limited thereto.
  • the immunoglobulin Fc region of the monomer may include an amino acid sequence of SEQ ID NO: 17, but is not limited thereto.
  • the term "hinge region” refers to a site located in the heavy chain to form a dimer of the immunoglobulin Fc region through an inter disulfide bond.
  • the multispecific antibody of the present invention may comprise 1) a CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, or 5) a combination of one or more of a CH1 domain, a CH2 domain, a CH3 domain or a CH4 domain with an immunoglobulin hinge region (or a part of the hinge region), but is not limited thereto.
  • the Fc region may include, but is not limited to, a hinge region, a CH2 domain, and a CH3 domain.
  • the Fc regions may form a dimer with each other due to a disulfide bond in the hinge region included in the Fc region. More specifically, the multispecific antibody may be, but is not limited to, a dimer formed symmetrically by monomers in which a lysosomal transporting receptor binding region, an Fc region, and a target protein binding region are connected to each other.
  • the hinge region may include an amino acid sequence of SEQ ID NO: 18, but is not limited thereto.
  • target protein binding region in the present invention refers to a region that binds to a target protein to be degraded by the multispecific antibody of the present invention, and may include a target protein binding site that can bind to a target protein. Specifically, the target protein binding region of the present invention may specifically bind to a target protein, but is not limited thereto.
  • the target protein binding region of the present invention may include, but is not limited to, a target protein binding site that can bind to a target protein.
  • the target protein binding site include, but are not limited to, an antibody capable of binding to a target protein, a variable region or fragment thereof including an antigen (target protein) binding site of the antibody, an antigen-binding site, a peptide capable of specifically binding to a target protein, a nanobody, a single domain antibody, a nucleic acid, and an aptamer.
  • the target protein binding region of the present invention may include, but is not limited to, a heavy chain variable region of an antibody against a target protein, a heavy chain constant region, a light chain constant region, and a light chain variable region.
  • the multispecific antibody of the present invention may comprise two or more target protein binding regions that can specifically bind to two or more target proteins. Specifically, when there are two or more target proteins, the multispecific antibody may comprise two or more target protein binding regions that can specifically bind to the respective target proteins, and the respective target protein binding regions may be connected to each other and connected to the N-terminus of the Fc region, but the multispecific antibody is not limited.
  • the lysosomal transporting receptor binding region and the target protein binding region are connected to different termini of the Fc region.
  • the multispecific antibody of the present invention may be a multispecific antibody in which the lysosomal transporting receptor binding region includes a VH or VHH domain (lysosomal transporting receptor binding region in the form of a domain), the target protein binding region includes a heavy chain variable region, a heavy chain constant region, a light chain constant region and a light chain variable region, and the lysosomal transporting receptor binding region and the target protein binding region are connected to the C-terminus and N-terminus of the Fc region, respectively.
  • the lysosomal transporting receptor binding region includes a VH or VHH domain (lysosomal transporting receptor binding region in the form of a domain)
  • the target protein binding region includes a heavy chain variable region, a heavy chain constant region, a light chain constant region and a light chain variable region
  • the lysosomal transporting receptor binding region and the target protein binding region are connected to the C-terminus and N-terminus of the Fc region, respectively.
  • Such a multispecific antibody comprising a lysosomal transporting receptor binding region in the form of a domain has superior target protein degradation compared to a multispecific antibody comprising a lysosomal transporting receptor binding region that is not in the form of a domain but in the form of a single-chain variable fragment (scFv).
  • scFv single-chain variable fragment
  • the multispecific antibody of the present invention in which the lysosomal transporting receptor binding region and the target protein binding region are connected to different termini of the Fc region, is referred to as a "Type C multispecific antibody” or a C-type “Lportbody”.
  • Type C multispecific antibody or a C-type “Lportbody”.
  • An example of this includes, but is not limited to, Type C in FIG. 1.
  • the lysosomal transporting receptor binding region may be connected to the C-terminus of the Fc region, the target protein binding region may be connected to the N-terminus of the Fc region, and the Fc region may include a hinge region at the N-terminus.
  • a lysosomal transporting receptor binding region in the form of a domain may be connected to the C-terminus of the Fc region, which includes the hinge region-CH2 domain-CH3 domain from the N-terminus, and a target protein binding region including a heavy chain variable region, a heavy chain constant region, a light chain constant region, and a light chain variable region may be connected to the N-terminal hinge region of the Fc region, but the multispecific antibody is not limited thereto.
  • the lysosomal transporting receptor binding region, the Fc region, and the target protein binding region may be directly connected or connected via a linker.
  • the lysosomal transporting receptor binding region and the Fc region and the Fc region and the target protein binding region may be directly connected or connected via a linker or may additionally include another protein moiety, but the connection is not limited thereto.
  • any method and any location known in the art may be used without limitation as long as the structure or activity of the protein being connected is not altered.
  • the Fc region and the target protein binding region may be directly connected by a covalent bond, but are not limited thereto.
  • the lysosomal transporting receptor binding region and the Fc region of the present invention may be connected via a linker, but are not limited thereto.
  • any linker may be included in the present invention without limitation as long as it is a peptide linker or non-peptide linker used in the art.
  • the peptide linker may include one or more amino acids, for example, 1 to 1000 amino acids, specifically 1 to 100 amino acids, more specifically 1 to 50 amino acids, but is not particularly limited thereto.
  • the peptide linker according to the present invention may be a peptide linker composed of glycine (G), serine (S) and threonine (T), and may have a sequence of GGGGSGGGGT (SEQ ID NO: 1), GGGGSGGGGS (SEQ ID NO: 2), GGGGSGGGGSGGT (SEQ ID NO: 3), or GGGGSGGGGSGGGGS (SEQ ID NO: 4) as a specific example, but is not limited thereto.
  • the peptide linker may include any peptide linker known in the art, for example, a [GS]x linker, a [GGGS]x linker, and a [GGGGS]x linker, where x may be a natural number 1 or more (for example, 1, 2, 3, 4, 5, or more).
  • the peptide linker of the present invention may include, but is not limited to, a site recognizable by a restriction enzyme (for example, the KpnI enzyme).
  • the non-peptide linker is not limited in kind as long as it can connect the heavy chain variable region and the target protein binding region.
  • the non-peptide linker may be selected from, but is not limited to, lipid polymers, biodegradable polymers, chitins, or oligonucleotides.
  • the multispecific antibody of the present invention may bind to a lysosomal transporting receptor and a target protein, respectively, to induce endocytosis and degradation by a lysosome of the target protein, but is not limited thereto.
  • Still another aspect of the present invention provides a multispecific antibody comprising an antibody or an antigen-binding fragment thereof that specifically binds to mannose 6-phosphate receptor (M6PR).
  • M6PR mannose 6-phosphate receptor
  • the antibody or antigen-binding fragment thereof may include, but is not limited to, the following complementarity determining regions (CDRs):
  • CDR-H1 including an amino acid sequence of SEQ ID NO: 11
  • CDR-H2 including an amino acid sequence of SEQ ID NO: 12
  • CDR-H3 including an amino acid sequence of SEQ ID NO: 13;
  • CDR-H1, CDR-H2, and CDR-H3 sequentially represent the CDRs included in the heavy chain variable region.
  • the polynucleotide encoding the multispecific antibody of the present invention can be readily isolated and sequenced using conventional procedures.
  • the polynucleotide encoding the multispecific antibody of the present invention may be, but is not limited to, codon optimized.
  • codon optimized codon optimized.
  • a polynucleotide sequence of which a part is deleted, modified, substituted, conservatively substituted or added is also included within the scope of the polynucleotide of the present invention as long as it is a polynucleotide sequence capable of encoding the sequence of the multispecific antibody according to the present invention or a polypeptide having homology or identity therewith.
  • Still another aspect embodying the present invention provides a vector comprising a polynucleotide encoding the multispecific antibody.
  • Still another aspect embodying the present invention provides a cell comprising the multispecific antibody of the present invention; a polynucleotide encoding the same; or a vector encoding the polynucleotide; or a cell transformed with the vector.
  • the expression vector comprising a polynucleotide encoding the multispecific antibody may be an expression vector that includes a polynucleotide encoding the lysosomal targeting receptor binding region of the multispecific antibody and a polynucleotide encoding the target protein binding region of the multispecific antibody, but is not limited thereto.
  • the pharmaceutical composition according to the present invention may be applied to different target diseases and patients depending on the kind of target protein targeted by the multispecific antibody. Those skilled in the art can determine the multispecific antibody by appropriately selecting the target protein and target protein binding region depending on the disease to be treated and the patient. As an example, the pharmaceutical composition of the present invention may be a pharmaceutical composition for preventing or treating cancer, but is not limited thereto.
  • the multispecific antibody of the present invention can function in various tissues and has versatility.
  • compositions to be prepared as liquid solutions saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these ingredients may be used as a sterile and biocompatible substance, and other common additives such as antioxidants, buffers, and bacteriostatic agents may be added if needed.
  • the pharmaceutical composition may be prepared as an injectable formulation such as an aqueous solution, suspension, or emulsion, pills, capsules, granules, or tablets by additionally adding diluents, dispersants, surfactants, binders and lubricants.
  • injectable formulation such as an aqueous solution, suspension, or emulsion
  • pills, capsules, granules, or tablets by additionally adding diluents, dispersants, surfactants, binders and lubricants.
  • the pharmaceutical composition may be various oral or parenteral formulations.
  • the preparation is prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants usually used.
  • Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations are prepared by mixing one or more compounds with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, and gelatin.
  • lubricants such as magnesium stearate and talc are also used.
  • Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and various excipients, such as humectants, sweeteners, flavoring agents, and preservatives, may be contained in addition to the commonly used simple diluents, such as water and liquid paraffin.
  • Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories, and propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like may be used as non-aqueous solvents and suspending agents.
  • suppository bases Witepsol, Macrogol, Tween 61, cocoa butter, laurin butter, and glycerogelatin may be used.
  • the pharmaceutical composition may have any one formulation selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral liquids, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories.
  • Still another aspect of the present invention provides a method for producing a multispecific antibody, comprising culturing a cell that comprises any one or more of the multispecific antibody of the present invention; a polynucleotide encoding the same; or a vector comprising the polynucleotide or is transformed with a vector comprising the polynucleotide in a medium.
  • a method for producing a multispecific antibody comprising culturing a cell that comprises any one or more of the multispecific antibody of the present invention; a polynucleotide encoding the same; or a vector comprising the polynucleotide or is transformed with a vector comprising the polynucleotide in a medium.
  • the culture process, medium, culture temperature, pH and the like may be appropriately controlled by suitable methods known in the art.
  • the multispecific antibody, polynucleotide, vector, cell, and transformation are as described previously.
  • the method for producing a multispecific antibody of the present invention may comprise preparing the cell and preparing a medium for culturing the cell.
  • the method for producing a multispecific antibody of the present invention may further comprise recovering the multispecific antibody from the medium or cell.
  • the method for producing a multispecific antibody of the present invention may further comprise purifying the multispecific antibody.
  • the purifying step may be performed by any one or more of column chromatography, affinity chromatography, anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, reversed phase column chromatography, ultrafiltration, diafiltration, salting out (examples: ammonium sulfate precipitation and sodium phosphate precipitation), solvent precipitation (examples: protein fraction precipitation using acetone, ethanol and the like), dialysis, or gel filtration, but is not limited thereto.
  • the purifying step may be performed two or more times if necessary, and purification may be performed by different methods in the respective purification steps.
  • Still another aspect of the present invention provides a use of the multispecific antibody for the degradation of a target protein.
  • the multispecific antibody is as described previously.
  • lysosomal transporting receptors As lysosomal transporting receptors (LTRs), APLP2 and M6PR that were systemically expressed and capable of transporting target proteins to lysosomes were selected.
  • EGFR a cell membrane protein overexpressed in cancer cells
  • TNF ⁇ a water-soluble protein
  • the multispecific antibodies according to the present invention were constructed into Types A, B, and C having three different characteristics (FIG. 1).
  • Type A has an asymmetric structure, containing one binding region for each target.
  • Ajou University KiH technology in which antibody HC mutations were introduced, was used (Choi, et al. (2015). Mol Immunol, 65:377-83.).
  • LC light chain is composed of Fab and VH (H) domains.
  • Type B and Type C have a symmetric structure based on wild-type Fc. Types B and C are similar in that they contain two binding regions for each target, but differ in the directionality of the binding regions (proximal or distal).
  • Example 2-1 Construction of Type A multispecific antibodies
  • PBS buffer was added into a viva spin tube (30K MWCO, Sartorious), and the membrane was equilibrated by centrifugation at 3500 rpm for 5 minutes.
  • PBS and the eluted sample in the first purification process were added together in the same volume, centrifugation was performed at 3500 rpm for 10 minutes, and the sample was repeatedly concentrated to be about 2 mL, and collected.
  • gel filtration the Hiload 16/600 superdex 200 column (Cytiva) was equilibrated with PBS buffer in a volume to be 1 time (120 mL) or more the column volume at a flow rate of 1.0 ml/min using the AKTA prime system.
  • the concentrated sample was allowed to pass through the column, and the profile of FPLC in the sample, containing the targets to be separated by size, was analyzed.
  • the profile curve observed at a wavelength of 280 nm in the gel filtration was analyzed and the peak corresponding to the target was fractionated by comparison with the reference molecular size.
  • the fractions of the first and second purification processes were confirmed using SDS-PAGE.
  • the sample and the 4x reducing sample buffer were mixed at a 3 : 1 volume ratio, heated at 90°C for 5 minutes, and then cooled, then the prepared sample was loaded by 10 ⁇ l per well on a 4% to 12% Bis-Tris gel, and electrophoresis was performed at 130 V for 60 minutes.
  • Expi293F cells were prepared at a concentration of 3 ⁇ 10 6 cells/mL in the Expi293 expression medium (37°C, 8% CO 2 , 125 rpm, viability ⁇ 95%). These cells were transformed with 1 ⁇ g of DNA per 1 ml according to the manufacturer's protocol.
  • the expressed supernatant was filtered and purified using Protein A PhyTip Columns (Biotage). Bound antibodies were eluted using an amine-based elution buffer at pH 2.8 or a sodium citrate elution buffer at pH 3.5. The eluted solution was neutralized by adding 1 M Tris-HCL solution at pH 8.8. The concentration was measured at a wavelength of 280 nm, and the purity was determined using SE-HPLC. As a result, a sample having a purity of 70% or more based on the main peak was used.
  • the Type C multispecific antibodies produced at this time are multispecific antibodies in which the lysosomal transporting receptor binding region includes a VH or VHH domain (lysosomal transporting receptor binding region in the form of a domain) and the target protein binding region includes a heavy chain variable region, a heavy chain constant region, a light chain constant region, and a light chain variable region.
  • the lysosomal transporting receptor binding region and the target protein binding region are connected on opposite sides based on the heavy chain constant region.
  • the sequence of the Fc region of the monomer used in the multispecific antibodies of the present invention is SEQ ID NO. 17, the sequence of the hinge region is SEQ ID NO. 18, and the sequences of the heavy chain constant region and light chain constant region are SEQ ID NOs: 19 and 20, respectively.
  • Example 11 Comparison of EGFR degradation of M6PR-mediated A/C-type Lportbodies
  • A/C-type Lportbody antibodies were produced using the clone sequences identified in Example 9, and the EGFR degradation thereof was examined.
  • the DLD-1 cell line was adjusted to 2 ⁇ 10 5 cells/300 ⁇ L in a culture medium (RPMI1640 (Gibco, A10491-01) supplemented with 10% fetal bovine serum (Gibco, 16000-044) and anti-anti 1X), dispensed into a 6-well plate, cultured for 24 hours, and then treated with the Lportbody clone.
  • the concentrations of the test substances were all 10 nM and the test substance was mixed with the culture medium for treatment.
  • Example 12 Comparison of EGFR degradation of C-type Lportbodies depending on form of lysosomal transporting receptor binding region
  • the EGFR degradation of the multispecific antibody of the present invention having a lysosomal transporting receptor binding region that binds to M6PR while having a VHH domain form and a multispecific antibody having a lysosomal transporting receptor binding region in the form of scFv was examined (FIG. 3).
  • the multispecific antibodies of the present invention had superior EGFR degradation compared to multispecific antibodies (Lytac-3C7, Lytac-6D1) having a lysosomal transporting receptor binding region in the form of scFv.
  • the Type C form of the multispecific antibody of the present invention has excellent ability to bind to both a lysosomal transporting receptor and a target protein, in particular, a multispecific antibody having a lysosomal transporting receptor binding region that binds to a lysosomal transporting receptor in the form of a domain has remarkably superior target protein degradation. This suggests that the multispecific antibody platform of the present invention can be utilized to develop therapeutic agents through degradation of target proteins.

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Abstract

Un anticorps multispécifique de la présente invention permet la dégradation d'une protéine cible d'intérêt dans des lysosomes et peut être utilisé dans le développement d'agents thérapeutiques.
PCT/KR2025/000209 2024-01-05 2025-01-06 Anticorps multispécifique pour dégradation lysosomale Pending WO2025147169A1 (fr)

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Citations (4)

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WO2020132100A1 (fr) * 2018-12-19 2020-06-25 The Board Of Trustees Of The Leland Stanford Junior University Molécules bi-fonctionnelles pour le ciblage des lysosomes, compositions et méthodes associées
WO2020185069A1 (fr) * 2019-03-08 2020-09-17 Linxis B.V. Internalisation de molécules de liaison ciblant des récepteurs impliqués dans la prolifération cellulaire ou la différenciation cellulaire
WO2022271981A2 (fr) * 2021-06-23 2022-12-29 Lycia Therapeutics, Inc. Composés bifonctionnels contenant des polypeptides d'igf-2
WO2023016828A2 (fr) * 2021-07-30 2023-02-16 Vib Vzw Liants du récepteur mannose-6-phosphate indépendants des cations pour la dégradation ciblée de protéines

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JP7328990B2 (ja) * 2018-04-30 2023-08-17 リジェネロン・ファーマシューティカルズ・インコーポレイテッド Ｈｅｒ２及び／またはａｐｌｐ２に結合する抗体及び二重特異性抗原結合分子、ならびにそれらのコンジュゲート及び使用

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WO2020132100A1 (fr) * 2018-12-19 2020-06-25 The Board Of Trustees Of The Leland Stanford Junior University Molécules bi-fonctionnelles pour le ciblage des lysosomes, compositions et méthodes associées
WO2020185069A1 (fr) * 2019-03-08 2020-09-17 Linxis B.V. Internalisation de molécules de liaison ciblant des récepteurs impliqués dans la prolifération cellulaire ou la différenciation cellulaire
WO2022271981A2 (fr) * 2021-06-23 2022-12-29 Lycia Therapeutics, Inc. Composés bifonctionnels contenant des polypeptides d'igf-2
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