WO2024091079A1 - Protéine de fusion perméable à la barrière hémato-encéphalique et ses utilisations - Google Patents

Protéine de fusion perméable à la barrière hémato-encéphalique et ses utilisations Download PDF

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WO2024091079A1
WO2024091079A1 PCT/KR2023/016928 KR2023016928W WO2024091079A1 WO 2024091079 A1 WO2024091079 A1 WO 2024091079A1 KR 2023016928 W KR2023016928 W KR 2023016928W WO 2024091079 A1 WO2024091079 A1 WO 2024091079A1
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blood
fusion protein
brain
brain barrier
antibody
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Korean (ko)
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김한주
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주식회사 아임뉴런
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • the present invention relates to a blood-brain barrier permeable fusion protein and its use, and relates to a fusion protein comprising an IgG antibody and a helical region binding moiety of a transferrin receptor linked to the terminus of the IgG antibody, and its medicinal use.
  • This patent application claims priority to Korean Patent Application No. 10-2022-0141617 filed with the Korean Intellectual Property Office on October 28, 2022, the disclosure of which is incorporated herein by reference.
  • the blood-brain barrier is a blood vessel barrier that isolates the brain and blood, and serves to isolate the central nervous system, including the brain, from potentially dangerous substances in the blood.
  • the blood-brain barrier is a cerebrovascular-centered structure composed of cells such as brain endothelial cells, astrocytes, and pericytes, and is distributed throughout the brain blood vessels within brain tissue. there is.
  • the endothelial cells of the blood-brain barrier are tightly bound together by tight junctions, and astrocytes and end feet surround the blood vessels, allowing substances flowing along the blood inside the brain blood vessels to penetrate the blood vessel barrier. This forms a barrier that selectively inhibits absorption/delivery into brain tissue.
  • This barrier-like structure selectively allows or inhibits penetration of substances depending on the type and size of the substance.
  • substances In the case of water and oxygen, which are essential for maintaining life, they can pass through the blood-brain barrier by diffusion, and energy
  • they In the case of amino acids or glucose used as sources, they can be transferred from blood to brain tissue by active transport.
  • the transport of toxic substances and pathogens that can potentially affect the brain is not only inhibited by the blood-brain barrier, but even if they penetrate, they are returned to the blood through the pumping action of cells and transferred to brain tissue. It plays a role in protecting brain tissue by preventing its absorption.
  • the blood-brain barrier with this structure acts as a major obstacle in the pharmacological treatment of diseases associated with brain dysfunction, such as Alzheimer's disease, Parkinson's disease, and brain cancer.
  • diseases associated with brain dysfunction such as Alzheimer's disease, Parkinson's disease, and brain cancer.
  • the present inventors designed a fusion protein in which an IgG antibody and a helical region binding moiety of a tetravalent transferrin receptor are linked to the terminal of the IgG antibody.
  • the fusion protein has improved permeability to the blood-brain barrier and improved delivery efficiency to brain tissue due to the specific transferrin receptor cluster expression pattern in the cerebrovascular region and the specific interaction with the cluster in the cerebrovascular region.
  • IgG antibodies were selectively distributed at a high level in brain tissue compared to other tissues.
  • one aspect includes IgG antibodies; And a blood-brain barrier permeable fusion protein containing a helical region binding moiety of a tetravalent transferrin receptor (TfR) linked to the C-terminal region of the light chain and the C-terminal region of the heavy chain of the IgG antibody as an active ingredient.
  • a pharmaceutical composition for preventing or treating diseases associated with brain dysfunction including:
  • One aspect is an IgG antibody; And a blood-brain barrier permeable fusion protein containing a helical region binding moiety of a tetravalent transferrin receptor (TfR) linked to the C-terminal region of the light chain and the C-terminal region of the heavy chain of the IgG antibody as an active ingredient.
  • a pharmaceutical composition for preventing or treating diseases associated with brain dysfunction including:
  • Another aspect includes medicinal use of the pharmaceutical composition for the prevention or treatment of diseases associated with brain dysfunction; Alternatively, it provides a method of treating a disease associated with brain dysfunction, comprising administering the pharmaceutical composition to a subject.
  • BBB Blood-brain barrier
  • blood-brain barrier refers to strictly controlling the movement of substances such as ions, molecules, and pathogens present in the blood into brain tissue, and maintaining life-sustaining substances such as amino acids and glucose. It refers to a structure centered on the brain blood vessels that enables selective absorption of essential ingredients.
  • the blood-brain barrier is composed of cells such as blood-brain barrier endothelial cells, astrocytes, and pericytes. The cells are arranged in a form that shares a common basement membrane, and on one side of the blood-brain barrier, tightly connected endothelial cells are distributed, and on the other side, astrocytes surrounding blood vessels are distributed.
  • the blood-brain barrier endothelial cells make up the walls of capillaries and are connected by very strong and complex tight junctions.
  • This structure forms a physical barrier that inhibits the simple diffusion of most substances, including average to large sized molecules such as insulin.
  • astrocytes are a type of glial cell of the central nervous system and affect brain endothelial function, blood flow, and ionic balance through interactions with brain blood vessels. Astrocytes use processes called end feet to surround blood vessels at one end and come into close contact with neurons at the synapse at the other end.
  • the blood-brain barrier with this structure not only protects brain tissue from potentially dangerous substances in the blood, but also prevents substances effective in treating diseases from being delivered to brain tissue, acting as a major obstacle in pharmacological treatment. do. Therefore, technology that improves permeability to the blood-brain barrier can improve the applicability or efficacy of therapeutic agents in the field of medicine targeting brain tissue.
  • the term “IgG antibody” refers to an immunoglobulin molecule that is immunologically reactive with a specific antigen, and refers to a protein molecule that specifically recognizes the antigen and serves as a receptor for the antigen.
  • the antibody has heavy and light chains, with each heavy and light chain comprising a constant region and a variable region.
  • the variable regions of the light and heavy chains include three variable regions called complementarity-determining regions (CDRs) and four framework regions.
  • CDR mainly functions to bind to the epitope of the antigen.
  • the CDRs of each chain are typically called CDR1, CDR2, and CDR3 sequentially starting from the N-terminus, and are also identified by the chain on which a particular CDR is located.
  • the antibody may include polyclonal antibodies, monoclonal antibodies, full-length antibodies, and antibody fragments or antigen-binding fragments containing an antigen-binding domain.
  • a full-length antibody is structured with two full-length light chains and two full-length heavy chains, with each light chain connected to the heavy chain by a disulfide bond.
  • the full-length antibody may preferably be IgG, and its subtypes may include IgG1, IgG2, IgG3, and IgG4.
  • the antibody may be a natural antibody or a recombinant antibody.
  • Natural antibodies refer to antibodies that have not been genetically modified, and the risk of immunogenicity that genetically modified antibodies may have in vivo may be significantly lower.
  • Recombinant antibodies refer to antibodies that have been genetically engineered, and have the ability to add antigen binding capacity or desired characteristics through genetic manipulation.
  • antibody fragment refers to a fragment that possesses an antigen-binding function, including Fab, F(ab'), F(ab') 2 , Fv, single chain Fv (scFv) ), and functional antibody fragments such as scFv-Fc bivalent molecules, or combinations thereof, etc.
  • the antigen-binding fragment may include, but is not limited to, the following, and structures of IgG-like bispecific antibodies known in the art may be employed without limitation: (1) Fab, antibody molecule A fragment comprising a monovalent antigen-binding fragment of and which can be produced by digesting the entire antibody with the enzyme papain to yield portions of a crude light chain and one heavy chain; (2) Fab', a fragment of the antibody molecule that can be obtained by treating the whole antibody with pepsin followed by reduction to yield portions of the crude light and heavy chains; Two Fab' fragments obtained per antibody molecule; (3) (Fab') 2 , a fragment of the antibody that can be obtained by treating the whole antibody with the enzyme pepsin without subsequent reduction and a dimer of two Fab' fragments joined together by two disulfide bonds; (4) Fv, a genetically engineered fragment comprising the variable region of the light chain and the variable region of the heavy chain expressed as two chains; (5) single chain antibodies (SCA or scFv), genetically engineered fragment
  • the antibody or antibody fragment is an immunoglobulin molecule possessing an antigen-binding function, such as a monoclonal antibody, multispecific antibody, human antibody, humanized antibody, mouse antibody, chimeric antibody, scFV, single chain antibody, Fab fragment, F (ab') fragment, F(ab') 2 , Disulfide-linked Fvs (sdFV), scFv fragments, scFv-Fc fragments, Fv fragments, diabodies, triabodies, tetrabodies, etc.
  • an antigen-binding function such as a monoclonal antibody, multispecific antibody, human antibody, humanized antibody, mouse antibody, chimeric antibody, scFV, single chain antibody, Fab fragment, F (ab') fragment, F(ab') 2 , Disulfide-linked Fvs (sdFV), scFv fragments, scFv-Fc fragments, Fv fragments, diabodies, triabodies
  • Fab has a structure that includes the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region (CH1) of the heavy chain, and has one antigen binding site.
  • Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain.
  • the F(ab') 2 antibody is produced when the cysteine residue in the hinge region of Fab' forms a disulfide bond.
  • Fv is the smallest antibody fragment containing only the heavy chain variable region and light chain variable region.
  • Two-chain Fv two-chain Fv (two-chain Fv) consists of a heavy chain variable region and a light chain variable region connected by a non-covalent bond.
  • antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of the whole antibody with papain, and F(ab') 2 fragment can be obtained by digestion with pepsin), and gene It can also be produced through recombinant technology.
  • proteolytic enzymes for example, Fab can be obtained by restriction digestion of the whole antibody with papain, and F(ab') 2 fragment can be obtained by digestion with pepsin
  • gene It can also be produced through recombinant technology.
  • the IgG antibody is an object that forms a fusion with the helical region binding moiety of the transferrin receptor, and each of the light chain C-terminus and the heavy chain C-terminus constituting the IgG antibody is connected to or has a helical region binding moiety of the transferrin receptor. It may be connected.
  • These IgG antibodies have enhanced selective permeability of the blood-brain barrier and can be distributed at a relatively high level in brain tissue compared to other organs and cells.
  • the IgG antibody may be an antibody or antigen-binding fragment capable of binding to a target antigen known in the art, applicable to treating, alleviating, or detecting diseases associated with brain dysfunction, for example, It may be a pharmaceutically active substance for treating or alleviating diseases associated with brain dysfunction.
  • the IgG antibody may be used to treat Alzheimer's disease; dementia with Lewy bodies; frontotemporal dementia; tangle only dementia; Parkinson's disease; cognitive dysfunction; multiple sclerosis; amyotrophic lateral sclerosis (ALS); traumatic brain injury; progressive supranuclear palsy; corticobasal degeneration; globular glial tauopathy; aging-related tau astrogliopathy; Chronic Traumatic Encephalopathy (CTE); brain cancer, including primary CNS lymphoma (PCNSL), glioma, neuroblastoma, glioblastoma multiforma, meningioma, and brain metastases; Pick's disease; anti-IgLON5-related tauopathy; Guadeloupean parkinsonism; nodding syndrome; pain; epilepsy; autism; stroke; Guillain-Barre Syndrome (GBS); Creutzfeldt-Jakob disease (CJD); Huntington's disease; progressive multifocal leukoencephalopathy (PML
  • Transferrin receptor TfR
  • transferrin receptor refers to a membrane glycoprotein expressed on the surface of cells that mediates intracellular uptake of iron from transferrin, a plasma glycoprotein, and is expressed in normal cells of various tissues. It has been reported that it is not only widely distributed, but also expressed in large amounts in activated immune cells and tumor cells. Therefore, transferrin receptor-mediated delivery technology requires not only effective delivery to brain tissue, but also low-level delivery to organs other than brain tissue or normal cells. In fact, it has been reported that transferrin receptor-mediated transporters or therapeutic agents can cause red blood cell-related toxicities, including decreased reticulocyte count, severe coma, intermittent limb stiffness, general stiffness, hemolysis, and hemoglobinuria.
  • extracellular domain (ectodomain) of the transferrin receptor is divided into an apical domain, a helical domain, and a protease-like domain, and these are known to have different binding affinities to target substances during receptor-mediated transcytosis.
  • the transferrin receptor can be obtained from known databases such as GenBank of NCBI (National Center for Biotechnology Information).
  • the transferrin receptor may be composed of the amino acid sequence of SEQ ID NO: 1, but It is not limited.
  • transferrin receptor cluster refers to a cluster in which a plurality of transferrin receptors exist densely in a local area, and the transferrin receptor cluster is distributed in tissues or cells such as blood vessels in the form of a plurality of transferrin receptors clustered together. can do.
  • transferrin receptor clusters present in the brain vascular region may have a specific expression pattern.
  • the specific expression pattern refers to a state in which transferrin receptors are distributed at a remarkably dense level in local areas where clusters are formed compared to other organ tissues or cells, and these brain tissue-specific clusters and transferrin within the clusters
  • the expression pattern of the receptor may be closely correlated with high permeability to the blood-brain barrier and selective delivery to brain tissue.
  • binding moiety of the helical region of the transferrin receptor refers to a functional unit that interacts with the receptor present on the surface of the cells constituting the blood-brain barrier, and specifically, the helical region of the region constituting the transferrin receptor. It may refer to a moiety that has effective binding affinity for the region.
  • the helical region binding moiety of the transferrin receptor reflects the expression pattern of the transferrin receptor cluster specific to brain tissue blood vessels and may be composed of a tetravalent form.
  • the helical region of the transferrin receptor is a functional/structural region exposed to the outside to form an effective quadrivalent bond between a plurality of transferrin receptors densely distributed in a cluster of blood vessels in brain tissue and a fusion protein, which acts on the blood-brain barrier. It may contribute to high permeability and selective delivery to brain tissue. Therefore, the binding moiety capable of binding to the helical region of the transferrin receptor is linked to an IgG antibody, which can not only significantly improve the blood-brain barrier permeability of the IgG antibody, but also selectively deliver/absorb into brain tissue. It can be given functionality that makes it possible.
  • the binding moiety is intended to form a complex/fusion by interacting with the transferrin receptor cluster distributed in the brain tissue vascular region, and may have binding affinity with a plurality of transferrin receptors in the local area where the cluster is formed, specifically. , It may have a binding affinity with at least one amino acid selected from the helical region of the transferrin receptor, more specifically, the region from the 606th amino acid to the 665th amino acid based on the transferrin receptor of SEQ ID NO: 1.
  • the helical region binding moiety of the transferrin receptor may be linked to the C-terminus of the light chain and the C-terminus of the heavy chain of the IgG antibody, whereby the helical region binding moiety of the transferrin receptor is 4 It can be linked to an IgG antibody in a false form to exert the desired functionality.
  • the helical region binding moiety of the transferrin receptor may have a length of 6 to 250 amino acids.
  • the helical region binding moiety of the transferrin receptor may have a length of 6 to 240 amino acids, or 6 to 220 amino acids.
  • the length of the helical region binding moiety of the transferrin receptor is, for example, 10 to 48, 10 to 44, 10 to 40, 10 to 36, 10 to 32, 10 to 28, 10 to 24.
  • 10 to 20, 10 to 16, 10 to 12, 12 to 48, 12 to 44, 12 to 40, 12 to 36, 12 to 32, 12 to 28, 12 to 24 , 12 to 20, 12 to 16, 14 to 48, 14 to 44, 14 to 40, 14 to 36, 14 to 32, 14 to 28, 14 to 24, 14 to 20 It may be, but is not limited to, 14 to 16 amino acids in length.
  • the helical region binding moiety of the transferrin receptor may include an amino acid sequence in which the monomer sequence is repeated 2 to 5 times, 2 to 4 times, or 2 to 3 times.
  • the monomer sequences may be fused or connected by a linker peptide.
  • the linker peptide is 2 to 50 amino acids long, for example, 2 to 40 amino acids, 2 to 30 amino acids, 2 to 20 amino acids, 2 to 15 amino acids, 2 to 10 amino acids, 2 to 5 amino acids, 5 to 50 amino acids, It may be 5 to 40, 5 to 30, 5 to 20, 5 to 15, or 5 to 10 amino acids in length, but is not limited thereto.
  • the linker peptide may be, for example, (G l S m ) n (l is 2 to 8, m is 1 to 5, n is 1 to 5), (G d Se AS) f (d is 2 to 5) 8, e is 1 to 5, f is 1 to 5), (G 4 S) a (EAAAK) b (G 4 S) a (a, b are integers 1 to 4), [(G 4 S) p (EAAAK) q ] r (p, q, and r are integers from 1 to 4), but is not limited thereto.
  • the monomeric sequence constituting the helical region binding moiety of the transferrin receptor may include a cell-penetrating peptide.
  • the monomer sequence is exemplarily SEQ ID NO: 3 to SEQ ID NO: 43. It may be any one of the above, but as long as it is a monomeric sequence that has effective interaction (hydrogen bonding, electrostatic attraction, van der Waals force) with the helical region of the transferrin receptor, that is, effective binding affinity, it can be applied without limitation.
  • each of the helical region binding moieties of the plurality of transferrin receptors may be the same or different.
  • fusion protein refers to a protein formed through the combination of two or more originally separate proteins, or parts thereof, and may optionally include a linker or space located between the two or more proteins.
  • blood-brain barrier permeable fusion protein is a protein formed through the bond between an IgG antibody and the helical region binding moiety of a transferrin receptor, and penetrates the blood-brain barrier to selectively deliver an effective amount of IgG antibody to brain tissue.
  • it may be a general term for proteins that contain a functional structure that allows them to be absorbed.
  • the functional structure is modified to have effective binding affinity by reflecting the structure and expression pattern of the transferrin receptor cluster of vascular endothelial cells that are specifically distributed in the blood-brain barrier, and is capable of binding to the helical region of the transferrin receptor. It may include a tetravalent bond (link) between the moiety and the C-terminal regions of the heavy and light chains constituting the IgG antibody and the binding moiety.
  • the blood-brain barrier permeable fusion protein may include a functional structure in which a tetravalent helical region binding moiety is linked to the C-terminus of the light chain and the C-terminus of the heavy chain of the IgG antibody. there is.
  • connection between the C-terminal region of the light chain and the C-terminal region of the heavy chain of the IgG antibody and the helical region binding moiety is fused to the terminal region of the above-mentioned IgG antibody, or is connected by a linker peptide. It can be linked, and details about the linker peptide are as described above.
  • the blood-brain barrier permeable fusion protein may form a complex by binding to a transferrin receptor that forms a transferrin receptor cluster specifically distributed in blood vessels of the blood-brain barrier.
  • the blood-brain barrier permeable fusion protein may bind to a plurality of transferrin receptors that are densely distributed within the transferrin receptor cluster compared to other organ tissues or cells.
  • the functional structure was designed to reflect the effective binding affinity with the helical region of the transferrin receptor and the specific distribution/expression pattern of the transferrin receptor cluster distributed in the cerebrovascular region, and the C of the light chain in the IgG antibody
  • a fusion protein was prepared in which a plurality of helical region binding moieties of the transferrin receptor were linked to the -terminal and heavy chain C-terminal regions, that is, a total of four terminal regions.
  • the preparation containing the fusion protein was administered intravenously, not only was the delivery of the fusion protein containing the IgG antibody to the brain tissue significantly enhanced, but also the IgG antibody was distributed at a high level in the brain tissue compared to other organs.
  • the fusion protein has a function derived from a tetravalent moiety having an effective binding affinity for the helical region of the transferrin receptor and a functional structure containing the same, regardless of the type of IgG antibody and the specific sequence of the binding moiety. It was found that the same effect as high permeability to the blood-brain barrier was demonstrated. Therefore, in one embodiment, a functional structure is provided that is linked to the binding moiety and can contribute to improving the blood-brain barrier permeability of the IgG antibody, while inducing selective delivery to brain tissue and reducing the side effects of antibody-based drugs. newly identified.
  • the blood-brain barrier permeable fusion protein includes a first binding moiety linked to the C-terminal region of the light chain of the IgG antibody; and a second binding moiety linked to the heavy chain C-terminal region of the IgG antibody.
  • the blood-brain barrier permeable fusion protein may be formulated and administered, and the administration may be performed by a non-limiting method of administering a conventional fusion protein or antibody-based formulation, for example, intravenous administration. , intramuscular administration, subcutaneous administration, intraperitoneal administration, ocular administration, intrathecal administration, intracerebroventricular administration, intranasal administration, etc.
  • the fusion protein can provide an appropriate level of interaction or binding affinity with a partial region of the receptor present on the surface of the cells constituting the blood-brain barrier, that is, the helical region of the transferrin receptor.
  • the fusion partner IgG antibody
  • prevention refers to all actions that suppress or delay the onset of diseases associated with brain dysfunction by administering the pharmaceutical composition.
  • treatment refers to any action in which symptoms of a disease associated with brain dysfunction are improved or beneficially changed by administration of the pharmaceutical composition.
  • Disease associated with brain dysfunction which is a disease to be prevented or treated by the pharmaceutical composition, includes Alzheimer's dementia; Lewy Body Dementia; frontotemporal dementia; Neurofibrillary tangles - predominant dementia; Parkinson's disease; cognitive dysfunction; multiple sclerosis; amyotrophic lateral sclerosis; traumatic brain injury; Progressive supranuclear palsy; Corticobasal degeneration; glial tauopathy; Age-related tau astrogliopathy; chronic traumatic encephalopathy; brain cancer, including primary central nervous system lymphoma, glioma, neuroblastoma, glioblastoma, meningioma, and brain metastasis; Pick's disease; anti-IgLON5 associated tauopathy; Guadeloupe Parkinsonism; Nohing Syndrome; ache; epilepsy; autism; stroke; Guillian-Barré syndrome; Creutzfeldt-Jakob disease; Huntington's disease; Progressive multifocal leukoencephalopathy; depression
  • the helical region binding moiety is linked to or added to the C-terminal region of each IgG antibody, and does not cause any modification to the CDR region or variable region of the IgG antibody. additionally shown. Therefore, the high penetrating ability across the blood-brain barrier and the selective delivery efficacy to brain tissue confirmed in one example may lead to improved treatment effects for diseases associated with brain dysfunction.
  • examples of the above-described therapeutic effects include reduction of beta-amyloid aggregates, reduction of abnormal phosphorylation of tau protein, improvement of local field potential, improvement of cognitive function and spatial perception ability, and synaptic function of neurons.
  • the efficacy of recovery, activation of phagocytosis of microglial cells, and improvement of reactive astrogliosis were experimentally demonstrated.
  • the pharmaceutical composition i.e., a pharmaceutical preparation comprising a fusion protein, comprises the antibody having the desired degree of purity in any pharmaceutically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized preparation or aqueous solution.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; Antioxidants such as ascorbic acid and methionine; Preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexamethylamine 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; Proteins such as serum albumin, gelatin, or immunoglobulin; Hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or
  • the pharmaceutical compositions may contain additional active ingredients, optionally ingredients with complementary activities that do not adversely affect each other.
  • the type and effective amount of the pharmaceutical composition are determined, for example, depending on the amount of antibody present in the preparation and the clinical parameters of the subject.
  • the active ingredients are placed in microcapsules prepared, for example, by droplet formation techniques or by interfacial polymerization, for example hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, for colloidal drug delivery. They can be delivered in systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in encapsulated form by macroemulsions.
  • compositions can be formulated, dosed, and administered in a manner consistent with medical practice. Factors to consider in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to the physician.
  • Antibodies may, but need not, be formulated with one or more agents currently used for the prevention or treatment of disease.
  • the effective amount of the other agent will depend on the amount of antibody present in the agent, the type of disorder or treatment, and other factors. They are generally administered in the same dosages and routes of administration as described herein, or from about 1 to 99% of the dosages described herein, or by any dosage and any route empirically/clinically determined to be appropriate. It is used.
  • the method of treating diseases associated with brain dysfunction may use the pharmaceutical composition alone or in combination with other agents.
  • the pharmaceutical composition can be co-administered with at least one additional therapeutic agent.
  • additional therapeutic agents are effective therapeutic agents in the treatment of diseases associated with brain dysfunction and include, but are not limited to: cholinesterase inhibitors (e.g. donepezil, galantamine, lovastigmine and tacrine), NMDA.
  • Receptor antagonists e.g.
  • amyloid beta peptide aggregation inhibitors antioxidants, ⁇ -secretase modulators, nerve growth factor (NGF) mimics or NGF gene therapy agents, PPAR ⁇ agonists, HMS-CoA reduction Enzyme inhibitors (statins), ampakines, calcium channel blockers, GABA receptor antagonists, glycogen synthase kinase inhibitors, intravenous immunoglobulins, muscarinic receptor agonists, nicotinic receptor modulators, active or passive amyloid beta peptides. Immunization, phosphodiesterase inhibitors, serotonin receptor antagonists and anti-amyloid beta peptide antibodies.
  • the appropriate dosage of the pharmaceutical composition depends on the type of disease being treated, the severity and course of the disease, and the antibody level. Whether administered for prophylactic or therapeutic purposes will be determined based on the previously administered therapy, the patient's clinical history and response to antibodies, and the judgment of the attending physician.
  • the antibody is suitably administered to the patient at once or over a series of treatments, and for the purpose of the present invention, the antibody may be provided in the form of a fusion protein bound to the helical region binding moiety of the tetravalent transferrin receptor.
  • approximately 1 ⁇ g/kg to 100 mg/kg (e.g., 0.1 mg/kg to 100 mg/kg) of the antibody or fusion protein may be administered, for example, by one or more separate administrations; or it may be an initial candidate dose for administration to a patient, whether by continuous infusion.
  • a typical daily dosage may range from about 1 ⁇ g/kg to 100 mg/kg or more depending on the factors mentioned above. For repeated administration over a period of several days or longer, depending on the condition, treatment will generally continue until the required suppression of disease symptoms occurs.
  • One exemplary dosage of antibody or fusion protein would range from about 0.05 mg/kg to about 100 mg/kg.
  • one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg, 20 mg/kg (or any combination thereof) may be administered to the patient.
  • the dosage may be administered intermittently, for example weekly or every three weeks (e.g., such that the patient is administered a dose of the antibody or fusion protein of about 2 mg/kg to about 20 mg/kg, or for example about 6 mg/kg). ) can be administered.
  • An initial higher loading dose may be followed by one or more lower doses.
  • the term "individual” refers to a subject in need of treatment for a disease, and more specifically, mammals such as humans or non-human primates, mice, dogs, cats, horses, and cattle. means.
  • the blood-brain barrier permeable fusion protein when a preparation containing the fusion protein is administered intravenously, delivery of IgG antibodies in brain tissue is significantly enhanced due to effective interaction between the binding moiety and the transferrin receptor. Confirmed.
  • IgG antibodies when a preparation containing the fusion protein is administered intravenously, IgG antibodies can be selectively delivered at a high level to brain tissue compared to other organs and cells, which is excellent. It shows biosafety, and effective therapeutic efficacy was demonstrated through experiments using animal models.
  • the fusion protein can be used as an active ingredient in pharmaceutical compositions for the medical field requiring selective drug delivery to brain tissue, for example, for the prevention or treatment of diseases associated with brain dysfunction. .
  • Figure 1 shows the results of confirming the thermodynamic structural stability of the binding between helical region binding moiety #01 and the transferrin receptor according to one embodiment.
  • Figure 2 shows the results of confirming the thermodynamic structural stability of the binding between helical region binding moieties #2, #3, #5, #6, #7, or #8 and the transferrin receptor according to one embodiment.
  • Figure 3 shows the results of confirming the thermodynamic structural stability of the binding between helical region binding moieties #9, #10, #11, #12, #13, or #14 and the transferrin receptor according to one embodiment.
  • Figure 4 shows the results of confirming the thermodynamic structural stability of the binding between helical region binding moieties #15, #16, #17, #18, #19, or #20 and the transferrin receptor according to one embodiment.
  • Figure 5 shows the results of confirming the thermodynamic structural stability of the binding between helical region binding moieties #21, #22, #23, or #24 and the transferrin receptor according to one embodiment.
  • Figure 6 shows the results of confirming the thermodynamic structural stability of the binding between helical region binding moieties #25, #27, #30, #32, #33, or #34 and the transferrin receptor according to one embodiment.
  • Figure 7 shows the results of confirming the thermodynamic structural stability of the binding between helical region binding moiety #36, #38, or #39 and the transferrin receptor according to one embodiment.
  • Figure 8 shows the results of confirming the intracellular delivery level of helical region binding moiety #01 according to one embodiment using a human brain endothelial cell line.
  • Figure 9 shows the results of confirming the intracellular delivery level of helical region binding moiety #04 according to one embodiment using a human brain endothelial cell line.
  • Figure 10 shows the results of confirming the intracellular delivery level of helical region binding moiety #16, #19, or #20 according to one embodiment using a human brain endothelial cell line.
  • Figure 11 shows the results of confirming the intracellular delivery level of helical region binding moiety #25, #26, or #27 according to one embodiment using a human brain endothelial cell line.
  • Figure 12 shows the results of confirming the intracellular delivery level of helical region binding moiety #28, #29, or #31 according to one embodiment using a human brain endothelial cell line.
  • Figure 13 shows the results of confirming the intracellular delivery level of helical region binding moiety #34, #35, #36, or #37 according to one embodiment using a human brain endothelial cell line.
  • Figure 14 shows the results of confirming the intracellular delivery level of helical region binding moiety #38, #40, or #41 according to one embodiment using a human brain endothelial cell line.
  • Figure 15 shows the results of confirming the level of IgG1 antibodies in brain tissue over time using a human IgG1 ELISA kit after intravenous administration of the blood-brain barrier permeable fusion protein F3#01 according to an example to an animal model.
  • Figure 16 shows the level of IgG1 antibody in brain tissue after intravenous administration of blood-brain barrier permeable fusion protein F3#02, F3#03, F3#04, F3#05, or F3#06 according to one embodiment to an animal model. This is a result confirmed using a human IgG1 ELISA kit.
  • Figure 17 shows the level of IgG1 antibody in brain tissue after intravenous administration of blood-brain barrier permeable fusion protein F3#07, F3#08, F3#09, F3#10, or F3#11 according to one embodiment to an animal model. This is a result confirmed using a human IgG1 ELISA kit.
  • Figure 18 shows the level of IgG1 antibody in brain tissue using human IgG1 ELISA after intravenous administration of blood-brain barrier permeable fusion protein F3#12, F3#13, F3#14, or F3#15 according to an embodiment to an animal model. This is the result confirmed using the kit.
  • Figure 19 shows the level of IgG1 antibody in brain tissue after intravenous administration of blood-brain barrier permeable fusion protein F3#16, F3#17, F3#18, F3#19, or F3#20 according to one embodiment to an animal model. This is a result confirmed using a human IgG1 ELISA kit.
  • Figure 20 shows the level of IgG1 antibody in brain tissue using human IgG1 ELISA after intravenous administration of blood-brain barrier permeable fusion protein F3#21, F3#22, F3#23, or F3#24 according to an embodiment to an animal model. This is the result confirmed using the kit.
  • Figure 21 shows the level of IgG1 antibody in brain tissue using a human IgG1 ELISA kit after intravenous administration of blood-brain barrier permeable fusion protein F3#25, F3#26, or F3#27 according to an embodiment to an animal model. This is the confirmed result.
  • Figure 22 shows the level of IgG1 antibodies in brain tissue after intravenous administration of blood-brain barrier permeable fusion proteins F3#28, F3#29, F3#30, F3#31, or F3#32 according to one embodiment to an animal model. This is a result confirmed using a human IgG1 ELISA kit.
  • Figure 23 shows the level of IgG1 antibody in brain tissue after intravenous administration of blood-brain barrier permeable fusion protein F3#33, F3#34, F3#35, F3#36, or F3#37 according to one embodiment to an animal model. This is a result confirmed using a human IgG1 ELISA kit.
  • Figure 24 shows the level of IgG1 antibody in brain tissue using human IgG1 ELISA after intravenous administration of blood-brain barrier permeable fusion protein F3#38, F3#39, F3#40, or F3#41 according to one embodiment to an animal model. This is the result confirmed using the kit.
  • Figure 25 shows the level of IgG1 antibody in ISF after intravenous administration of blood-brain barrier permeable fusion protein F3#25-Tau containing an IgG1 antibody specifically binding to Tau according to an example to an animal model using human IgG1 ELISA. It was confirmed using a kit, and the results were expressed as a multiple compared to the control group.
  • Figure 26 shows the IgG1 antibody in the ISF after intravenous administration of the blood-brain barrier permeable fusion protein F3#27-Tau or F3#36-Tau containing an IgG1 antibody specifically binding to Tau according to an example to an animal model. The level of was confirmed using a human IgG1 ELISA kit, and the results were expressed as multiples compared to the control group.
  • Figure 27 shows the level of IgG1 antibody in brain tissue after intravenous administration of blood-brain barrier permeable fusion protein F3#25-PD1 containing an IgG1 antibody that specifically binds to PD1 according to an embodiment to an animal model. It was confirmed using an ELISA kit, and the results were expressed as a multiple compared to the control group.
  • Figure 28 shows the level of IgG1 antibody in brain tissue after intravenous administration of the blood-brain barrier permeable fusion protein F3#25-HER2 containing an IgG1 antibody that specifically binds to HER2 according to an embodiment to an animal model. It was confirmed using an ELISA kit, and the results were expressed as a multiple compared to the control group.
  • Figure 29 shows the level of IgG1 antibody in brain tissue after intravenous administration of the blood-brain barrier permeable fusion protein F3#25-A ⁇ containing an IgG1 antibody that specifically binds to A ⁇ according to one embodiment to an animal model. It was confirmed using an ELISA kit, and the results were expressed as a multiple compared to the control group.
  • Figure 30 shows the results of confirming the distribution level of IgG1 antibodies in each organ using a human IgG1 ELISA kit after intravenous administration of the blood-brain barrier permeable fusion protein F3#01 according to an example to an animal model.
  • Figure 31 shows the results of confirming the distribution level of IgG1 antibodies in each organ using a human IgG1 ELISA kit after intravenous administration of the blood-brain barrier permeable fusion protein F3#01 according to an example to an animal model.
  • Figure 32 shows the results of intravenously administering blood-brain barrier permeable fusion protein F3#01 to an animal model according to an example, obtaining blood, and confirming the percentage (%) of reticulocytes among total red blood cells.
  • Figure 33 shows the results of confirming the concentration of the fusion protein in plasma after intravenous administration of the blood-brain barrier permeable fusion protein F3#01 according to an example to an animal model.
  • Figure 34 shows the results of calculating the blood-to-plasma ratio after treating plasma and blood samples with the blood-brain barrier permeable fusion protein F3#01 according to an example.
  • Figure 35 shows the results of confirming the change in binding affinity of IgG1 antibody to PD-L1 according to the presence or absence of binding moiety #01 in blood-brain barrier permeable fusion proteins F3#01 and F3'#01 according to an embodiment.
  • Figure 36 shows the results of confirming the level of insoluble A ⁇ present in the subiculum region through immunohistostaining after intravenous administration of the blood-brain barrier permeable fusion protein F3#01 according to an example to 3xTg mice.
  • Figure 37 shows the results of confirming the level of insoluble A ⁇ present in the cortex, hippocampus, subiculum, and dentate gyrus regions through immunohistostaining after intravenous administration of blood-brain barrier permeable fusion protein F3#01 according to an embodiment to 5xFAD mice. am.
  • Figure 38 shows the results of confirming the total A ⁇ level present in the subiculum region through immunohistostaining after intravenous administration of the blood-brain barrier permeable fusion protein F3#01 according to an example to 3xTg mice.
  • Figure 39 shows the results of confirming the total A ⁇ level present in the cortex and dentate gyrus regions through immunohistostaining after intravenous administration of the blood-brain barrier permeable fusion protein F3#01 according to an example to 5xFAD mice.
  • Figure 40 shows the results of confirming the level of pTau231 present in the subiculum and CA1 region after intravenous administration of the blood-brain barrier permeable fusion protein F3#01 according to an example to 3xTg mice.
  • Figure 41 shows the results of confirming the level of pTau181 present in the subiculum and CA1 region after intravenous administration of the blood-brain barrier permeable fusion protein F3#01 according to an example to 3xTg mice.
  • Figure 42 shows the results of confirming changes in basal biological local field potential after intravenous administration of the blood-brain barrier permeable fusion protein F3#01 according to an example to 3xTg mice.
  • Figure 43 shows the results of confirming whisker stimulation-induced local field potential changes in the living body after intravenous administration of the blood-brain barrier permeable fusion protein F3#01 according to an example to 3xTg mice.
  • Figure 44 shows the results of analyzing whisker stimulation-induced changes in biolocal field potential by separating nerve activity by frequency band after intravenous administration of blood-brain barrier permeable fusion protein F3#01 to 3xTg mice according to an example.
  • Figure 45 shows the results of evaluating cognitive function and spatial perception ability after intravenous administration of blood-brain barrier permeable fusion protein F3#01 to 3xTg mice according to an example.
  • Figure 46 shows the results of evaluating cognitive function and spatial perception ability after intravenous administration of blood-brain barrier permeable fusion protein F3#01 according to an example to 5xFAD mice.
  • Figure 47 shows the results of confirming the expression levels of Synaptophysin and PDS95 proteins in brain tissue after intravenous administration of the blood-brain barrier permeable fusion protein F3#01 according to an example to 3xTg mice.
  • Figure 48 shows the results of confirming the expression levels of Synaptophysin and PDS95 proteins in brain tissue after intravenous administration of the blood-brain barrier permeable fusion protein F3#01 according to an example to 5xFAD mice.
  • Figure 49 shows the results of confirming the level of activated microglial cells that phagocytosed A ⁇ present in the hippocampus region of brain tissue after intravenous administration of the blood-brain barrier permeable fusion protein F3#01 according to an example to 3xTg mice.
  • Figure 50 shows the results of confirming the level of anti-glial fibrillary acidic protein (GFAP) positive area present in the subiculum area after intravenous administration of the blood-brain barrier permeable fusion protein F3#01 according to an example to 3xTg mice.
  • GFAP anti-glial fibrillary acidic protein
  • a fusion protein with improved blood-brain barrier permeability.
  • a fusion protein was prepared in which the binding moiety of the helical region of the transferrin receptor was linked to the C-terminal region of the light chain and the C-terminal region of the heavy chain in an IgG1 antibody, that is, a total of four terminal regions.
  • a total of 24 binding moieties were derived as binding moieties for the helical region of the transferrin receptor, each having binding characteristics for the helical region but each having a different amino acid sequence (first binding moiety group).
  • helical region binding moiety of the transferrin receptor a total of 17 binding moieties maintain a certain level of sequence identity with the binding moiety of SEQ ID NO: 3, but some of the amino acid sequences are substituted, inserted, or deleted. was further derived (second binding moiety group).
  • a blood-brain barrier permeable fusion protein in which the binding moiety of the helical region of the transferrin receptor was linked to the four terminal regions described above in the IgG1 antibody was prepared as follows. Specifically, a 1 mL sample was collected with a pipette from a flask containing cells, and the cell mass was measured. Afterwards, if the cell survival rate is more than 95% and the cell mass is more than 4 ⁇ 6 ⁇ 106 cells/mL, add culture medium stored in an incubator at 37°C and culture it so that the cell mass level is 9 ⁇ 106 cells/mL. Ready. Afterwards, transfection was performed on the prepared cells according to the following experimental conditions.
  • the feed media and enhancer were treated on the 1st day of culture, and the feed media was treated and cultured on the 5th day of culture.
  • Transfected cells were obtained when the cell viability was below 70% or when 8 days had elapsed from the date of transfection.
  • the sample containing the transfected cells was centrifuged at 4500 rpm and 25°C for 15 minutes, and the supernatant was recovered therefrom.
  • the supernatant was filtered using a 0.22 ⁇ m filter.
  • the filtrate was purified using purification techniques such as affinity chromatography and size exclusion chromatography to obtain each fusion protein.
  • the purified antibody was analyzed by SEC-HPLC (size exclusion high performance liquid chromatography), SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and mass spectrometry to confirm the purification results.
  • a fusion protein containing a second binding moiety group was prepared in the same manner as above. Meanwhile, in the fusion protein according to this example, details about the helical region binding moiety of the transferrin receptor, IgG1 antibody, etc. are as follows.
  • binding moieties #01 to #41 according to one embodiment was sought to be confirmed through binding to the helical region of the transferrin receptor and evaluating the level of delivery into human brain endothelial cells through this.
  • Helical domain binding moieties #01, #02, #03, #05 to #25, #27, #30, #32, #33, #34, #36, #38, #39 and transferrin according to one embodiment. Binding between receptors (transferrin receptor: TfR) was confirmed through docking simulation. The structure of the binding moiety was modeled using the RosettaRelax program, and the position expected to interact with the transferrin receptor helical region was modeled using structural information and thermodynamic calculations. Afterwards, a docking simulation was performed using the RosettaDocking program to randomly change the position of the binding moiety and calculate the interaction with the helical region to find the most stable position. 20,000 simulations were performed for each sequence number, and the resulting data were analyzed based on homology to the initial modeling structure and thermodynamic structural stability.
  • HCMEC/D3 cells which are human brain endothelial cells that constitute the human blood-brain barrier, through inter-domain interaction.
  • the hCMEC/D3 cells were cultured under conditions of 37°C and 5% CO 2 using synthetic endothelial Cell Basal Medium 2 (EBM2) culture medium containing growth factors.
  • EBM2 synthetic endothelial Cell Basal Medium 2
  • the cells were separated, 4 ⁇ 10 3 cells were added to 40 ⁇ L of culture medium, distributed in a 384 well plate, centrifuged for 10 seconds, and incubated at 37°C, 5% CO 2 The cells were allowed to attach to the plate by culturing under these conditions for more than 18 hours. Meanwhile, 500 ⁇ L of 1x PBS was added to 1 mg of the binding moiety peptide, mixed, and the concentration was measured using an ultraviolet-visible spectrometer. The peptide was then diluted to a final concentration of 200 ⁇ M and stored at 4°C. Afterwards, the 200 ⁇ M concentration of the peptide was diluted in EBM2 culture medium at a concentration 5 times the treatment concentration.
  • binding moiety #01 was confirmed.
  • binding moieties #04, #16, #19, #20, #25 to #29, #31, #34 to #38, #40, and #41 are also human. Effective intracellular delivery to brain endothelial cell lines was confirmed.
  • a preparation containing 20 mg/kg of fusion protein F3#01 was intravenously administered to C57BL/6 mice through the caudal vein, and a group administered only IgG1 antibody intravenously was used as a control group.
  • the mouse was anesthetized and blood was collected from the blood vessels inside the eyes or the abdominal vein. Afterwards, blood was removed by perfusion with physiological saline. Subsequently, the brain of the mouse was extracted, and the extracted brain tissue was rapidly frozen with liquid nitrogen and stored in a deep freezer until use. Meanwhile, the extracted brain tissue was homogenized using a protein extraction solution, and then the homogenized brain tissue sample was dissolved at 4°C using a rotating mixer.
  • the dissolved brain tissue sample was centrifuged, and the supernatant was obtained to prepare a brain tissue lysate.
  • the level of IgG1 antibodies in brain tissue lysate was measured using a human IgG1 ELISA kit. Specifically, a standard (STD) was prepared by diluting to each concentration using a protein extraction solution, and a sample (SPL) containing brain tissue lysate was prepared according to each dilution ratio using a protein extraction solution. 50uL of this was added to each well. Afterwards, an additional 50 uL of the Ab cocktail contained in the ELISA kit was added to each well, and then reacted at 4°C.
  • the level of IgG1 antibodies in the brain tissue of the F3#01 administration group was observed to be significantly higher than that of the control group until 14 days after administration.
  • the F3#01 administration group showed a high level of delivery ranging from about 25 times to about 320 times compared to the control group.
  • the level of absorption of IgG1 antibodies into brain tissue by intravenous administration of blood-brain barrier permeable fusion proteins F3#02 to F3#41 according to one embodiment was measured in the same manner as (1) in Experimental Example 1-2, after intravenous administration. Evaluation was made after 2 or 4 days. Meanwhile, as a control group, a group administered intravenously only IgG1 antibody was used.
  • fusion proteins F3#02 of Examples 2 to 24 were prepared using first binding moiety groups that possessed binding properties to the helical region but had different amino acid sequences. It was confirmed that the level of IgG1 antibodies in brain tissue was significantly increased in all groups administered F3#24 to F3#24 compared to the control group.
  • a second binding moiety group is used that maintains a certain level of sequence identity with the binding moiety of SEQ ID NO: 3, but in which some of the amino acid sequences are substituted, inserted, or deleted. It was confirmed that the levels of IgG1 antibodies in brain tissue were significantly increased in the groups administered with the fusion proteins F3#025 to F3#41 prepared in Examples 25 to 41, as compared to the control group.
  • the fusion protein has a function derived from a moiety with effective binding affinity for the helical region of the transferrin receptor, high permeability to the blood-brain barrier, and brain tissue of IgG1 antibodies. It was found that the same effect as the high transmission of .
  • the IgG1 antibody is an IgG1 antibody (anti-Tau) that binds to Tau.
  • the helical region binding moiety of the transferrin receptor was prepared as a blood-brain barrier permeable fusion protein in the same manner as in Example 1 by applying binding moiety #25, #27, or #36 (F3#25-Tau, F3# 27-Tau, F3#36-Tau).
  • the skin on the head of the mouse was incised, and a drill was used to make a hole in the skull adjacent to the hippocampus area.
  • a guide cannula was inserted into the formed skull perforation and fixed using resin.
  • the incised skin was sutured to prevent the skull perforation area from being exposed to the outside, and the mouse was allowed to recover for two weeks.
  • a preparation containing 20 mg/kg of fusion protein (F3#25-Tau, F3#27-Tau, F3#36-Tau) was intravenously administered to the recovered mouse through the caudal vein.
  • the mouse was anesthetized and an activated probe for detecting IgG1 antibodies was inserted into the probe guide cannula of the mouse. Thereafter, while flowing cerebrospinal fluid (CSF) containing BSA at a constant rate, a sample of interstitial fluid (ISF) containing the fusion protein was collected and stored at -20°C. Afterwards, the level of IgG1 antibodies in the ISF sample was measured using a human IgG1 ELISA kit. Specifically, standards (STD) and samples (SPL) diluted to each concentration were prepared using N.S sample buffer, and then 50uL of them were added to each well.
  • CSF cerebrospinal fluid
  • ISF interstitial fluid
  • the IgG1 antibody is an IgG1 antibody (anti-PD1) that binds to PD1.
  • a blood-brain barrier permeable fusion protein was prepared in the same manner as in Example 1 by applying binding moiety #25 as the helical region binding moiety of the transferrin receptor (F3#25-PD1). Thereafter, the level of absorption of IgG1 antibody into brain tissue by intravenous administration of the prepared fusion protein was evaluated in the same manner as (1) of Experimental Example 1-3. Meanwhile, as a control group, a group to which only IgG1 antibody was added was used.
  • the IgG1 antibody is an IgG1 antibody (anti-HER2) that binds to HER2.
  • a blood-brain barrier permeable fusion protein was prepared in the same manner as Example 1 by applying binding moiety #25 as the helical region binding moiety of the transferrin receptor (F3#25-HER2). Thereafter, the level of absorption of IgG1 antibody into brain tissue by intravenous administration of the prepared fusion protein was evaluated in the same manner as (1) of Experimental Example 1-3. Meanwhile, as a control group, a group to which only IgG1 antibody was added was used.
  • the IgG1 antibody is an IgG1 antibody (anti-A ⁇ ) that binds to A ⁇ .
  • a blood-brain barrier permeable fusion protein was prepared in the same manner as Example 1 by applying binding moiety #25 as the helical region binding moiety of the transferrin receptor (F3#25-A ⁇ ). Thereafter, the level of absorption of IgG1 antibody into brain tissue by intravenous administration of the prepared fusion protein was evaluated in the same manner as (1) of Experimental Example 1-3. Meanwhile, as a control group, a group to which only IgG1 antibody was added was used.
  • the fusion protein has a function derived from a moiety having an effective binding affinity for the helical region of the transferrin receptor and a functional structure containing the same, and is independent of the type of IgG1 antibody and is capable of activating cerebrovascular fluids. It was found that effects such as high permeability to the barrier and high delivery of IgG1 antibodies to brain tissue were exhibited.
  • the level of distribution of IgG1 antibodies to each organ was evaluated by intravenous administration of the blood-brain barrier permeable fusion protein F3#01 according to an example. Specifically, a preparation containing 20 mg/kg of fusion protein F3#01 was intravenously administered to C57BL/6 mice through the caudal vein, and a group administered only IgG1 antibody intravenously was used as a control group. Then, 1 or 4 days after this, the level of IgG1 antibodies in a total of 6 organs (brain, lung, spleen, kidney, liver, muscle) was measured in (1) and (1) of Experimental Example 1-2 above. Measurements were made in the same manner and compared.
  • the distribution level of IgG1 antibodies by organ is as shown in Figure 30, and the blood-brain barrier permeable fusion protein F3
  • the distribution level of IgG1 antibodies by organ is shown in Figure 31.
  • the IgG1 antibody administered intravenously as a control group was observed to have the highest distribution in the lungs and a relatively low distribution in the brain tissue among the organs, whereas the F3#01 administered group had a very high level in the brain tissue compared to other organs. It was confirmed that IgG1 antibodies were distributed.
  • the fusion protein containing the functional structure is selectively distributed to brain tissue when administered intravenously, thereby reducing the distribution of IgG antibodies in tissues other than brain tissue, thereby reducing the side effects of antibody-based drugs. It was found that it can contribute to reducing .
  • the effect on reticulocytes one of the cells in which the transferrin receptor is expressed in large quantities, was sought to be confirmed.
  • a preparation containing 20 mg/kg or 50 mg/kg of fusion protein F3#01 was intravenously administered to C57BL/6 mice through the caudal vein.
  • 50 ⁇ l of blood sample was obtained from the blood vessel inside the eye of the mouse using a heparinized capillary tube.
  • the proportion of reticulocytes among total red blood cells was similar to that of the control group.
  • a preparation containing 20 mg/kg of fusion protein F3#01 was intravenously administered to mice through the caudal vein.
  • Blood samples were obtained from the mice 30 minutes, 120 minutes, 360 minutes, 1 day, 2 days, 4 days, 7 days, or 14 days after intravenous administration of the fusion protein to the mice.
  • the plasma sample was separated by centrifugation, pretreatment for liquid chromatography mass spectrometry (LC-MS) was performed, and then LC-MS analysis was performed on the sample.
  • LC-MS liquid chromatography mass spectrometry
  • a group administered intravenously an IgG1 antibody was used as a control group.
  • the plasma PK profile of the group administered the blood-brain barrier permeable fusion protein F3#01 according to one embodiment was similar to the control group, and the above experimental results showed that several This indicates that the distribution of the fusion protein to the organ is minimal.
  • blood-to-plasma ratio peak of blood supernatant area ratio/peak area ratio of plasma
  • the plasma and blood supernatant samples were mixed with a PBS solution containing a surfactant and magnetic beads, the mixture was cultured, and the culture was washed twice with PBS containing a surfactant.
  • RapiGest surfactant and dithiothreitol were added, incubated at 60°C for 50 minutes, and left at room temperature for 10 minutes. Afterwards, 1) iodoacetic acid was added and cultured in the dark at room temperature for 30 minutes, 2) trypsin was added and cultured at 60°C for 24 hours, 3) HCl was added and cultured at 37°C. Incubation was performed sequentially for 30 minutes.
  • the culture was centrifuged to obtain the supernatant, trypsin was added, and the blood-to-plasma ratio (peak area ratio of blood supernatant / peak area ratio of plasma) was measured using LC-MS. Calculated. Meanwhile, an IgG1 antibody-treated group was used as a control group.
  • the blood-to-plasma ratio of the group administered the blood-brain barrier permeable fusion protein F3#01 according to one embodiment also showed a similar level to that of the control group, and the above experimental results are as follows. It was found that this was due to the fact that no bond was formed between the reticulocyte cell containing the transferrin receptor and the fusion protein.
  • the blood-brain barrier permeable fusion protein according to one embodiment does not have a high level of transferrin receptor-mediated delivery in organs other than brain tissue, and from this, selective delivery to brain tissue was confirmed.
  • the purpose was to determine the effect of linking with the binding moiety on the reactivity of the IgG1 antibody, that is, its ability to bind to the target.
  • the fusion proteins F3#01 and F3'#01 had IgG1 antibody reactivity, that is, binding affinity to PD-L1, at a level similar to that of the control group. It was confirmed that it represents.
  • the blood-brain barrier permeable fusion protein F3#01 according to one example was administered intravenously to 3xTg mice or 5xFAD mice at a concentration of 20 mg/kg. After 28 days, blood was removed by perfusion with physiological saline, the brain was removed from the mouse, and a 40 um thick brain tissue slice was prepared using a Cryostat (Leica Biosystems, CM1950).
  • an antibody that binds to A ⁇ (Biolegend, 6E10, #803001, 1:1000) was added to the washed brain tissue slice, reacted at 4°C for 16 hours, and then washed using a PBS solution.
  • a fluorescently labeled secondary antibody (anti-mouse Alexa568, Invitrogen, A10037, 1:1000) that recognizes antibodies bound to A ⁇ was added to the washed brain tissue slice, reacted at room temperature for 2 hours, and then added with PBS. Washed using a solution. Afterwards, samples of the immunostained brain tissue sections were prepared and the total A ⁇ level was evaluated.
  • Imaging of all stained samples was performed using the THUNDER imaging system (Leica), and image analysis was performed using the THUNDER imaging system (Leica). Meanwhile, a group administered intravenously with Vehicle (Vehicle) was used as a control group, and a group administered only anti-PD-L1 intravenously (anti-PD-L1) was used as a comparison group.
  • Vehicle Vehicle
  • anti-PD-L1 anti-PD-L1
  • the areas containing total A ⁇ in the cortex and dentate gyrus of the blood-brain barrier permeable fusion protein F3#01-administered group decreased by about 30% and 40%, respectively, compared to the Vehicle-administered group, resulting in anti-PD-L1 It was confirmed that the reduction level of total A ⁇ was improved compared to the administration group. The above results indicate that the level of reduction of beta-amyloid aggregates in the F3#01 administered group was improved compared to the anti-PD-L1 administered group.
  • the blood-brain barrier permeable fusion protein F3#01 was intravenously administered to 3xTg mice at a concentration of 20 mg/kg. After 28 days, blood was removed by perfusion with physiological saline, the brain was removed from the mouse, and a 40 um thick brain tissue slice was prepared using a Cryostat (Leica Biosystems, CM1950).
  • a fluorescently labeled secondary antibody (anti-mouse Alexa568, Invitrogen, A10037, 1:1000) that recognizes the antibody bound to pTau231 was added to the washed brain tissue section, reacted at room temperature for 2 hours, and then added with PBS. Washed using a solution. Afterwards, samples of the immunostained brain tissue sections were prepared to evaluate the level of pTau231. In addition, the antibody binding to pTau231 was changed to an antibody binding to pTau181 (Invitrogen, MN1050, 1:200), immunostaining was performed in the same manner as above, and a specimen was prepared for the immunostained brain tissue section. The level of pTau181 was evaluated.
  • Imaging of all stained samples was performed using the THUNDER imaging system (Leica), and image analysis was performed using the THUNDER imaging system (Leica). Meanwhile, a group administered intravenously with Vehicle (Vehicle) was used as a control group, and a group administered only anti-PD-L1 intravenously (anti-PD-L1) was used as a comparison group.
  • Vehicle Vehicle
  • anti-PD-L1 anti-PD-L1
  • the blood-brain barrier permeable fusion protein F3#01 was intravenously administered to 3xTg mice at a concentration of 20 mg/kg.
  • craniotomy surgery to remove part of the skin and skull was performed on 3xTg mice and wild-type mice, and a probe was inserted deep into the brain. It was made possible.
  • the mouse was anesthetized and a single electrode with 0.5 M ⁇ impedance (FHC, custom-made) was used to target layer II/III of the somatosensory cortex in the brain. It was placed using a micromanipulator (Eppendorf) within the active area of the right barrel cortex at a depth of 250 ⁇ m. Afterwards, Plexon's electrophysiology recording system was connected to electrodes to continuously measure the activity of the right barrel cortex inside the brain. All electrophysiological activities were recorded at a frequency of 40 kHz, and raw data was filtered and stored in the range of 0.5 to 200 Hz.
  • FHC micromanipulator
  • a self-made air puff whisker stimulator was connected to the Master9 electrical signal generator and set to stimulate the whiskers 64 times with the same frequency for 16 seconds.
  • Local field potential changes in the barrel cortex were recorded for at least 20 whisker stimulations for each animal.
  • the body's local field potential signals were analyzed using the self-written MATLAB software code using the open source Chronux 2,12 toolbox.
  • Vehicle was administered intravenously to wild-type C57BL/6 mice (WT), as a comparison group, Vehicle was administered intravenously to 3xTg mice (Vehicle), and only anti-PD-L1 was administered intravenously to 3xTg mice. group (anti-PD-L1) was used.
  • Vehicle was intravenously administered between wild-type C57BL/6 mice (WT) and 3xTg mice (Vehicle, anti-PD-L1, F3#01), an Alzheimer's animal model, and to 3xTg mice.
  • WT wild-type C57BL/6 mice
  • 3xTg mice 3xTg mice
  • Significant differences in basal biolocalized field potential were observed between the group (Vehicle) and the group administered anti-PD-L1 or blood-brain barrier permeable fusion protein F3#01 to 3xTg mice (anti-PD-L1, F3#01). I could't do it.
  • the raw local field potential results were compared by calculating the average value of all whisker stimulation results within each administration group, and as a result, the Vehicle administration group targeting the Alzheimer's animal model showed whisker stimulation compared to the WT group.
  • the level of increased neural activity was low. While this trend was also observed in the anti-PD-L1 administered group, a relatively high level of increased neural activity upon whisker stimulation was observed in the blood-brain barrier permeable fusion protein F3#01 administered group according to one embodiment.
  • the Vehicle administration group and the anti-PD-L1 administration group targeting the Alzheimer's animal model were compared to the WT group.
  • Neurological activity was significantly decreased.
  • the group administered with blood-brain barrier permeable fusion protein F3#01 according to one embodiment, neural activity was significantly increased compared to the Vehicle administered group and the anti-PD-L1 administered group, and among these, beta and gamma waves were at the level of the WT group. Neurological activity was restored.
  • the above results indicate that the level of improvement in local field potential in the F3#01 administration group was improved compared to the anti-PD-L1 administration group.
  • the purpose was to determine the effect of intravenous administration of the blood-brain barrier permeable fusion protein according to one embodiment on improving cognitive function and spatial perception ability.
  • the blood-brain barrier permeable fusion protein F3#01 according to one embodiment was intravenously administered to 3xTg mice (15 months old) or 5xFAD mice (7 months old) at a concentration of 20 mg/kg.
  • adaptation training to the experimenter's hand was performed for 20 minutes every day for 5 days to help the mouse adapt to the experimenter's hand.
  • the animals were left in the open field space for 15 minutes to undergo spatial adaptation training to prevent new stimuli and stress-related reactions to the open field space.
  • the memory analysis method for all objects is based on the experimental animal's exploration of the object.
  • Object exploration is defined as counting the time when the experimental animal's nose is pointed at a distance of 1.5 cm or less from the object, and the discrimination rate is calculated as It was calculated using the formula below and quantified through preference for new things.
  • Vehicle was administered intravenously to wild-type C57BL/6 mice (WT), and as a comparison group, Vehicle was administered intravenously to 3xTg mice or 5xFAD mice (Vehicle), and anti-PD was administered to 3xTg mice or 5xFAD mice.
  • a group administered intravenously only -L1 (anti-PD-L1) was used.
  • A is the exploration time for a new object
  • B is the object exploration time in the process of becoming familiar with the experimental object
  • C is the exploration time for all objects. However, the time to climb on the object or chew or lick the object during the test is excluded from the exploration time. .
  • the Vehicle-administered group and the anti-PD-L1-administered group targeting an Alzheimer's animal model showed cognitive improvement compared to wild-type C57BL/6 mice (WT). While functional and spatial perception abilities were significantly reduced, the cognitive function and spatial perception abilities of the group administered with blood-brain barrier permeable fusion protein F3#01 according to one embodiment recovered to the level of the WT group. The above results indicate that the efficacy of improving cognitive function and spatial perception ability in the F3#01 administration group was improved compared to the anti-PD-L1 administration group.
  • the purpose was to determine the effect of intravenous administration of the blood-brain barrier permeable fusion protein according to one embodiment on the functionality of neuronal junctions, that is, on the recovery of synaptic function of neurons.
  • the blood-brain barrier permeable fusion protein F3#01 according to one embodiment was intravenously administered to 3xTg mice (15 months old) or 5xFAD mice (7 months old) at a concentration of 20 mg/kg. After 1 month, blood was collected from the orbital vein or abdominal vein of the mouse, the brain was extracted from the mouse, the extracted brain tissue was rapidly frozen with liquid nitrogen, and then deep frozen until use. It was stored in . Meanwhile, total protein was extracted from the extracted brain tissue using RIPA buffer.
  • the extracted total protein was mixed with sample buffer, denatured, loaded on an SDS-PAGE gel, and then transferred to a PVDF membrane.
  • Synaptophysin or PSD95 protein was detected on the membrane using a primary antibody that detects Synaptophysin, PSD95, and beta-actin, and the intensity was measured using Image J software.
  • Vehicle was administered intravenously to wild-type C57BL/6 mice (WT), and as a comparison group, Vehicle was administered intravenously to 3xTg mice or 5xFAD mice (Vehicle), and anti-PD was administered to 3xTg mice or 5xFAD mice.
  • a group administered intravenously only -L1 (anti-PD-L1) was used.
  • the purpose was to determine the effect of the blood-brain barrier permeable fusion protein according to one example on the phagocytosis of microglial cells.
  • the blood-brain barrier permeable fusion protein F3#01 according to one example was administered intravenously to 3xTg mice (15 months old) at a concentration of 20 mg/kg.
  • the mice were intraperitoneally injected with Methoxy-XO4, which stains A ⁇ aggregates, and then perfused with sodium chloride perfusion solution (saline) to remove blood.
  • the brain of the mouse was removed, the hippocampus region was separated from the extracted brain tissue, and cell lysate for flow cytometry was prepared.
  • APC fluorescence-attached CD11b antibody (Miltenyi biotec, 130-113-793, 1:100) and propidium iodide (Sigma, P4864) were added to the cell lysate for the isolated hippocampal region.
  • the reaction was performed at 4°C for 40 minutes, cell staining was performed, and then the cells were washed using BSA solution. Thereafter, flow cytometry was performed on the stained sample using a FACS Aria Fusion flow cytometer (BD Biosciences). In the flow cytometry analysis, the number of CD11b-expressing cells (microglia) and the number of Methoxy .
  • Vehicle was administered intravenously to wild-type C57BL/6 mice (WT)
  • WT wild-type C57BL/6 mice
  • Vehicle was administered intravenously to 3xTg mice (Vehicle), and only anti-PD-L1 was administered intravenously to 3xTg mice.
  • group (anti-PD-L1) was used.
  • the purpose was to determine the effect of the blood-brain barrier permeability fusion protein according to an example on improving reactive astrogliosis.
  • the purpose was to determine the effect of the blood-brain barrier permeable fusion protein according to one example on the phagocytosis of microglial cells.
  • the blood-brain barrier permeable fusion protein F3#01 according to one example was administered intravenously to 3xTg mice (15 months old) at a concentration of 20 mg/kg. After 28 days, blood was removed by perfusion with physiological saline, the brain was removed from the mouse, and a 40 um thick brain tissue slice was prepared using a Cryostat (Leica Biosystems, CM1950).
  • a fluorescently labeled secondary antibody (anti-mouse Alexa568, ThermoFisher Scientific, A10037, 1:400) that recognizes the primary antibody was added to the washed brain tissue slice, reacted at room temperature for 10 minutes, and then added with PBS. Washed using a solution. Afterwards, nuclear counterstaining solution (Sigma, 4',6-Diamidino-2-Phenylindole, DAPI, 1:1,000, MBD0015) was added to the washed brain tissue section, reacted at room temperature for 10 minutes, and then added with PBS. Washed using a solution. Afterwards, a specimen of the immunostained brain tissue section was prepared and the ratio of GFAP-positive areas was evaluated.
  • the fusion protein has functions derived from a moiety having an effective binding affinity for the helical region of the transferrin receptor and a functional structure containing the same, and has high permeability to the blood-brain barrier and IgG1 antibody. It was found to have the same effect as high delivery to brain tissue, and through this, it was found to improve the treatment efficacy for diseases related to brain dysfunction targeting brain tissue.

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Abstract

La présente invention concerne une protéine de fusion perméable à la barrière hémato-encéphalique et son utilisation. La présente invention concerne une composition pharmaceutique comprenant une protéine de fusion perméable à la barrière hémato-encéphalique en tant que principe actif pour la prévention ou le traitement de maladies associées à un dysfonctionnement cérébral, la protéine de fusion perméable à la barrière hémato-encéphalique comprenant : un anticorps IgG ; et une fraction de liaison pour la région hélicoïdale du récepteur de transferrine tétravalent (TfR), reliée à la région C-terminale de chaîne légère et à la région C-terminale de chaîne lourde dans l'anticorps IgG.
PCT/KR2023/016928 2022-10-28 2023-10-27 Protéine de fusion perméable à la barrière hémato-encéphalique et ses utilisations WO2024091079A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6963807B2 (en) * 2000-09-08 2005-11-08 Oxford Glycosciences (Uk) Ltd. Automated identification of peptides
US7473531B1 (en) * 2003-08-08 2009-01-06 Colora Corporation Pancreatic cancer targets and uses thereof
KR20150039798A (ko) * 2012-08-29 2015-04-13 에프. 호프만-라 로슈 아게 혈액 뇌 장벽 셔틀
KR20190099470A (ko) * 2016-12-26 2019-08-27 제이씨알 파마 가부시키가이샤 혈액뇌관문을 통과하는 신규한 항인간 트랜스페린 수용체 항체
KR20210074279A (ko) * 2018-08-22 2021-06-21 데날리 테라퓨틱스 인크. 항-her2 폴리펩타이드 및 이의 사용방법
KR20230133248A (ko) * 2021-12-31 2023-09-19 주식회사 아임뉴런 뇌혈관장벽 투과성 융합 단백질 및 이의 용도

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6963807B2 (en) * 2000-09-08 2005-11-08 Oxford Glycosciences (Uk) Ltd. Automated identification of peptides
US7473531B1 (en) * 2003-08-08 2009-01-06 Colora Corporation Pancreatic cancer targets and uses thereof
KR20150039798A (ko) * 2012-08-29 2015-04-13 에프. 호프만-라 로슈 아게 혈액 뇌 장벽 셔틀
KR20190099470A (ko) * 2016-12-26 2019-08-27 제이씨알 파마 가부시키가이샤 혈액뇌관문을 통과하는 신규한 항인간 트랜스페린 수용체 항체
KR20210074279A (ko) * 2018-08-22 2021-06-21 데날리 테라퓨틱스 인크. 항-her2 폴리펩타이드 및 이의 사용방법
KR20230133248A (ko) * 2021-12-31 2023-09-19 주식회사 아임뉴런 뇌혈관장벽 투과성 융합 단백질 및 이의 용도

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