WO2023210585A1 - 標的化剤 - Google Patents

標的化剤 Download PDF

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Publication number
WO2023210585A1
WO2023210585A1 PCT/JP2023/016125 JP2023016125W WO2023210585A1 WO 2023210585 A1 WO2023210585 A1 WO 2023210585A1 JP 2023016125 W JP2023016125 W JP 2023016125W WO 2023210585 A1 WO2023210585 A1 WO 2023210585A1
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Prior art keywords
antibody
seq
targeting agent
amino acid
domain
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English (en)
French (fr)
Japanese (ja)
Inventor
法弘 湯本
大二郎 猪俣
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Jiksak Bioengineering Inc
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Jiksak Bioengineering Inc
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Priority to US18/858,923 priority Critical patent/US20250277028A1/en
Priority to AU2023259903A priority patent/AU2023259903A1/en
Priority to JP2024517316A priority patent/JP7849074B2/ja
Priority to EP23796325.1A priority patent/EP4516316A4/en
Publication of WO2023210585A1 publication Critical patent/WO2023210585A1/ja
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6843—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a targeting agent for motor neurons, an agent for visualizing motor neurons, a composition containing the same, a targeting method using the same, a method for visualizing motor neurons, a method for preventing or treating a condition or disease, etc. .
  • Nerves are composed of central nerves and peripheral nerves, and regulate various emotions, muscles, and the functions of internal organs. Such neurological functions may deteriorate due to nerve damage, disease, aging, etc. In such cases, mental and physical health may be impaired, and the ability to lead a social life may be severely affected. Therefore, maintaining and improving neurological function can be said to be an extremely important issue as it is directly linked to maintaining and improving quality of life (QOL).
  • QOL quality of life
  • the central nerve and peripheral nerve are each composed of nerve cells, and these cells exchange signals with each other via synapses.
  • a synapse is a junction that includes a gap formed between the axon terminal of a neuron (presynaptic part) and the dendrite of another neuron or a cell such as a skeletal muscle or organ (postsynaptic part). , chemicals released from the presynapse transmit signals by binding to receptors present at the postsynapse. Synapse formation is triggered by interactions between specific membrane proteins expressed pre- and post-synaptic.
  • Patent Document 1 describes that a specific peptide has a dendrite outgrowth-promoting effect and a synapse formation-promoting effect in primary cultured cortical neuron cells (PCN), and that such a peptide is used for the treatment of mild cognitive impairment or early dementia. It is described that it is used.
  • Patent Document 2 describes that C-terminal fragment ⁇ (CTF ⁇ ), which is generated when amyloid precursor protein (APP) is cleaved by ⁇ -secretase, promotes synapse formation, and that CTF ⁇ can be used to treat neurodegenerative diseases, etc. It is described that it is used for.
  • CTF ⁇ C-terminal fragment ⁇
  • Patent Document 3 describes a method for culturing motor neurons having a presynaptic region using microbeads on which an LRRTM molecule or a fusion protein containing the molecule is immobilized.
  • an object of the present invention is to provide a means for targeting a substance to motor neurons and a means for visualizing the targeted site.
  • the present inventors focused on the fact that a specific protein is expressed on the membrane of synaptic vesicles, and as a result of intensive studies to solve the above problems, the intravesicular domain (N-terminal). Furthermore, the present inventors discovered that similar effects can be obtained by using antibodies that bind to other membrane proteins of synaptic vesicles, leading to the completion of the present invention.
  • the present invention includes the following.
  • a targeting agent for motor neurons comprising an antibody capable of binding to the intravesicular domain of a membrane protein present in synaptic vesicles of motor neurons.
  • the targeting agent according to [1] further comprising a labeling substance and/or a physiologically active substance.
  • the targeting agent according to [2] comprising a conjugate of the antibody and the labeling substance and/or the physiologically active substance.
  • the membrane protein includes any one protein selected from the group consisting of synaptotagmin 2, synaptic vesicle glycoprotein 2A, synaptogyrin 1, synaptophysin, and synaptotagmin 1, [1] to [4]
  • the targeting agent according to any one of.
  • the intravesicular domain has an amino acid sequence represented by any one selected from the group consisting of SEQ ID NOs: 3, 7-12, 15, 16, 19, 20, and 23; SEQ ID NOs: 3, 7-12, An amino acid sequence in which one or more amino acids are added, deleted, and/or substituted in any one of the amino acid sequences selected from the group consisting of 15, 16, 19, 20, and 23; or SEQ ID NO: 3 , [5] or [6], consisting of an amino acid sequence having 90% or more sequence identity with any one of the amino acid sequences selected from the group consisting of , 7 to 12, 15, 16, 19, 20, and 23.
  • the targeting agent described in. [8] The targeting agent according to any one of [2] to [7], wherein the labeling substance is a fluorescent molecule.
  • the physiologically active substance is one or more selected from the group consisting of a synapse formation promoter, a synapse maintenance agent, a muscle enhancer, and a nerve cell function modifying agent.
  • Targeting agents as described.
  • a pharmaceutical composition comprising the targeting agent according to any one of [2] to [11].
  • a method for targeting a labeling substance and/or a physiologically active substance to a motor neuron comprising the step of delivering a composition to a synapse of the motor neuron.
  • a condition or disease comprising the step of contacting the targeting agent according to any one of [2] to [11] with a motor neuron, and the step of delivering the targeting agent to the synapse of the motor neuron. prevention or treatment methods.
  • the prevention or treatment method according to [15] wherein the condition or disease is a condition or disease exhibiting a decline in neurological function.
  • the prevention or treatment method according to [15] or [18], wherein the condition or disease is a neurological disease or a neuromuscular disease.
  • a composition for targeting motor neurons comprising an antibody capable of binding to the intravesicular domain of a membrane protein present in synaptic vesicles of motor neurons.
  • a desired substance can be targeted to motor neurons using an antibody.
  • desired substances can be delivered to motor neurons (eg, synaptic vesicles within the cells).
  • motor neurons eg, synaptic vesicles within the cells.
  • the substance is a physiologically active substance or a therapeutic agent, symptoms and/or diseases caused by motor neuron abnormalities can be treated.
  • motor neurons can be visualized.
  • FIG. 1 is a diagram schematically showing the experimental procedure for presynaptic induction and antibody delivery using LRRTM2 beads.
  • nerve axons grow from the neurospheres.
  • LRRTM2 beads by seeding LRRTM2 beads there, a presynapse is induced from the elongated nerve axon to the LRRTM2 bead surface.
  • a normal rabbit IgG antibody (control) or an anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) was introduced into the plate in which presynapses were induced in this way, and the cells were stimulated with chemicals.
  • FIG. 2-1 is a diagram showing a fluorescence image when an antibody delivery experiment using spontaneous activity was performed at an antibody concentration of 1 ⁇ g/mL.
  • a and B show the localization of the administered antibody when normal rabbit IgG antibody (A) and anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) (B) were used as antibodies, respectively.
  • This is a fluorescence image showing.
  • the scale bar is common to A and B and indicates 100 ⁇ m.
  • Figure 2-2 shows a fluorescence image when a spontaneous activity delivery experiment of anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) was carried out at an antibody concentration of 1 ⁇ g/mL.
  • a and B are fluorescent images showing the localization of anti-SYT2 N-terminal antibody in LRRTM2 beads (A) where nerve axons are not densely packed and LRRTM2 beads (B) where nerve axons are densely packed, respectively.
  • the dashed circle indicates the position of LRRTM2 beads.
  • FIG. 3 is a diagram showing a fluorescence image when an antibody delivery experiment using spontaneous activity was performed at an antibody concentration of 10 ⁇ g/mL.
  • a and B show the localization of the administered antibody when normal rabbit IgG antibody (A) and anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) (B) were used as antibodies, respectively.
  • This is a fluorescence image showing.
  • the scale bar is common to A and B and indicates 100 ⁇ m.
  • FIG. 4 is a diagram showing fluorescence images obtained when an antibody delivery experiment was performed by stimulating nerve cells with 4-aminopyridine for 10 minutes.
  • a and B show the localization of the administered antibody when normal rabbit IgG antibody (A) and anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) (B) were used as antibodies, respectively.
  • This is a fluorescence image showing.
  • the scale bar is common to A and B and indicates 100 ⁇ m.
  • FIG. 5 is a diagram showing fluorescence images when an antibody delivery experiment was performed by stimulating nerve cells with 4-aminopyridine for 30 minutes.
  • a and B show the localization of the administered antibody when normal rabbit IgG antibody (A) and anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) (B) were used as antibodies, respectively.
  • This is a fluorescence image showing. In the figure, the scale bar is common to A and B and indicates 100 ⁇ m.
  • FIG. 6 shows fluorescence images at the neuromuscular junction 12 hours after administration of antibodies by tail vein injection.
  • a and B show the localization of the administered antibody when normal rabbit IgG antibody (A) and anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) (B) were used as antibodies, respectively.
  • ⁇ -BgtX refers to a stained image showing an ⁇ -bungarotoxin signal
  • SYN1 refers to a stained image showing a synapsin 1 signal.
  • the arrowhead indicates the location of the neuromuscular junction where the anti-SYT2 N-terminal antibody was delivered, and the scale bar is common to each image and indicates 50 ⁇ m.
  • FIG. 7 shows fluorescence images at the neuromuscular junction 72 hours after administration of antibodies by tail vein injection.
  • a and B show the localization of the administered antibody when normal rabbit IgG antibody (A) and anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) (B) were used as antibodies, respectively.
  • This is a fluorescence image showing.
  • ⁇ -BgtX refers to a stained image showing an ⁇ -bungarotoxin signal
  • SYN1 refers to a stained image showing a synapsin 1 signal.
  • the arrowhead indicates the location of the neuromuscular junction where the anti-SYT2 N-terminal antibody was delivered, and the scale bar is common to each image and indicates 50 ⁇ m.
  • FIG. 8 shows fluorescence images at the neuromuscular junction 12 hours after administration of antibodies by intraperitoneal injection.
  • a and B show the localization of the administered antibody when normal rabbit IgG antibody (A) and anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) (B) were used as antibodies, respectively.
  • This is a fluorescence image showing.
  • ⁇ -BgtX refers to a stained image showing an ⁇ -bungarotoxin signal
  • SYN1 refers to a stained image showing a synapsin 1 signal.
  • FIG. 9 shows fluorescence images at the neuromuscular junction 72 hours after administration of antibodies by intraperitoneal injection.
  • a and B show the localization of the administered antibody when normal rabbit IgG antibody (A) and anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) (B) were used as antibodies, respectively. This is a fluorescence image showing.
  • ⁇ -BgtX refers to a stained image showing an ⁇ -bungarotoxin signal
  • SYN1 refers to a stained image showing a synapsin 1 signal.
  • the arrowhead indicates the location of the neuromuscular junction where the anti-SYT2 N-terminal antibody was delivered, and the scale bar is common to each image and indicates 50 ⁇ m.
  • FIG. 10 shows electron microscopy images of neuromuscular junctions 72 hours after administration of antibodies by tail vein injection.
  • a and B show the localization of the administered antibody when normal rabbit IgG antibody (A) and anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) (B) were used as antibodies, respectively.
  • FIG. 11 shows an electron microscopy image of the neuromuscular junction 72 hours after administration of anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) by tail vein injection.
  • A is an electron microscope image of the same field of view as FIG. 10B
  • B is an electron microscope image in which the area surrounded by the solid black line in A is enlarged.
  • black dots indicate the positions of antibodies.
  • the area surrounded by white lines indicates nerve axon terminals of motor neurons
  • the area surrounded by black dashed lines indicates gastrocnemius muscle cells, which are skeletal muscles
  • the scale bar indicates 2 ⁇ m.
  • a to c represent synaptic vesicles that do not contain antibodies (a), synaptic vesicles that contain antibodies (b), and mitochondria (c), respectively.
  • the scale bar indicates 500 nm.
  • ChAT refers to a stained image showing the signal of choline acetyltransferase.
  • the dashed line indicates the boundary between the spinal ventral horn and the white matter, and the spinal ventral horn is located to the left of the dashed line.
  • Arrowheads illustrate the location of cell bodies of motor neurons to which anti-SYT2 N-terminal antibodies were delivered, scale bars are common to each image and indicate 100 ⁇ m.
  • FIG. 13 is a diagram showing an enlarged fluorescence image of the ventral horn of the spinal cord 72 hours after administration of normal rabbit IgG antibody by tail vein injection.
  • ChAT refers to a stained image showing the signal of choline acetyltransferase.
  • the scale bar is common to each image and indicates 100 ⁇ m.
  • FIG. 14 is a diagram showing an enlarged fluorescent image of the ventral horn of the spinal cord 72 hours after administration of anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) by tail vein injection.
  • ChAT refers to a stained image showing the signal of choline acetyltransferase.
  • FIG. 15 is a diagram showing fluorescence images in the ventral horn of the spinal cord 6 to 72 hours after administration of anti-synaptotagmin 2 intravesicular domain (N-terminus) antibody (anti-SYT2 N-terminus antibody) by tail vein injection.
  • the fluorescent signal shown in the figure is a signal based on anti-SYT2 N-terminal antibody.
  • the scale bar is common to each image and indicates 100 ⁇ m.
  • FIG. 16 is a diagram showing fluorescence images of the anterior horn of the spinal cord 120 to 240 hours after administration of anti-synaptotagmin 2 intravesicular domain (N-terminus) antibody (anti-SYT2 N-terminus antibody) by tail vein injection.
  • the fluorescent signal shown in the figure is a signal based on anti-SYT2 N-terminal antibody.
  • the scale bar is common to each image and indicates 100 ⁇ m.
  • FIG. 17 is a diagram showing fluorescence images in a control antibody group into which a conjugate of a normal rabbit antibody and monomethyl auristatin E (MMAE) was introduced.
  • the fluorescent signal shown in the figure is a signal based on ⁇ III tubulin (Tuj1).
  • FIG. 18 is a diagram showing fluorescence images of the SYT2 antibody group into which a conjugate of anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) and monomethyl auristatin E (MMAE) was introduced.
  • the fluorescent signal shown in the figure is a signal based on ⁇ III tubulin (Tuj1).
  • Figures B and C are diagrams showing enlarged images of the white frame area in Figure A.
  • FIG. 19 is a diagram showing a graph quantifying the pharmacological effects based on monomethyl auristatin E (MMAE).
  • MMAE monomethyl auristatin E
  • the relative axon volume is a value normalized to the result obtained when a conjugate of a normal rabbit antibody and MMAE was used as a control ("Cont. IgG-MMAE" in the figure) as 100%.
  • the dashed line indicates the position where the relative axon amount is 100%
  • MMAE is the single introduction group in which MMAE was introduced alone
  • ⁇ -SYT2 IgG-MMAE is the anti-synaptotagmin 2 intravesicular domain (N-terminal )
  • error bars indicate standard errors
  • "*” indicates p ⁇ 0.05
  • *** indicates p ⁇ 0.001.
  • FIG. 3 is a diagram showing a fluorescence image in a firing group.
  • the fluorescent signal shown in the figure is a signal based on ⁇ III tubulin (Tuj1).
  • the scale bar indicates 1 mm.
  • Figure 21 shows that a conjugate of anti-synaptotagmin 2 intravesicular domain (N-terminus) antibody (anti-SYT2 N-terminus antibody) and monomethyl auristatin E (MMAE) was introduced into neurons under conditions that do not induce presynaptic formation.
  • N-terminus anti-synaptotagmin 2 intravesicular domain
  • MMAE monomethyl auristatin E
  • FIG. 3 is a diagram showing fluorescence images in the endocytosis inhibition group cultured.
  • the fluorescent signal shown in the figure is a signal based on ⁇ III tubulin (Tuj1).
  • the scale bar indicates 1 mm.
  • MMAE monomethyl auristatin E
  • anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody anti-SYT2 N-terminal antibody.
  • the relative axon mass is a value normalized to 100% of the synaptic non-formation group in which neurons were cultured under conditions that do not induce the formation of presynapses.
  • FIG. 24 is a diagram showing a graph quantifying the pharmacological effects based on monomethyl auristatin E (MMAE) delivered by various antibodies.
  • the present invention provides targeting agents for motor neurons (referred to as “intravesicular domain antibodies”) that are capable of binding to intravesicular domains of membrane proteins present in synaptic vesicles of motor neurons (referred to as “intravesicular domain antibodies”). (referred to as “targeting agent of the present invention”).
  • Membrane protein refers to a protein that exists on biological membranes.
  • the “membrane protein” in the present invention particularly refers to a protein present on the membrane of synaptic vesicles.
  • Membrane proteins in the present invention include transmembrane proteins, membrane surface proteins, and lipid-modified proteins. Both superficial membrane proteins and lipid-modified proteins are proteins that do not have transmembrane domains.
  • the type of membrane protein in the targeting agent of the present invention is not particularly limited as long as it contains an intravesicular domain, but is preferably a transmembrane protein.
  • the term "intravesicular domain" of a protein refers to a protein region in a membrane protein that is exposed to the lumen of a synaptic vesicle.
  • the length of the intravesicular domain of the membrane protein in the targeting agent of the present invention is not particularly limited.
  • continuous vesicles with 1 or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 7 or more amino acids, 8 or more amino acids, 9 or more amino acids, 10 or more amino acids, or 11 or more amino acids.
  • Membrane proteins with internal domains can be used.
  • a transmembrane protein having a continuous intravesicular domain of 4 or more amino acids can be suitably used.
  • the lumen of a synaptic vesicle refers to the space that exists on the opposite side of the synaptic vesicle from the cytoplasm.
  • the lumen of the synaptic vesicle communicates with the extracellular space when the synaptic vesicle fuses with the cell membrane. Therefore, the intravesicular domain of the membrane protein in the targeting agent of the present invention is exposed outside the cell when the synaptic vesicle fuses with the cell membrane.
  • synaptotagmin 2 as a membrane protein as an example.
  • the present invention relates to a targeting agent for motor neurons, which includes an antibody capable of binding to the intravesicular domain (N-terminal portion) of synaptotagmin 2 (referred to as "anti-SYT2 N-terminal antibody”).
  • synaptotagmin 2 refers to one of the membrane proteins belonging to the synaptotagmin family.
  • the synaptotagmin family includes 17 proteins in mammals, among which synaptotagmin 2 is mainly expressed on the presynaptic vesicle membrane of the neuromuscular junction in peripheral nerves, and is a calcium ion Proteins that promote the fusion of synaptic vesicles and cell membranes in a dependent manner (e.g., Rickman, Colin, et al., Journal of Biological Chemistry 279.13 (2004): 12574-12579., Stephanie Bauche, et al., Neurol). Genet. 2020 Dec 3;6(6):e534. doi: 10.1212).
  • Synaptotagmin 2 is a single-transmembrane protein that contains an intravesicular domain, a transmembrane domain, and a cytoplasmic domain in order from the N-terminus.
  • the C-terminal cytoplasmic domain has a tandem C2 domain with calcium ion binding ability, and it is known that this cytoplasmic domain is primarily responsible for membrane fusion-related functions.
  • the intravesicular domain of synaptotagmin 2 is exposed in the lumen of synaptic vesicles.
  • the lumen of the synaptic vesicle connects with the extracellular space, and the intravesicular domain of synaptotagmin 2 is temporarily exposed to the outside of the cell. do.
  • the cell membrane portion containing synaptotagmin 2 is collected into the cell by endocytosis as a synaptic vesicle membrane and reused as a synaptic vesicle.
  • the intravesicular domain of synaptotagmin 2 is exposed again to the synaptic vesicle lumen.
  • an exemplary human synaptotagmin 2 is a protein consisting of 419 amino acids whose amino acid sequence is represented by SEQ ID NO: 1.
  • the position of each domain is, for example, in SEQ ID NO: 1, the intravesicular domain is the region shown by the amino acid sequence from positions 1 to 62, the transmembrane domain is the region shown by the amino acid sequence from positions 63 to 83, and the cytoplasmic domain is the region indicated by the amino acid sequence from position 84 to position 419.
  • An exemplary mouse synaptotagmin 2 is a protein consisting of 422 amino acids whose amino acid sequence is represented by SEQ ID NO: 2.
  • the positions of each domain are as follows: the intravesicular domain is the region shown by the amino acid sequence from positions 1 to 60, the transmembrane domain is the region shown by the amino acid sequence from positions 61 to 87, and the cytoplasmic domain. is the region indicated by the amino acid sequence from position 88 to position 422.
  • Sequence information on synaptotagmin 2 of other organisms can be easily obtained from known databases such as the NCBI database.
  • the intravesicular domain of synaptotagmin 2 refers to all or part of the intravesicular domain present in the N-terminal portion of synaptotagmin 2.
  • the entire intravesicular domain includes, for example, the region shown by the amino acid sequence from positions 1 to 62 in SEQ ID NO: 1 (SEQ ID NO: 3), and the amino acid sequence from positions 1 to 60 in SEQ ID NO: 2 (SEQ ID NO: 4). Includes the area indicated by .
  • a part of the intravesicular domain includes, for example, a region shown by a partial sequence of arbitrary length in SEQ ID NOs: 3 and 4.
  • one or more amino acids Included is the N-terminal portion of synaptotagmin 2 having a mutant amino acid sequence with substitutions, insertions, deletions, and/or additions.
  • “plurality” refers to a number of 2 or more, for example, 2 to 6, 2 to 5, 2 to 4, or 2 to 3, preferably 2.
  • an antibody capable of binding to any membrane protein other than synaptotagmin 2 exemplified above can be used.
  • specific membrane proteins in the targeting agent of the present invention include, for example, synaptotagmin family, major facilitator superfamily, synaptic vesicle glycoprotein 2 family, synaptogyrin family, synaptophysin/synaptobrevin. family, synaptobrevin family, vesicular amine transporter family, solute carrier family 5, vesicular glutamate transporter family, amino acid/polyamine transporter family, secreted carrier-associated membrane protein family, glutamate transporter subfamily, autophagy-related Examples include proteins belonging to any one family selected from the group consisting of protein 9 family, sugar transporter family, and vacuolar ATPase subunit S1 family.
  • membrane proteins in the targeting agent of the present invention include, in addition to synaptotagmin 2, for example, synaptic vesicle glycoprotein 2A, synaptogyrin 1, synaptophysin, synaptotagmin 1, synaptogyrin 3, and synaptogyrin 3.
  • synaptotagmin 2A for example, synaptic vesicle glycoprotein 2A, synaptogyrin 1, synaptophysin, synaptotagmin 1, synaptogyrin 3, and synaptogyrin 3.
  • vesicular acetylcholine transporter high affinity choline transporter, vesicular glutamate transporter 1, vesicular glutamate transporter 3, vesicular GABA transporter, synaptic vesicle glycoprotein 2B, synaptic vesicle glycoprotein 2C, vesicle-associated membrane protein 1, synaptogyrin 4, synaptotagmin 4, synaptotagmin 7, secreted carrier-associated membrane protein 5, synaptic vesicle glycoprotein 2-associated protein (SVOP), excitatory amino acid transporter 3, autophagy-related protein 9A, glucose trans Examples include porter 4 and ATPase H+ transport accessory protein 1.
  • the membrane protein in the targeting agent of the present invention includes any one protein selected from the group consisting of synaptotagmin 2, synaptic vesicle glycoprotein 2A, synaptogyrin 1, synaptophysin, and synaptotagmin 1.
  • the present invention provides an antibody capable of binding to the intravesicular domain of any one protein selected from the group consisting of synaptotagmin 2, synaptic vesicle glycoprotein 2A, synaptogyrin 1, synaptophysin, and synaptotagmin 1.
  • a targeting agent comprising:
  • Synaptic vesicle glycoprotein 2A refers to one of the 12 transmembrane proteins belonging to the synaptic vesicle glycoprotein 2 family of the major facilitator superfamily. This protein is thought to be involved in the control of regulatory secretion in neurons and endocrine cells, and to promote low-frequency neurotransmission in resting neurons. Synaptic vesicle glycoprotein 2A is also known by other names such as KIAA0736, SV2, and SLC22B1. Exemplary synaptic vesicle glycoprotein 2A is a human-derived protein consisting of 742 amino acids whose amino acid sequence is represented by SEQ ID NO: 6.
  • the intravesicular domain of synaptic vesicle glycoprotein 2A refers to all or part of the intravesicular domain of synaptic vesicle glycoprotein 2A.
  • the entire intravesicular domain includes, for example, the region shown by the amino acid sequence from position 191 to position 205 in SEQ ID NO: 6 (sequence: PSAEKDMCLSDSNKG; SEQ ID NO: 7), the amino acid sequence from position 255 to position 262 ( Sequence: YGTFLFCR; SEQ ID NO: 8), region indicated by the amino acid sequence from position 316 to position 334 (sequence: PHYGWSFQMGSAYQFHSWR; SEQ ID NO: 9), amino acid sequence from position 469 to position 598 (sequence: PDMIRHLQAVDYASRTKVFPGERVEHVTFNFTLENQIHRGGQYFNDKFIGLRLKSVSFEDSLFEECYFEDVTSSNTFFRNCTFINTVFYNTDLFEYKFV
  • a part of the intravesicular domain includes, for example, a region represented by a partial sequence of any length in SEQ ID NOs: 7 to 12.
  • the length of the partial sequence is as described for anti-SYT2 N-terminal antibody. More specifically, for example, in the region shown by the amino acid sequence from position 451 to position 550 of SEQ ID NO: 6 (SEQ ID NO: 13: MGVWFTMSFSYYGLTVWFPDMIRHLQAVDYASRTKVFPGERVEHVTFNFTLENQIHRGGQYFNDKFIGLRLKSVSFEDSLFEECYFEDVTSSNTFFRNCT) and the amino acid sequence of SEQ ID NO: 13, one or more amino acids are substituted, Included are protein regions of synaptic vesicle glycoprotein 2A that have insertions, deletions, and/or additions of mutant amino acid sequences.
  • Synaptogyrin 1 refers to one of the four transmembrane proteins belonging to the synaptogyrin family. This protein is present in presynaptic vesicles of neurons and is thought to be involved in regulated exocytosis, synaptic vesicle formation and maturation, and synaptic plasticity.
  • An exemplary synaptogyrin 1 is a human-derived protein whose amino acid sequence is represented by SEQ ID NO: 14 and consists of 233 amino acids.
  • the intravesicular domain of synaptogyrin 1 refers to all or part of the intravesicular domain of synaptogyrin 1.
  • the entire intravesicular domain includes, for example, the region shown by the amino acid sequence from position 45 to position 71 in SEQ ID NO: 14 (sequence: NEGYLNSASEGEEFCIYNRNPNACSYG; SEQ ID NO: 15), the amino acid sequence from position 125 to position 148 ( Sequence: YLANQWQVSKPKDNPLNEGTDAAR; SEQ ID NO: 16) is included.
  • a part of the intravesicular domain includes, for example, a region shown by a partial sequence of arbitrary length in SEQ ID NOs: 15 and 16.
  • the length of the partial sequence is as described for anti-SYT2 N-terminal antibody. More specifically, for example, in the region shown by the amino acid sequence from position 130 to position 146 of SEQ ID NO: 14 (SEQ ID NO: 17: WQVSKPKDNPLNEGTDA) and the amino acid sequence of SEQ ID NO: 14, one or more amino acids are substituted, Included are protein regions of synaptogyrin 1 that have insertions, deletions, and/or additions of mutant amino acid sequences.
  • Synaptophysin refers to one of the four transmembrane proteins belonging to the synaptophysin/synaptobrevin family. This protein is thought to be involved in the organization of vesicle membrane components, targeting of vesicles to the cell membrane, and control of synaptic plasticity. Synaptophysin is also known by other names such as MRX96, tumor synaptic vesicle protein P38, MRXSYP, and XLID96.
  • An exemplary synaptophysin is a human-derived protein whose amino acid sequence consists of 313 amino acids represented by SEQ ID NO: 18.
  • the intravesicular domain of synaptophysin refers to all or part of the intravesicular domain of synaptophysin.
  • the entire intravesicular domain includes, for example, the region shown by the amino acid sequence from position 50 to position 106 in SEQ ID NO: 18 (Sequence: ELQLSVDCANKTESDLSIEVEFEYPFRLHQVYFDAPTCRGGTTKVFLVGDYSSSAEF; SEQ ID NO: 19), the amino acid sequence from position 162 to position 199 (Sequence: ELQLSVDCANKTESDLSIEVEFEYPFRLHQVYFDAPTCRGGTTKVFLVGDYSSSAEF; Sequence: KGLSDVKMATDPENIIKEMPVCRQTGNTCKELRDPVTS; SEQ ID NO: 20) is included.
  • a part of the intravesicular domain includes, for example, a region shown by a partial sequence of arbitrary length in SEQ ID NOs: 19 and 20.
  • the length of the partial sequence is as described for anti-SYT2 N-terminal antibody. More specifically, for example, in the region shown by the amino acid sequence from position 178 to position 190 of SEQ ID NO: 18 (SEQ ID NO: 21: CRQTGNTCKELRD) and the amino acid sequence of SEQ ID NO: 21, one or more amino acids are substituted, Included are protein regions of synaptophysin that have insertions, deletions, and/or additions with variant amino acid sequences.
  • Synaptotagmin 1 refers to one of the single-transmembrane proteins belonging to the synaptotagmin family. This protein is thought to be one of the proteins that promotes the fusion of synaptic vesicles and cell membranes in a calcium ion-dependent manner. Synaptotagmin 1 is also known by other names such as P65, SVP65, SYT, and BAGOS. Exemplary synaptotagmin 1 is a human-derived protein whose amino acid sequence consists of 422 amino acids represented by SEQ ID NO: 22.
  • the intravesicular domain of synaptotagmin 1 refers to all or part of the intravesicular domain of synaptotagmin 1.
  • the entire intravesicular domain includes, for example, the region shown by the amino acid sequence from position 1 to position 57 in SEQ ID NO: 22 (sequence: MVSESHHEALAAPPVTTVATVLPSNATEPASPGEGKEDAFSKLKEKFMNELHKIPLP; SEQ ID NO: 23).
  • a part of the intravesicular domain includes, for example, a region shown by a partial sequence of arbitrary length in SEQ ID NO: 23. The length of the partial sequence is as described for anti-SYT2 N-terminal antibody.
  • SEQ ID NO: 24 MVSASRPE
  • amino acid sequence of SEQ ID NO: 24 one or more amino acids are substituted, Included are N-terminal portions of synaptotagmin 1 having insertions, deletions, and/or additions of mutant amino acid sequences.
  • Synaptogyrin 3 refers to one of the four transmembrane proteins belonging to the synaptogyrin family. This protein is thought to be involved in regulated exocytosis, dopamine recycling, etc.
  • An exemplary synaptogyrin 3 is a human-derived protein consisting of 229 amino acids whose amino acid sequence is represented by SEQ ID NO: 25.
  • the intravesicular domain of synaptogyrin 3 refers to all or part of the intravesicular domain of synaptogyrin 3.
  • the entire intravesicular domain includes, for example, the region shown by the amino acid sequence from position 51 to position 69 in SEQ ID NO: 25 (sequence: TDSGPELRCVFNGNAGACR; SEQ ID NO: 26), the amino acid sequence from position 126 to position 147 ( Sequence: LTNQWQRTAPGPATTQAGDAAR; SEQ ID NO: 27) is included.
  • a part of the intravesicular domain includes, for example, a region shown by a partial sequence of arbitrary length in SEQ ID NOs: 26 and 27. The length of the partial sequence is as described for anti-SYT2 N-terminal antibody.
  • Vesicular Acetylcholine Transporter refers to one of the 12 transmembrane membrane proteins belonging to the vesicular amine transporter family. This protein is a transmembrane protein that transports acetylcholine into secretory vesicles and releases it outside the cell. Vesicular acetylcholine transporter is also known by other names such as VACHT, SLC18A3, and CMS21. Specifically, an exemplary vesicular acetylcholine transporter is a protein of human origin consisting of 532 amino acids whose amino acid sequence is represented by SEQ ID NO: 29.
  • the intravesicular domain of the vesicular acetylcholine transporter refers to all or part of the intravesicular domain of the vesicular acetylcholine transporter.
  • the entire intravesicular domain includes, for example, the region shown by the amino acid sequence from position 55 to position 125 in SEQ ID NO: 29 (sequence: PIVPDYIAHMRGGGEGPTRTPEVWEPTLPLPTPANASAYTANTSASPTAAWPAGSALRPRYPTESEDVKIG; SEQ ID NO: 30), the amino acid sequence from position 174 to position 182 ( Sequence: DYATLFAAR; SEQ ID NO: 31), region indicated by the amino acid sequence from position 235 to position 242 (sequence: LYEFAGKR; SEQ ID NO: 32), amino acid sequence from position 311 to position 325 (sequence: TIATWMKHTMAASEW; SEQ ID NO: 33), the region indicated by the amino acid sequence from position 378 to
  • High Affinity Choline Transporter 1 refers to one of the 13 transmembrane proteins belonging to solute carrier family 5. This protein is a sodium ion- and chloride ion-dependent transmembrane transporter for the high affinity uptake of choline from the extracellular space for acetylcholine synthesis. High affinity choline transporter is also known by other names such as SLC5A7, HCHT, and CHT.
  • An exemplary high-affinity choline transporter is a human-derived protein whose amino acid sequence consists of 580 amino acids represented by SEQ ID NO: 36.
  • the intravesicular domain of high affinity choline transporter 1 refers to all or part of the intravesicular domain of high affinity choline transporter 1.
  • the entire intravesicular domain includes, for example, the region shown by the amino acid sequence from position 1 to position 6 in SEQ ID NO: 36 (sequence: MAFHVE; SEQ ID NO: 37), the amino acid sequence from position 70 to position 81 ( Sequence: GTAEAVYVPGYG; SEQ ID NO: 38), region indicated by the amino acid sequence from position 147 to position 164 (sequence: GEMFWAAAIFSALGATISVIIDVDMHIS; SEQ ID NO: 39), amino acid sequence from position 213 to position 237 (sequence: ADIGFTAVHAKYQKPWLGTVDSSEV; SEQ ID NO: 40), the region indicated by the amino acid sequence from position 296 to position 317 (sequence: ASTDWNQTAYGLPDPKTTEEAD; SEQ ID NO: 41),
  • VGLUT1 Vesicular Glutamate Transporter 1 (VGLUT1) refers to one of the 12 transmembrane proteins belonging to the vesicular glutamate transporter family. This protein is considered to be a multifunctional symport transporter that transports multiple types of ions, including sodium and phosphate ions, as well as L-glutamate and chloride ions. Vesicular glutamate transporter 1 is also known by other names such as SLC17A7 and BNPI. Exemplary vesicular glutamate transporter 1 is a human-derived protein whose amino acid sequence consists of 560 amino acids represented by SEQ ID NO: 44.
  • the intravesicular domain of vesicular glutamate transporter 1 refers to all or part of the intravesicular domain of vesicular glutamate transporter 1.
  • the entire intravesicular domain includes, for example, the region shown by the amino acid sequence from position 85 to position 116 in SEQ ID NO: 44 (sequence: VAIVSMVNNSTTHRGGHVVVQKAQFSWDPETV; SEQ ID NO: 45), the amino acid sequence from position 162 to position 169 (sequence: VAIVSMVNNSTTHRGGHVVVQKAQFSWDPETV; The region shown by the amino acid sequence from position 230 to position 236 (sequence: QYSGWSS; SEQ ID NO: 47), the amino acid sequence from position 324 to position 341 (sequence: SQPAYFEEVFGFEISKVG; SEQ ID NO: 48), region indicated by the amino acid sequence from position 400 to position 401 (sequence: SK), amino acid sequence from position
  • VGLUT3 Vesicular Glutamate Transporter 3 (VGLUT3) refers to one of the 10 transmembrane proteins belonging to the vesicular glutamate transporter family. This protein is considered to be a multifunctional uniporter that transports multiple types of ions, including sodium and phosphate ions, as well as L-glutamic acid and chloride ions. Vesicular glutamate transporter 3 is also known by other names such as SLC17A8 and DFNA25. Exemplary vesicular glutamate transporter 3 is a human-derived protein whose amino acid sequence consists of 589 amino acids represented by SEQ ID NO: 50.
  • the intravesicular domain of vesicular glutamate transporter 3 refers to all or part of the intravesicular domain of vesicular glutamate transporter 3.
  • the entire intravesicular domain includes, for example, the region shown by the amino acid sequence from position 98 to position 130 in SEQ ID NO: 50 (sequence: VAIVEMVNNSTVYVDGKPEIQTAQFNWDPETVG; SEQ ID NO: 51), the amino acid sequence from position 175 to position 182 ( The region shown by the amino acid sequence from position 243 to position 249 (sequence: QYIGWSS; sequence number 53), the amino acid sequence from position 336 to position 353 (sequence: SQPAYFEEVFGFAISKVG; SEQ ID NO: 54), region indicated by the amino acid sequence from position 412 to position 413 (sequence: TK), amino acid sequence from position 469 to position 481 (sequence: GAMTRHKTREEWQ; SEQ ID NO: 55
  • Vesicular GABA Transporter refers to one of the 10 transmembrane proteins that belong to the amino acid/polyamine transporter family. This protein is an antiporter that exchanges 4-aminobutanoic acid or glycine in the cytoplasm with protons in the vesicle and transports it into the vesicle for secretion from nerve terminals. Vesicular GABA transporter is also known by other names such as VIAAT, SLC32A1, and BA122O1.1.
  • An exemplary vesicular GABA transporter is a human-derived protein whose amino acid sequence consists of 525 amino acids represented by SEQ ID NO: 56.
  • intravesicular domain of a vesicular GABA transporter refers to all or part of the intravesicular domain of a vesicular GABA transporter.
  • the entire intravesicular domain includes, for example, the region shown by the amino acid sequence from position 154 to position 204 in SEQ ID NO: 56 (sequence: FAAVVCCYTGKILIACLYEENEDGEVVRVRDSYVAIANACCAPRFPTLGGR; SEQ ID NO: 57), the amino acid sequence from position 287 to position 305 (sequence: FAAVVCCYTGKILIACLYEENEDGEVVRVRDSYVAIANACCAPRFPTLGGR; Sequence: SRARDWAWEKVKFYIDVKK; SEQ ID NO: 58), region indicated by the amino acid sequence from position 363 to position 383 (sequence: ADETKEVITDNLPGSIRAVVN; SEQ ID NO: 59), amino acid sequence from position 460 to position 461 (sequence:
  • a part of the intravesicular domain includes, for example, a region shown by a partial sequence of arbitrary length in SEQ ID NOs: 57 to 60.
  • the length of the partial sequence is as described for anti-SYT2 N-terminal antibody.
  • Synaptic vesicle glycoprotein 2B refers to one of the 12 transmembrane proteins belonging to the synaptic vesicle glycoprotein 2 family of the major facilitator superfamily. This protein is involved in the control of regulatory secretion in neurons and endocrine cells, and has been suggested to function as a protein receptor for botulinum neurotoxin E in neurons.
  • Synaptic vesicle glycoprotein 2B is also known by other names such as KIAA0735, HsT19680, and SLC22B2.
  • Exemplary synaptic vesicle glycoprotein 2B is a human-derived protein whose amino acid sequence consists of 683 amino acids represented by SEQ ID NO: 61.
  • the intravesicular domain of synaptic vesicle glycoprotein 2B refers to all or part of the intravesicular domain of synaptic vesicle glycoprotein 2B.
  • the entire intravesicular domain includes, for example, the region shown by the amino acid sequence from position 130 to position 148 in SEQ ID NO: 61 (sequence: SFALPSAEKDMCLSSSSKKG; SEQ ID NO: 62), the amino acid sequence from position 204 to position 205 ( The region shown by the amino acid sequence from positions 259 to 277 (Sequence: PHYGWGFSMGTNYHFHSWR; SEQ ID NO: 63), the amino acid sequence from positions 412 to 535 (PDMIRYFQDEEYKSKMKVFFGEHVYGATINFTMENQIHQHGKLVNDKFTRMYFKHVLFEDTFDECYFEDVTSTDTYFKNCTIESTIFYNTDLYE) HKFINCRFINSTFLEQKEGCHMDLEQDND; Sequ
  • a part of the intravesicular domain includes, for example, a region represented by a partial sequence of any length in SEQ ID NOs: 62 to 66.
  • the length of the partial sequence is as described for anti-SYT2 N-terminal antibody.
  • Synaptic vesicle glycoprotein 2C refers to one of the 12 transmembrane proteins belonging to the synaptic vesicle glycoprotein 2 family of the major facilitator superfamily. This protein is thought to be involved in the control of regulatory secretion in nerve cells and endocrine cells, and to be involved in neurotransmitter transport and transmembrane transport. Synaptic vesicle glycoprotein 2C is also known by other names such as SLC22B3 and KIAA1054. Exemplary synaptic vesicle glycoprotein 2C is a protein of human origin consisting of 727 amino acids whose amino acid sequence is represented by SEQ ID NO: 67.
  • the intravesicular domain of synaptic vesicle glycoprotein 2C refers to all or part of the intravesicular domain of synaptic vesicle glycoprotein 2C.
  • the entire intravesicular domain includes, for example, the region shown by the amino acid sequence from position 176 to position 191 in SEQ ID NO: 67 (sequence: LPSAETDLCIPNSGSG; SEQ ID NO: 68), the amino acid sequence at position 248 (sequence: R) The region shown by the amino acid sequence from positions 302 to 320 (Sequence: PHYGWSFSMGSAYQFHSWR; SEQ ID NO: 69), the amino acid sequence from positions 459 to 578 (Sequence: KPLQSDEYALLTRNVERDKYANFTINFTMENQIHTGMEYDNGRFIGVKFKSVTFKDSVFKSCTFEDVTSVNTYFKNCTFIDTVFDNTDFEPYKFIDSEFK NCSFFHNKTGCQIT
  • VAMP1 Vehicle Associated Membrane Protein 1
  • VAMP1 refers to one of the single-transmembrane proteins belonging to the synaptobrevin family. This protein is thought to be involved in targeting and/or membrane fusion of transport vesicles to cell membranes and the like. Vesicle-associated membrane protein 1 is also known by other names such as SYB1, CMS25, SPAX1, and synaptobrevin 1. Exemplary vesicle-associated membrane protein 1 is a human-derived protein whose amino acid sequence consists of 118 amino acids represented by SEQ ID NO: 73.
  • the intravesicular domain of vesicle-associated membrane protein 1 refers to all or part of the intravesicular domain of vesicle-associated membrane protein 1.
  • the entire intravesicular domain includes, for example, the region shown by the amino acid sequence from position 117 to position 118 in SEQ ID NO: 73 (sequence: FT).
  • Synaptogyrin 4 refers to one of the four transmembrane proteins belonging to the synaptogyrin family. This protein, like other proteins in the same family, has four transmembrane domains.
  • An exemplary synaptogyrin 4 is a human-derived protein whose amino acid sequence is represented by SEQ ID NO: 74 and consists of 234 amino acids.
  • the intravesicular domain of synaptogyrin 4" refers to all or part of the intravesicular domain of synaptogyrin 4.
  • the entire intravesicular domain includes, for example, the region shown by the amino acid sequence from position 46 to position 65 in SEQ ID NO: 74 (sequence: YQNKMESPQLHCILNSNSVA; SEQ ID NO: 75), the amino acid sequence from position 125 to position 144 ( Sequence: ANQWQHSPPKEFLLGSSSAQ; SEQ ID NO: 76) is included.
  • a part of the intravesicular domain includes, for example, a region shown by a partial sequence of arbitrary length in SEQ ID NOs: 75 and 76. The length of the partial sequence is as described for anti-SYT2 N-terminal antibody.
  • Synaptotagmin 4 refers to one of the single-transmembrane proteins belonging to the synaptotagmin family. This protein is thought to be involved in the movement of dense-core vesicles in nerve cells through interaction with KIF1A. Synaptotagmin 4 is also known by other names such as KIAA1342 and HsT1192. Exemplary synaptotagmin 4 is a human-derived protein consisting of 425 amino acids whose amino acid sequence is represented by SEQ ID NO: 77.
  • the intravesicular domain of synaptotagmin 4" refers to all or part of the intravesicular domain of synaptotagmin 4.
  • the entire intravesicular domain includes, for example, the region shown by the amino acid sequence from position 1 to position 16 in SEQ ID NO: 77 (Sequence: MAPITTSREEFDEIPT; SEQ ID NO: 78).
  • a part of the intravesicular domain includes, for example, a region shown by a partial sequence of arbitrary length in SEQ ID NO: 78. The length of the partial sequence is as described for anti-SYT2 N-terminal antibody.
  • Synaptotagmin 7 refers to one of the single-transmembrane proteins belonging to the synaptotagmin family. This protein is thought to be one of the proteins that promotes the fusion of secretory vesicles or synaptic vesicles with the cell membrane in a calcium ion-dependent manner. Synaptotagmin 7 is also known by other names such as IPCA-7, PCNAP7, and MGC150517. Exemplary synaptotagmin 7 is a human-derived protein whose amino acid sequence consists of 403 amino acids represented by SEQ ID NO: 79.
  • the intravesicular domain of synaptotagmin 7 refers to all or part of the intravesicular domain of synaptotagmin 7.
  • the entire intravesicular domain includes, for example, the region shown by the amino acid sequence from position 1 to position 16 in SEQ ID NO: 79 (sequence: MYRDPEAASPGAPSRD; SEQ ID NO: 80).
  • a part of the intravesicular domain includes, for example, a region shown by a partial sequence of arbitrary length in SEQ ID NO: 80. The length of the partial sequence is as described for anti-SYT2 N-terminal antibody.
  • Secretory Carrier Membrane Protein 5 refers to one of the four transmembrane proteins belonging to the secretory carrier-associated membrane protein family. This protein is thought to be involved in calcium-dependent exocytosis of cytokines and the like. Secreted carrier-associated membrane protein 5 is also known by other names such as MGC24969 and HSCAMP5. Exemplary secreted carrier-related membrane protein 5 is a human-derived protein whose amino acid sequence is represented by SEQ ID NO: 81 and consists of 235 amino acids.
  • the intravesicular domain of the secreted carrier-associated membrane protein 5" refers to all or part of the intravesicular domain of the secreted carrier-associated membrane protein 5.
  • the entire intravesicular domain includes, for example, the region shown by the amino acid sequence from position 61 to position 67 in SEQ ID NO: 81 (sequence: WLIGGGG; SEQ ID NO: 82), the amino acid sequence from position 126 to position 148 ( Sequence: IPGWGVCGWIATISFFGTNIGSA; SEQ ID NO: 83) is included.
  • a part of the intravesicular domain includes, for example, a region shown by a partial sequence of arbitrary length in SEQ ID NOs: 82 and 83. The length of the partial sequence is as described for anti-SYT2 N-terminal antibody.
  • Synaptic Vesicle 2-Related Protein refers to one of the 12 transmembrane proteins belonging to the synaptic vesicle glycoprotein 2 family of the major facilitator superfamily. This protein is thought to have transmembrane transporter activity. Synaptic vesicle glycoprotein 2-related protein is also known by other names such as SLC22B4, DKFZp761H039, and SCF22B4. An exemplary synaptic vesicle glycoprotein 2-related protein is a human-derived protein whose amino acid sequence is represented by SEQ ID NO: 84 and consists of 548 amino acids.
  • the intravesicular domain of the synaptic vesicle glycoprotein 2-related protein refers to all or part of the intravesicular domain of the synaptic vesicle glycoprotein 2-related protein.
  • the entire intravesicular domain includes, for example, the region shown by the amino acid sequence from position 109 to position 122 in SEQ ID NO: 84 (sequence: PQLHCEWRLPSWQV; SEQ ID NO: 85), the amino acid sequence from position 178 to position 180 ( The region shown by the amino acid sequence from positions 231 to 238 (sequence: VMPSLGWR; SEQ ID NO: 86), the amino acid sequence from positions 338 to 373 (sequence: TTELFQAGDVCGISSRKKAVEAKCSLACEYLSEEDY; SEQ ID NO: 87), the region shown by the amino acid sequence from position 423 to position 424 (sequence: RN), the region shown by the amino acid sequence from position 479 to position 489 (sequence
  • EAAT3 Excitatory Amino Acid Transporter 3
  • SLC1A1, HEAAC1, and EAAC1 Excitatory Amino Acid Transporter 3
  • Exemplary excitatory amino acid transporter 3 is a human-derived protein whose amino acid sequence consists of 524 amino acids represented by SEQ ID NO: 89.
  • the intravesicular domain of excitatory amino acid transporter 3 refers to all or part of the intravesicular domain of excitatory amino acid transporter 3.
  • the entire intravesicular domain includes, for example, the region shown by the amino acid sequence from position 39 to position 61 in SEQ ID NO: 89 (Sequence: REHSNLSTLEKFYFAFPGEILMR; SEQ ID NO: 90), the amino acid sequence from position 115 to position 205 ( Sequence: SIKPGVTQKVGEIARTGSTPEVSTVDAMLDLIRNMFPENLVQACFQQYKTKREEVKPPSDPEMNMTEESFTAVMTTAISKNKTKEYKIVGM; SEQ ID NO: 91), region indicated by the amino acid sequence from position 267 to position 286 (sequence: AGKIIEVEDWEIFRKLGLYM; SEQ ID NO: 92), amino acid sequence from position 381 to position 393 (sequence: : It includes the region shown by IAQLND
  • Autophagy-Related Protein 9A refers to one of the five transmembrane proteins belonging to the autophagy-related protein 9 family. This protein is a phospholipid scramblase that participates in autophagy by modifying the phospholipid composition on the autophagosome membrane and mediating its expansion. Autophagy-related protein 9A is also known by other names such as APG9L1, FLJ22169, and MATG9. Exemplary autophagy-related protein 9A is a human-derived protein whose amino acid sequence is represented by SEQ ID NO: 95 and consists of 839 amino acids.
  • the intravesicular domain of autophagy-related protein 9A refers to all or part of the intravesicular domain of autophagy-related protein 9A.
  • the entire intravesicular domain includes, for example, the region shown by the amino acid sequence from position 85 to position 128 in SEQ ID NO: 95 (sequence: SCVDYDILFANKMVNHSLHPTEPVKVTLPDAFLPAQVCSARIQE; SEQ ID NO: 96), the amino acid sequence from position 398 to position 406 (sequence: SCVDYDILFANKMVNHSLHPTEPVKVTLPDAFLPAQVCSARIQE; Sequence: DEDVLAVEH; SEQ ID NO: 97) is included.
  • a part of the intravesicular domain includes, for example, a region shown by a partial sequence of arbitrary length in SEQ ID NOs: 96 and 97.
  • the length of the partial sequence is as described for anti-SYT2 N-terminal antibody.
  • Glucose Transporter Type 4 refers to one of the 12 transmembrane proteins belonging to the sugar transporter family. This protein is an insulin-controlled glucose transporter and plays an important role in the removal of glucose from the systemic circulation. Glucose transporter 4 is also known by other names such as SLC2A4. Exemplary glucose transporter 4 is a human-derived protein whose amino acid sequence consists of 509 amino acids represented by SEQ ID NO: 98.
  • the intravesicular domain of glucose transporter 4" refers to all or part of the intravesicular domain of glucose transporter 4.
  • the entire intravesicular domain includes, for example, the region shown by the amino acid sequence from position 46 to position 81 in SEQ ID NO: 98 (sequence: NAPQKVIEQSYNETWLGRQGPEGPSSIPPGTLTTLW; SEQ ID NO: 99), the amino acid sequence from position 133 to position 142 ( Sequence: ASYEMLILGR; SEQ ID NO: 100), region indicated by the amino acid sequence from position 193 to position 201 (sequence: ESLLGTASL; SEQ ID NO: 101), amino acid sequence from position 309 to position 323 (sequence: YSTSIFETAGVGQPA; SEQ ID NO: 102), a region indicated by the amino acid sequence from position 375 to position 384 (sequence: ERVPAMSYVS; SEQ ID NO: 103), and an amino acid sequence from position 439 to
  • ATPase H+ Transporting Accessory Protein 1 (ATPase H+ Transporting Accessory Protein 1: ATP6AP1) refers to one of the single-transmembrane proteins belonging to the vacuolar ATPase subunit S1 family. This protein is thought to be a subunit of the proton-transporting vacuole (type V)-ATPase protein complex required for acidification of the lumen of secretory vesicles. ATPase H+ transport accessory protein 1 is also known as VATPS1, XAP3, ATP6IP1, ATP6S1, etc.
  • Exemplary ATPase H+ transport accessory protein 1 is a protein of human origin consisting of 470 amino acids whose amino acid sequence is represented by SEQ ID NO: 105.
  • the intravesicular domain of ATPase H+ transport accessory protein 1 refers to all or part of the intravesicular domain of ATPase H+ transport accessory protein 1.
  • the entire intravesicular domain includes, for example, the amino acid sequence from position 42 to position 419 in SEQ ID NO: 105 (Sequence: EQQVPLVLWSSDRDLWAPAADTHEGHITSDLQLSTYLDPALELGPRNVLLFLQDKLSIEDFTAYGGVFGNKQDSAFSNLENALDLAPSSLVLPAVDWYAVSTLTTYLQEKLGASPLHVDLATLRELKLNASLPALLLIRLPYTASSGLMAPREVLTGNDEV IGQVLSTLKSEDVPYTAALTAVRPSRVARDVAVVAGGLGRQLLQKQPVSPVIHPPVSYNDTAPRILFWAQNFSVAYKDQWEDLTPLTFGVQELNLTGSFWNDSFARLSLTYERLFGTTVTFKFILANRLYP
  • the targeting agent of the present invention can use an antibody that can bind to a non-protein region of a membrane protein present in a vesicle that is exposed in the vesicle lumen.
  • non-protein regions include sugar chains, lipids, and the like.
  • antibodies capable of binding thereto can be used.
  • an antibody that can bind to the lipid anchor portion of a Ras-related protein such as Rab3a can be used.
  • antibodies capable of binding to hydrophobic domains present in the vesicle membrane of superficial membrane proteins can be used.
  • vesicle membranes of synapsin family proteins such as synapsin 1 (Synapsin 1: SYN1: SEQ ID NO: 107), synapsin 2 (Synapsin 2: SYN2: SEQ ID NO: 108), and synapsin 3 (Synapsin 3: SYN3: SEQ ID NO: 109).
  • Antibodies and the like that can bind to the hydrophobic domain present within the protein can be used.
  • the animal from which the membrane protein in this specification is derived is not particularly limited, but may be derived from, for example, various vertebrates and mammals as described below with respect to cells, and proteins derived from humans are preferred.
  • targeting agent refers to an agent for delivering a specific substance to a target.
  • a targeting agent by using a targeting agent, a desired substance (a labeling substance and/or a physiologically active substance) is transported to a motor neuron, particularly a motor neuron synapse.
  • a desired substance a labeling substance and/or a physiologically active substance
  • a motor neuron particularly a motor neuron synapse.
  • the targeting agent of the present invention allows the transported substance to exert physiological activity in the cytoplasm.
  • the transported desired substance (labeling substance and/or physiologically active substance) can permeate the synaptic vesicle membrane and migrate into the cytoplasm.
  • the transported desired substance labeling substance and/or physiologically active substance
  • the biological substance in the nucleus, the desired biological substance in the cytoplasm, or on the cytoplasmic membrane. can act on biological substances.
  • the biological substance on which the physiologically active substance acts is not particularly limited as long as it can exist on the cell membrane or inside the cell, and includes, for example, high molecular compounds such as proteins and nucleic acids, and low molecules such as lipids, sugars, amino acids, and nucleotides. It may be a compound, an ion such as a metal ion, or an atom.
  • biological substances in the nucleus include DNA, RNA (mRNA, siRNA, miRNA, etc.), transcription factors, nuclear receptors, etc.
  • biological substances in the cytoplasm include the above-mentioned nucleic acids.
  • Other examples include cytoskeleton, enzymes, and metal ions.
  • the biological substances on the cell membrane include lipids on the cell membrane, receptors on the cell membrane, enzymes coupled to the receptors, and cell adhesion factors.
  • motor neuron refers to a group of nerve cells that transmit central stimulation to skeletal muscles, which are effect organs.
  • Motor neurons generally include primary motor neurons, which are central nerve cells, and secondary motor neurons, which are peripheral nerve cells, and the motor neurons herein are secondary motor neurons.
  • the term "secondary motor neuron” refers to a motor neuron that has its cell body in the ventral horn of the spinal cord or brainstem and extends its axon to the junction with skeletal muscle.
  • motor neurons include, for example, ⁇ motor neurons, ⁇ motor neurons, ⁇ motor neurons, and the like.
  • spinal nerve cells having cell bodies in the anterior nucleus of the spinal cord some cranial nerves such as the oculomotor nerve, trochlear nerve, abducens nerve, facial nerve, and hypoglossal nerve are also included.
  • the motor neurons herein generally secrete acetylcholine as a neurotransmitter and are classified as cholinergic neurons. However, motor neurons that secrete neurotransmitters other than acetylcholine may also be used.
  • the muscle cells projected by the motor neurons herein are skeletal muscle cells.
  • skeletal muscle cells refer to cells that constitute striated muscles that move the skeleton or cells that have the phenotype thereof. Skeletal muscle cells as used herein broadly include muscle cells attached to bones and other muscle cells contained in skeletal muscles such as muscle spindles.
  • the types of skeletal muscles are not particularly limited, but include, for example, the diaphragm, vastus lateralis, vastus medialis, rectus femoris, vastus intermedius, biceps, tibialis anterior, tibialis posterior, gastrocnemius, soleus, and deltoid.
  • skeletal muscle cells in this specification refer to cultured cells differentiated in vitro from artificial stem cells (iPS cells, ES cells, etc.) and/or natural stem cells (mesenchymal stem cells, skeletal muscle stem cells, etc.). Also includes skeletal muscle cells.
  • the cell may be a cell derived from a vertebrate.
  • Vertebrates include fish, reptiles, amphibians, birds and mammals. Specific mammals include, for example, primates (eg, humans).
  • cells are derived from livestock (chickens, horses, cows, sheep, goats, pigs, etc.), pet animals (tropical fish, lizards, dogs, cats, rabbits, etc.), and experimental animals (frogs, mice, rats, monkeys, etc.) It can be a cell.
  • the cells do not need to be derived from one type of tissue, individual, or animal species, and may be a mixture of multiple types of cells.
  • the tissue from which the cells are derived and the health condition of the individual are not particularly limited.
  • a desired substance (labeled substance and/or physiologically active substance) is delivered to a motor neuron (e.g., an axon terminal, an axon, an axon of a motor neuron) via a motor neuron synapse. caruncle, cell body, dendrites, etc.).
  • a motor neuron e.g., an axon terminal, an axon, an axon of a motor neuron
  • syne refers to the connection between the axon terminal of a neuron and the dendrite of another neuron (in the case of the central nervous system) or cells of skeletal muscles, organs, etc. (in the case of the peripheral nervous system). refers to a joint that includes a gap formed between
  • a synapse may be a chemical synapse, such as an excitatory synapse, an inhibitory synapse, etc.
  • a synapse is a synapse formed between a neuron and another neuron (e.g., a synapse formed between an axon of a neuron and a dendrite of another neuron), Alternatively, it may be a synapse formed between a nerve cell and a cell of another type (such as a muscle cell), but preferably a synapse formed between a presynapse on a nerve cell and a postsynapse on a skeletal muscle cell ( (also called the "neuromuscular junction").
  • presynaptic area refers to the vast area formed at the axon terminal of a neuron at a synapse
  • postsynaptic area refers to the dendrites of another neuron or skeletal muscle
  • synaptic cleft refers to the space between the presynaptic region and the postsynaptic region.
  • synaptic vesicles refer to secretory vesicles present in the cytoplasm of presynaptic neurons.
  • synaptic vesicles include not only vesicles that contain neurotransmitters inside and fuse with the cell membrane in response to stimulation to release neurotransmitters into the synaptic cleft, but also Also included are vesicles retrieved into neuronal cells by cytosis (including bulk endocytosis).
  • the targeting agent of the present invention binds to the intravesicular domain of membrane proteins such as synaptotagmin 2 exposed on the cell membrane in the synaptic cleft, and is taken up into synaptic vesicles by endocytosis, thereby etc. can be delivered up to. Therefore, according to the targeting agent of the present invention, a desired substance (labeling substance and/or physiologically active substance) can be targeted to the inside of a motor neuron, particularly to the cell body of a motor neuron.
  • an antibody capable of binding to the intravesicular domain of a membrane protein refers to an antibody capable of specifically binding to the intravesicular domain of a membrane protein as an antigen. .
  • an antibody capable of binding to the intravesicular domain (N-terminal portion) of synaptotagmin 2 refers to an antibody that uses the intravesicular domain of synaptotagmin 2 as an antigen and is specific for it. Refers to antibodies that can bind to.
  • Intravesicular domain antibodies such as anti-SYT2 N-terminal antibodies include both monoclonal and polyclonal antibodies, but the included antibodies include IgG antibody molecules, IgM antibody molecules, or antigen-binding fragments and antigen-binding derivatives thereof. It can be.
  • antibodies include complete antibodies, Fab, Fab', F(ab') 2 fragments, and single chain antibody (scFv) fragments in which the heavy chain variable region (VH) and light chain variable region (VL) are linked by a linker. , scFv-Fc, sc(Fv) 2 , Fv, diabody, etc.
  • the antibody may be a human chimeric, humanized or human antibody, and when a conjugate or targeting agent of the invention is administered to a human, the antibody portion may be a human chimeric, humanized or human antibody. Preferably, it is an antibody.
  • the targeting agent of the present invention can contain antibodies capable of binding to multiple types of intravesicular domains.
  • these multiple types of intravesicular domains may be from the same membrane protein or from different membrane proteins.
  • Antibodies that can be used in the present invention further include derivatives that can be understood by those skilled in the art to the extent that antigen binding is not affected, such as derivatives that have been modified to facilitate antibody purification or increase stability. It will be done. As used herein, fragments and derivatives that retain binding to the intravesicular domain of synaptotagmin 2 are intended to be included in the term "antibody" unless the context indicates otherwise.
  • any molecule capable of binding to the target molecule can be used as the targeting agent or motor neuron visualization agent of the present invention.
  • molecules capable of binding to the target molecule include aptamers, cyclic peptides, receptors or ligands for the target molecule, or combinations thereof.
  • the targeting agent of the present invention includes antibodies capable of binding to the intravesicular domain of membrane proteins, including antibodies capable of binding to the intravesicular domain (N-terminal portion) of synaptotagmin 2, as well as desired substances (labeled and/or physiologically active substances).
  • labeling substance refers to a substance that emits a signal whose presence can be detected.
  • the labeling substance include luminescent labeling substances that emit light under specific conditions such as fluorescent molecules and chemiluminescent substances, acoustic labeling substances that emit sound waves such as photoacoustic effect probes, and radioactive labeling substances.
  • fluorescent molecules include, but are not limited to, fluorescent molecules such as fluorescent proteins, fluorescein and its derivatives, pyrene and its derivatives, and quantum dots.
  • chemiluminescent substances include enzymes such as peroxidase (HRP) and alkaline phosphatase (ALP).
  • radiolabeled substances examples include reagents containing 14 C, 3 H, 125 I, and the like.
  • the photoacoustic effect refers to a phenomenon in which thermoelastic waves are generated due to adiabatic expansion accompanying light absorption, and these thermoelastic waves can be detected as acoustic waves.
  • the photoacoustic effect probe include indocyanine green or its derivatives, curcumin derivatives, and choline derivatives. If the absorption characteristics of the antibody, labeling substance, and physiologically active substance used are known, it is not necessarily necessary to use a labeling substance for photoacoustic effects; for example, a luminescent labeling substance can be detected based on the photoacoustic effect. You may.
  • physiologically active substance refers to a substance that can directly or indirectly exert a physiological effect on living organisms or cells.
  • Examples include low-molecular compounds that can exert physiological effects on target motor neurons, functional middle molecules such as peptides and aptamers, and biopolymers such as antibodies, proteins such as enzymes, and nucleic acids such as DNA and RNA.
  • Examples include polymer compounds.
  • drugs or prodrugs such as synapse formation promoters, synapse maintainers, muscle enhancers, or nerve cell function modifying agents can be used as physiologically active substances.
  • physiological effect refers to an effect that brings about quantitative and/or qualitative changes in biomolecules such as proteins, DNA, and RNA.
  • physiological effects for example, the functions and properties of living organisms, organs, tissues, cells, etc. may change. For example, effects such as promotion or inhibition of synapse formation, improvement or prevention of decline in nerve function, improvement or prevention of nerve cell overactivity, etc. can be obtained.
  • the targeting agent of the present invention contains a desired substance (labeling substance and/or physiologically active substance), it binds to the intravesicular domain of membrane proteins, including antibodies capable of binding to the intravesicular domain of synaptotagmin 2.
  • a desired substance labeling substance and/or physiologically active substance
  • Possible antibodies and the desired substance are included, either uncovalently linked (eg, non-covalently linked) or covalently linked.
  • covalently linked an antibody capable of binding to the intravesicular domain of a membrane protein, including an antibody capable of binding to the intravesicular domain of synaptotagmin 2, and the desired substance may be conjugated ( conjugate of the present invention).
  • conjugate refers to a substance in which two or more molecules are linked by a covalent bond.
  • an antibody capable of binding to the intravesicular domain of a membrane protein is linked to a desired substance (labeling substance and/or physiologically active substance).
  • a desired substance labeling substance and/or physiologically active substance
  • an antibody capable of binding to the intravesicular domain of synaptotagmin 2 and a desired substance are linked.
  • the covalent bond and non-covalent bond between the antibody and the desired substance in the targeting agent of the present invention are such that the antibody capable of binding to the intravesicular domain of synaptotagmin 2 and the desired substance are linked to motor neurons. There is no particular limitation as long as the connection can reach the vicinity.
  • synapse formation promoting agent refers to a drug that has the effect of promoting the formation of presynaptic and/or postsynaptic sites. Promoting synapse formation includes, for example, increasing the bonding force of synapses, such as (i) increasing the surface area and/or volume of the presynaptic and/or posterior parts, (ii) expressing specifically in the presynaptic part. Increases and/or qualitative changes in the amount, density, accumulation rate, accumulation frequency, etc.
  • proteins e.g., synapsin 1 or synapsin 2, etc.
  • proteins specifically expressed in the postsynapse e.g., LRRTM family proteins
  • density e.g., density, accumulation rate, accumulation frequency, etc., and/or bringing about a qualitative change.
  • synaptic maintenance agent refers to a drug that has the effect of suppressing or assisting presynaptic and/or postsynaptic degeneration.
  • Synapse maintenance includes, for example, suppressing or assisting the weakening of synaptic junction strength, such as (i) suppressing or assisting the decrease in pre-synaptic and/or post-synaptic surface area and/or volume; ii) Decreasing the amount, density, accumulation rate, accumulation frequency, etc.
  • proteins specifically expressed in the presynaptic region e.g., synapsin 1 or synapsin 2, etc.
  • muscle-enhancing agent refers to a drug that has the effect of increasing muscle strength or suppressing muscle attenuation, or has the effect of promoting it.
  • the type of muscle enhancement is not particularly limited as long as muscle function is strengthened, but examples include increases in muscle surface area and/or volume, muscle bundles, muscle fibers, myofibrils, sarcomeres, muscle cells, etc. Actions that bring about an increase and/or qualitative change in the number, density, etc. of each element constituting the muscle, and/or a change in the expression level of a specific protein in the cells constituting the muscle, muscle mass and/or muscle strength (e.g. Examples include actions that increase the muscle mass or muscle strength of skeletal muscles, and actions that cause muscle attenuation due to these actions.
  • Specific synapse formation promoters, synapse maintainers, and muscle enhancers include, but are not limited to, the compounds disclosed in JP-A No. 2022-053535 (for example, thiamine and derivatives thereof), compounds disclosed in JP-A No. 2023-028848 (eg, atropine, busulfan, chromocarb, procainamide, udenafil, propyphenazone and derivatives thereof), and the like.
  • nerve cell function modifying agent refers to a drug that has the effect of changing or promoting the function exerted by nerve cells.
  • Functional modification of nerve cells is not particularly limited as long as the degree and/or properties of nerve cell function are changed, but examples include changes in electrophysiological properties (stimulus conduction and transmission characteristics, etc.) of nerve cells, gene expression, etc. These include changes in pattern, changes in morphological properties (elongation, retraction, and branching of neurites, formation and retraction of synapses, etc.), and the like.
  • Specific function modifiers include, but are not limited to, AP-1 inhibitors (for example, compounds disclosed in WO2020/196725 discovered by the present inventors), FUS inhibitors, and SOD1 inhibitors. agents, TDP-43 inhibitors (for example, anacardic acid compounds, etc.), KIF1A inhibitors, cytoskeleton modifying agents such as monomethyl auristatin E (MMAE), and the like.
  • AP-1 inhibitors for example, compounds disclosed in WO2020/196725 discovered by the present inventors
  • FUS inhibitors for example, FUS inhibitors
  • SOD1 inhibitors SOD1 inhibitors.
  • agents for example, anacardic acid compounds, etc.
  • KIF1A inhibitors for example, anacardic acid compounds, etc.
  • KIF1A inhibitors for example, anacardic acid compounds, etc.
  • cytoskeleton modifying agents such as monomethyl auristatin E (MMAE), and the like.
  • MMAE monomethyl auristatin E
  • cytoskeleton modifying agent refers to an agent that suppresses and/or promotes one or more selected from the group consisting of formation, maintenance, decomposition, branching, migration, and localization of the cytoskeleton.
  • the cytoskeleton-altering agent in this specification also includes agents that modify the formation of the cytoskeleton by acting on molecules other than the cytoskeleton.
  • the cytoskeleton includes microtubules, intermediate filaments, and actin filaments.
  • a drug that suppresses the formation and maintenance of the cytoskeleton specifically, for example, a microtubule polymerization inhibitor, etc. can be used.
  • an intravesicular domain antibody such as an anti-SYT2 N-terminal antibody and a labeling substance and/or a physiologically active substance may be directly covalently linked, or they may be linked via a linker or the like. They may also be indirectly connected.
  • the labeling substance and/or physiologically active substance preferably has a moiety that can bind to an intravesicular domain antibody such as an anti-SYT2 N-terminal antibody.
  • an intravesicular domain antibody such as an anti-SYT2 N-terminal antibody.
  • a labeling substance and/or a physiologically active substance covalently bound to an antibody capable of binding to an intravesicular domain antibody such as an anti-SYT2 N-terminal antibody can be used.
  • antibody capable of binding to anti-SYT2 N-terminal antibody and “antibody capable of binding to intravesicular domain antibody” are “anti-SYT2 N-terminal antibody” and “antibody capable of binding to intravesicular domain antibody”, except that the antigen is anti-SYT2 N-terminal antibody. According to the definition section of "Intravesicular domain antibody”.
  • the binding of a labeling substance and/or a physiologically active substance to a moiety that can bind to an intravesicular domain antibody such as an anti-SYT2 N-terminal antibody is similar to the binding in the targeting agent or conjugate of the present invention. Therefore, the moiety that can bind to an intravesicular domain antibody such as an anti-SYT2 N-terminal antibody and a labeling substance and/or a physiologically active substance may be linked non-covalently, or directly covalently. or may be indirectly linked via a linker or the like.
  • the targeting agent of the present invention contains a labeling substance and/or a physiologically active substance
  • the labeling substance and/or the physiologically active substance and the intravesicular domain antibody such as an anti-SYT2 N-terminal antibody are non-covalently linked.
  • a labeling substance and/or a physiologically active substance can be included in the targeting agent of the present invention in the form of a peptide complex.
  • the site where the labeling substance and/or bioactive substance binds to the intravesicular domain antibody such as the anti-SYT2 N-terminal antibody is determined by the binding site of the intravesicular domain antibody to the antigen, such as the binding of the anti-SYT2 N-terminal antibody to the SYT2 N-terminus. There is no particular limitation as long as the binding is not impaired. Specifically, for example, a labeling substance and/or a physiologically active substance can be bound to a site other than the hypervariable region (HVR) or a constant region.
  • HVR hypervariable region
  • a plurality of labeling substances and/or physiologically active substances may be included as long as they do not impair each other's functions.
  • it may contain a plurality of the same substances, or it may contain one or more different substances.
  • linkers that are suitably used in this field can be used as appropriate.
  • the structure and chain length of the linker can be appropriately selected within a range that does not impair the function of the resulting conjugate.
  • the linker may be configured to be cleavable after delivery to the synapse, for example.
  • the linker may also be configured such that it is not cleaved after transport to the synapse, for example.
  • the linker may be any linker commonly used in the art, and is not particularly limited, such as a peptide linker consisting of 5 to 25, preferably 10 to 20 amino acid residues, such as a GS linker. etc. can be suitably used.
  • Cleavable linkers may also be used, such as, for example, acid-labile linkers, photolabile linkers, peptidase-sensitive linkers, dimethyl linkers or disulfide-containing linkers.
  • Intravesicular domain antibodies bind to the intravesicular domain of membrane proteins, which are antigens, that are temporarily exposed on the cell surface due to the fusion of synaptic vesicles and cell membranes, and as a result of endocytosis of synaptic vesicles, the intravesicular domain of the membrane protein is temporarily exposed on the cell surface. Can be delivered into cells together with proteins.
  • the anti-SYT2 N-terminal antibody contained in the targeting agent of the present invention binds to the intravesicular domain of synaptotagmin 2, which is temporarily exposed on the cell surface due to the fusion of synaptic vesicles and the cell membrane, and induces endocytosis of synaptic vesicles.
  • the targeting agent or conjugate is preferably designed such that the label and/or bioactive substance is delivered to the synaptic vesicles of the targeted synapse.
  • the characteristics of compounds that can be delivered to synaptic vesicles are well known in the art. Since the diameter of endosomes in bulk endocytosis is 90 nm to 160 nm, the particle size of the targeting agent or conjugate of the present invention is determined by adjusting the average value (or median value) to, for example, 160 nm or less, 150 nm or less, 140 nm or less.
  • the diameter of synaptic vesicles is 40 nm to 60 nm
  • the average (or median) particle diameter of the conjugate of the present invention is, for example, 60 nm or less, 55 nm or less, 50 nm or less, 45 nm or less, It can be 40 nm or less, 35 nm or less, 30 nm or less, 25 nm or less, 23 nm or less, 20 nm or less, 18 nm or less, 15 nm or less, 14 nm or less, 13 nm or less, or 12 nm or less.
  • the overall particle size when the labeling substance and/or physiologically active substance is bound to the targeting agent can be within the above range.
  • the particle size may be designed to be large, for example, for the purpose of inhibiting endocytosis of synaptic vesicles or for the purpose of delivering a substance to the synaptic surface or synaptic cleft.
  • the particle size before separation may be designed to be large.
  • the conjugates or targeting agents of the invention need not be configured to cross the blood-brain barrier.
  • the conjugates or targeting agents of the invention do not cross the blood-brain barrier and therefore do not act on the central nervous system, but may act only at synapses present in the periphery.
  • the targeting effect on motor neurons via synapses can be achieved, for example, by administering the conjugate or targeting agent of the present invention containing a physiologically active substance to a subject such as a vertebrate (e.g., non-human mammal, human, or other vertebrate).
  • a subject such as a vertebrate (e.g., non-human mammal, human, or other vertebrate).
  • the determination can be made by administering the conjugate or targeting agent of the present invention to the subject's nerve cells and evaluating the physiological effects of the conjugate or targeting agent of the present invention.
  • the physiological effect can be evaluated, for example, by comparing the degree of physiological effect between a group administered with the conjugate or targeting agent of the present invention and a group not administered with the conjugate or targeting agent of the present invention, and/or This can be done by comparing the level of physiological effects between a group administered with a targeting agent and a group administered with a physiologically active substance alone.
  • LRRTM molecules such as the extracellular domain of LRRTM2
  • test substance has a targeting effect on motor neurons including synapses also depends on whether the test substance is localized in the presynaptic area induced by co-culture of neurons and microbeads. It can also be determined by checking.
  • LRRTM leucine-rich repeat transmembrane neuronal protein family protein
  • LRRTM family protein refers to a protein belonging to the LRRTM family.
  • the LRRTM family is a family of synaptic organizer proteins on the postsynaptic side and has the activity of inducing the formation of presynapses. In mammals including humans, four types of LRRTM family proteins have been reported: LRRTM1, LRRTM2, LRRTM3, and LRRTM4.
  • the LRRTM family protein used in microbeads may be any of them.
  • the present invention provides antibodies that can bind to the intravesicular domain of membrane proteins present in synaptic vesicles, such as antibodies that can bind to the intravesicular domain (N-terminal portion) of synaptotagmin 2 (referred to as "anti-SYT2 N-terminal antibody”).
  • the present invention relates to a conjugate of an antibody (referred to as an "intravesicular domain antibody”) and a labeling substance and/or a physiologically active substance.
  • the conjugate of the invention is, for example, taken up into synaptic vesicles of motor neurons and delivered to the motor neurons.
  • the present invention provides a targeting agent that is a visualization agent for motor neurons or their synapses (referred to as "visualization agent of the present invention").
  • the visualization agent of the present invention is a targeting agent containing a labeling substance for use in visualizing motor neurons or their synapses.
  • Visualizing motor neurons refers to making all or part of motor neurons detectable.
  • the portion to be visualized may be a random portion or a predetermined portion.
  • synapses can be visualized as predetermined parts.
  • the visualization agent of the present invention can be used as a synapse visualization agent.
  • “Visualizing a synapse” refers to making a presynaptic region and/or a postsynaptic region detectable. Therefore, in addition to a labeling substance that can be detected directly visually, any detectable labeling substance can be used in the visualization agent of the present invention.
  • the visualization agent of the present invention can visualize axon terminals, axons, axon hillocks, cell bodies, dendrites, etc. of motor neurons.
  • the visualization agent of the present invention can be used in vivo or in vitro. Detection of the signal of the labeling substance can be performed while the motor neurons are alive or after the motor neurons are fixed. Labeling substances suitable for detection of living motor neurons are known in the art. Examples include luminescent substances such as fluorescent substances, chemical or bioluminescent substances, acoustic substances such as photoacoustic probes, radioactive substances such as radioactive isotopes, or contrast agents known in the field of in vivo imaging.
  • the visualization agent of the present invention can be used for any purpose, e.g. to visualize the number, size or position of motor neurons or synapses (including neuromuscular junctions) or synaptic vesicles, or to visualize tissues in surgery or diagnosis. can be used to visualize the number, size or position of motor neurons or synapses (including neuromuscular junctions) or synaptic vesicles, or to visualize tissues in surgery or diagnosis. can be used to
  • the visualization agent of the present invention may be provided in the form of a kit together with other reagents, for example, reagents necessary for detecting the labeling substance contained in the visualization agent of the present invention.
  • the labeling substance is an enzyme or the like
  • its substrate can be provided together with the visualization agent of the present invention.
  • compositions of the invention
  • pharmaceutical compositions of the invention comprising conjugates or targeting agents of the invention.
  • composition or pharmaceutical composition of the present invention contains the targeting agent of the present invention containing a physiologically active substance.
  • the composition or pharmaceutical composition of the present invention may optionally contain additives such as carriers (solid or liquid carriers, etc.), excipients, surfactants, binders, and disintegrants. , lubricant, solubilizing agent, suspending agent, coating agent, coloring agent, preservative, buffering agent, pH adjuster), etc.
  • the additive can be appropriately selected depending on the dosage form of the composition or pharmaceutical composition.
  • composition or pharmaceutical composition of the present invention may be prepared in any dosage form, including, but not limited to, solid preparations, liquid preparations, gel preparations, and aerosol preparations. Note that when the composition or pharmaceutical composition is used as a liquid preparation, it can also be prepared as a dry product intended to be reconstituted with, for example, physiological saline immediately before use.
  • excipients include lactose, crystalline cellulose, starch, and the like.
  • binder include starch paste, gum arabic paste, hydroxypropylcellulose, and the like.
  • disintegrant include starch, celluloses, carbonates, and the like.
  • lubricant include wax and talc.
  • composition or pharmaceutical composition of the present invention contains a synapse formation promoting agent, a synapse maintaining agent, or a muscle strengthening agent as a physiologically active substance, it can improve or prevent a decline in nerve function by promoting synapse formation. . Therefore, the composition or pharmaceutical composition of the present invention can be used to improve or prevent a decline in nerve function, such as a decline in nerve function due to nerve damage, a decline in nerve function due to aging, or a decline in nerve function due to disease. or can be used to improve neurological function.
  • nerve damage refers to damage to a nerve at any location, and includes damage caused physically from outside the body and damage caused by internal factors such as cancer and tumors. include.
  • aging refers to various functional declines, morphological changes, external appearance changes, etc. that occur in individual organisms over time, and the processes thereof.
  • Frailty and sarcopenia are known as conditions that occur due to aging. Frailty refers to a state in which physical and mental vitality (motor function, cognitive function, etc.) declines with age, and while living functions are impaired due to the effects of multiple coexisting chronic diseases, the body and mind become fragile. means. Examples of decreased mental and physical vitality include cognitive dysfunction, dizziness, eating disorders, dysphagia, visual impairment, depression, anemia, hearing loss, delirium, infectious susceptibility, weight loss, and muscle mass loss. Chronic diseases include hypertension, heart disease, cerebrovascular disease, diabetes, respiratory disease, malignant tumor, and the like.
  • sarcopenia refers to a state in which skeletal muscle mass and muscle strength decrease due to aging, disease, or the like. Morphological changes such as synaptic detachment and partial or complete withdrawal of axons from postsynapses are observed in the neuromuscular junctions of aged mice. Changes in skeletal muscle mass are thought to be involved in the decrease in skeletal muscle mass in sarcopenia.
  • composition or pharmaceutical composition of the present invention can be used to improve or prevent the decline in neurological function due to aging, particularly in subjects who have or are at high risk of having frailty or sarcopenia.
  • diseases include, for example, neurological diseases and neuromuscular diseases.
  • nerve disease refers to a disease caused by damage to nerves such as central nerves or peripheral nerves, and for example refers to one or more diseases selected from the group consisting of: Alzheimer's disease, Parkinson's disease, etc.
  • Lewy body dementia Lewy body dementia, frontotemporal lobar degeneration, progressive supranuclear palsy, corticobasal degeneration, Huntington's disease, dystonia, prion disease, acanthocytic chorea, adrenoleukodystrophy, multiple system atrophy disease, spinocerebellar degeneration, amyotrophic lateral sclerosis, primary lateral sclerosis, spinobulbar muscular atrophy, spinal muscular atrophy, spastic paraplegia, syringomyelia, Charcot-Marie-Tooth disease, Frontotemporal dementia, epilepsy, schizophrenia, autism, autism spectrum disorder, etc.
  • neurodecular disease refers to a disease caused by any disorder in motor nerves, neuromuscular junctions, or muscle cells, such as one or more diseases selected from the group consisting of: Refers to: muscular dystrophy, myopathy, congenital myasthenic syndrome, hereditary periodic quadriplegia, myasthenia gravis, Lambert-Eaton syndrome, etc.
  • Amyotrophic lateral sclerosis is a disease in which primary motor nerves and secondary motor nerves selectively and progressively degenerate and disappear.
  • the initial pathological condition is when motor nerves are removed from skeletal muscles at the neuromuscular junction. known to be detached. Therefore, the composition or pharmaceutical composition of the present invention containing a synapse formation promoting agent or a synapse maintaining agent capable of promoting the formation of synapses or suppressing their regression between skeletal muscle and motor nerves is particularly suitable for muscle atrophy. It can be used to improve or prevent decline in neurological function due to lateral sclerosis.
  • the pharmaceutical composition of the present invention can be a pharmaceutical composition for treating amyotrophic lateral sclerosis, a pharmaceutical composition for treating spinal muscular atrophy, etc., containing the conjugate or targeting agent of the present invention. .
  • composition or pharmaceutical composition of the present invention containing a muscle-enhancing agent can improve or prevent a decrease in muscle strength by strengthening muscles. Therefore, the composition or pharmaceutical composition of the present invention containing a muscle-enhancing agent improves or prevents muscle weakness, such as muscle weakness after trauma or surgery, aging, or disease. It can be used for muscle function or to improve muscle function.
  • Trauma refers to damage to tissues or organs caused by external factors, and includes, for example, wounds, fractures, sprains, ruptured internal organs, burns, frostbite, and the like.
  • composition or pharmaceutical composition of the present invention containing a function-modifying agent can improve or prevent the decline or enhancement of nerve cells by modifying the function of motor neurons. Therefore, the composition or pharmaceutical composition of the present invention containing a function modifying agent can be used to improve or prevent overactivity of nerve cells, for example, due to a disease or condition, or to improve muscle tension, for example, due to aging. It can also be used to improve or prevent tremors caused by tremors, muscle tension caused by trauma or disease.
  • abnormal involuntary movements e.g. abnormal head movements, tremors, (painful) convulsions, muscle fasciculations, etc.
  • walking and abnormalities of locomotion e.g., ataxic gait, difficulty walking, etc.
  • other coordination disorders e.g., ataxia, etc.
  • other conditions related to the nervous system and musculoskeletal system e.g., tetany, abnormal reflexes, postural abnormalities
  • spasticity muscle hypertonia, myotonia, hypertonic deep tendon reflexes, dysphagia, etc.
  • disease refers to a pathological condition that can be classified according to identifiable symptoms or causes in a subject individual, and includes diseases and disorders.
  • condition refers to a pathological condition including identifiable symptoms in a target individual that does not correspond to a disease.
  • composition or pharmaceutical composition of the present invention may contain multiple targeting agents of the present invention, and may further contain other active ingredients.
  • Other active ingredients are not particularly limited as long as they do not impair the function of the targeting agent contained in the composition or pharmaceutical composition.
  • the membrane proteins to which each targeting agent can bind may be the same or different.
  • the same membrane protein can contain targeting agents including antibodies that can bind to different sites.
  • the labeling substances and/or physiologically active substances contained in these targeting agents may be different or the same.
  • the present invention relates to a method for targeting motor neurons or synapses.
  • antibodies capable of binding to the intravesicular domain of membrane proteins present in synaptic vesicles (anti-SYT2 N-terminal antibody), etc., which bind to the intravesicular domain of synaptotagmin 2 (anti-SYT2 N-terminal antibody), etc.
  • antibody By bringing the antibody (referred to as "antibody") into contact with the motor neuron, the antibody is incorporated into the motor neuron (particularly within the synaptic vesicle of the motor neuron), and the antibody is introduced into the motor neuron (e.g., the axon terminal of the motor neuron).
  • intravesicular domain antibodies including antibodies that bind to the intravesicular domain of synaptotagmin 2, are targeted to motor neurons or synapses.
  • a desired substance e.g., a labeling substance and/or a physiologically active substance
  • the conjugate is brought into contact with motor neurons. or by contacting the antibody and the desired substance capable of binding to the antibody separately with the motor neuron, the desired substance is introduced into the motor neuron (e.g., within the synaptic vesicle of the motor neuron). can be delivered to. Therefore, according to the present invention, a method for targeting motor neurons or synapses is provided, which method comprises contacting a cell such as a motor neuron with an antibody (optionally linked to a desired substance). include.
  • the present invention is a method for targeting a labeling substance and/or a physiologically active substance, which includes the steps of bringing the conjugate or targeting agent of the present invention into contact with a motor neuron, and delivering the targeting agent to a motor neuron synapse.
  • a method comprising:
  • the object of contact in the present invention is not particularly limited as long as it includes motor neurons. Contact may be performed, for example, with a single motor neuron as the target, or with a tissue containing cells other than motor neurons.
  • the targeting agent of the present invention primarily targets the anterior part of the motor neuron synapse at the neuromuscular junction, it may also target tissues that further include skeletal muscle cells to which motor neurons project.
  • the motor neurons in this method may include motor neurons of vertebrates (non-human mammals, humans, and other vertebrates).
  • the motor neurons in this method preferably include human motor neurons.
  • the present invention will be described below using human motor neurons as an example, but the method is not limited to human motor neurons.
  • Human motor neurons can be used without any restrictions regardless of their origin. Examples include, but are not limited to, primary culture of cells isolated from humans, cells isolated from humans and established as cell lines, and human motor neuron cells induced to differentiate from human-derived pluripotent stem cells. can.
  • the pluripotent stem cells from which human motor neurons are derived are preferably human-derived pluripotent stem cells.
  • Pluripotent stem cells used in the present invention refer to cells that have self-renewal ability, can be cultured in vitro, and have multipotency that can differentiate into cells constituting an individual. Specifically, examples include embryonic stem cells (ES cells), pluripotent stem cells (GS cells) derived from fetal primordial germ cells, and induced pluripotent stem cells (iPS cells) derived from somatic cells. Human-derived iPS cells or ES cells are preferably used in this method.
  • ES cells are often obtained from fertilized eggs, but they can also be obtained from sources other than fertilized eggs, such as adipose tissue, placenta, and testicular cells, and any ES cells are subject to the present invention.
  • sources other than fertilized eggs such as adipose tissue, placenta, and testicular cells, and any ES cells are subject to the present invention.
  • Methods for producing ES cells from sources other than fertilized eggs have been reported (eg, WO2003/046141), and these reports can be used with appropriate reference.
  • iPS cells are artificial stem cells derived from somatic cells that can be produced by introducing specific reprogramming factors into somatic cells in the form of nucleic acids or proteins, and are almost equivalent to ES cells. properties such as pluripotency and the ability to proliferate based on self-renewal. Examples of genes included in reprogramming factors include Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-MYC, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas.
  • the iPS cells that can be used in this method are preferably human-derived iPS cells, for example, human fibroblast-derived iPS cells.
  • the method is an in vitro method. In another embodiment, the method is an ex vivo method. In another embodiment, the method is an in vivo method. If the method is an in vivo method, the subject may be a mammal other than a human.
  • the method may further include the step of inducing synapse formation.
  • inducing synapse formation refers to causing the formation of a presynapse in the axon of a neuron, and/or in the dendrite of another neuron or in a cell such as a skeletal muscle or organ. Refers to causing the formation of postsynapses. Synapse formation can also be induced by co-culturing cells that are going to form a presynapse and cells that are going to be a postsynapse.
  • synapse formation can be induced by other methods, such as co-culturing motor neurons and beads coated with the extracellular domain of LRRTM2.
  • the step of inducing synapse formation may be performed simultaneously with or before the step of contacting the motor neuron with the targeting agent of the present invention.
  • the step of contacting the targeting agent of the present invention with motor neurons is performed by contacting the targeting agent of the present invention with a sample containing motor neurons.
  • the method of contact is not particularly limited as long as the motor neurons in the sample and the targeting agent can contact each other.
  • the targeting agent can be applied by sprinkling, spraying, dropping, painting directly onto the sample, by dipping the sample in the targeting agent, or a combination thereof.
  • the targeting agent may be applied by scattering, spraying, dropping, or coating the carrier.
  • the amount to be applied is not particularly limited, and can be set appropriately taking into consideration the number of motor neurons and other conditions.
  • the concentration of IgG antibody is 0.01 ⁇ g/mL or more, 0.1 ⁇ g/mL or more, 0.2 ⁇ g/mL or more, 0.5 ⁇ g/mL or more, 0.7 ⁇ g/mL or more, 0.9 ⁇ g/mL or more, 1 ⁇ g/mL or more, 2 ⁇ g /mL or more, 5 ⁇ g/mL or more, 7 ⁇ g/mL or more, 9 ⁇ g/mL or more, or 10 ⁇ g/mL or more can be applied.
  • the step of bringing the targeting agent of the present invention into contact with the test sample is performed by administering the targeting agent of the present invention to the subject.
  • the administration method is not particularly limited, but for example, local administration, enteral administration, and parenteral administration, specifically, administration on the skin, inhalation administration, enema administration, eye drops, ear drops, nasal administration, and intravaginal administration. , tube feeding, intravenous administration, transarterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intraosseous administration, intradermal administration, intrathecal (intrathecal) administration, intraperitoneal administration, intravesical administration, transdermal administration , transmucosal administration, epidural administration, intravitreal administration, etc.
  • the dosage is not particularly limited, and can be set appropriately taking into consideration the target animal species and other conditions.
  • a dose of 0.1 mg/kg or more, 0.5 mg/kg or more, 1 mg/kg or more, 2 mg/kg or more, 4 mg/kg or more, or 5 mg/kg or more per 1 kg of body weight. can do.
  • targeting agent it is not necessary that only one type of targeting agent is used in this step.
  • multiple types of targeting agents can be used together or separately.
  • a targeting agent that includes a conjugate and a targeting agent that does not include a labeling substance and/or a physiologically active substance can be used.
  • a labeling substance and a physiologically active substance may be used in combination.
  • the step of bringing the targeting agent of the present invention into contact with motor neurons can be performed multiple times. If this step is performed multiple times, the targeting agent and cell type used each time, as well as the method of application and administration, may be the same or different each time.
  • the targeting agent of the present invention When the conjugate or targeting agent of the present invention is brought into contact with motor neurons, the targeting agent of the present invention is taken up into the motor neurons via synaptic vesicles. When the targeting agent of the present invention is brought into contact with motor nerve cells, the motor nerve cells can be activated or their activity can be promoted. By activating motor neurons or promoting their activity, the efficiency of uptake of the conjugate or targeting agent of the present invention into motor neurons can be improved. Typically, once a conjugate or targeting agent of the invention is taken up into a synaptic vesicle, it is delivered directly to the cell body through the axon by retrograde transport.
  • the method of activating motor neurons is not particularly limited, but examples include methods of activating motor neurons spontaneously for a sufficient period of time, methods of activating motor neurons by artificial stimulation, or endocytosis of synaptic vesicles. Examples include methods of promoting tosis.
  • Methods for spontaneously activating motor neurons include, for example, placing motor neurons in an environment where they can be activated for a sufficient period of time.
  • the environments in which motor neurons can operate are well known in the art.
  • the time for activating motor neurons is not particularly limited, but for example, when the present method is an in vitro method. , 1 hour or more, 3 hours or more, 6 hours or more, 12 hours or more, 18 hours or more, or 24 hours or more.
  • this method is an in vivo method, for example, 1 hour or more, 3 hours or more, 6 hours or more, 12 hours or more, 18 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 60 hours or more , 72 hours or more, 100 hours or more, 120 hours or more, 150 hours or more, 168 hours or more, 200 hours or more, or 240 hours or more.
  • Methods for activating motor neurons or promoting their activity by artificial stimulation include, for example, placing motor neurons in an environment where motor neurons can be actively active for a sufficient period of time.
  • the method for activating or promoting the activity of motor neurons can include applying chemical stimulation and/or physical stimulation to motor neurons.
  • Stimulation for activating motor neurons is well known in the art.
  • Compounds used for chemical stimulation include, for example, potassium ion channel inhibitors such as amiodarone, tetraethylammonium, 4-aminopyridine, barium, and dendrotoxin, sodium channel agonists such as batrachotoxin, and calcium channels such as Bay K8644. Examples include agonists, high concentrations of potassium ions or neurotransmitters, or combinations thereof.
  • Examples of physical stimuli include temperature changes and the like.
  • the amount of the compound added is not particularly limited. For example, it can be added at a concentration of 1 ⁇ M or more, 10 ⁇ M or more, 50 ⁇ M or more, or 100 ⁇ M or more.
  • the stimulation time is not particularly limited, and can be set as appropriate in consideration of conditions such as the type and intensity of stimulation. For example, applying stimulation for 2 minutes or more, 3 minutes or more, 4 minutes or more, 5 minutes or more, 8 minutes or more, 9 minutes or more, 10 minutes or more, 20 minutes or more, 25 minutes or more, 30 minutes or more, or 1 hour or more. be able to.
  • the method of activating motor neurons or promoting their activity is a method of making a subject actively active (for example, a method of making a subject exercise or a method of activating brain activity). etc.) or by a method of promoting the activity of motor neurons using chemical substances or the like.
  • Compounds that promote motor nerve activity include compounds used for the chemical stimulation described above, and can be administered to the subject.
  • the compounds used for chemical stimulation may be administered at pharmaceutically acceptable concentrations or methods.
  • the above-mentioned compound can be administered to a subject so that the compound stimulation does not exhibit biotoxicity, but if biotoxicity occurs, it is not necessary to administer the above-mentioned compound to the subject.
  • the same level of effect is usually expected in a shorter period of time compared to a method that causes spontaneous firing.
  • the labeling substance and/or physiologically active substance and the targeting agent can be brought into contact with motor neurons together or separately.
  • the timing is not particularly limited as long as the labeling substance and/or the physiologically active substance can bind to the targeting agent.
  • the contact with the labeling substance and/or the physiologically active substance can be performed before the contact with the targeting agent or after the contact with the targeting agent.
  • Each contact method can be selected according to the above-mentioned contact methods. For example, the same method or different methods can be used for each contact.
  • This method may further include the step of confirming the success or failure of targeting, if necessary.
  • the targeting agent used in this method contains a physiologically active substance
  • targeting can be determined to be successful if a physiological effect is observed, for example, as described above in the section on targeting agents. I can do it.
  • the targeting agent used in this method contains a labeling substance
  • targeting is considered successful if a signal is detected according to the step of detecting the signal of the labeling substance in the visualization method described below. can be judged.
  • the method can be used as a method for preventing or treating a condition or disease.
  • the present invention provides a step of contacting the motor neuron with a targeting agent containing an antibody and a physiologically active substance capable of binding to the intravesicular domain of a membrane protein present in the synaptic vesicle of the motor neuron, and the target
  • the present invention relates to a method for preventing or treating a condition or disease, the method comprising the step of delivering a stimulant to the motor neuron synapse.
  • various conditions or diseases exemplified with respect to the pharmaceutical composition can be prevented or treated.
  • the contacting step of the method preferably includes administering the targeting agent and/or pharmaceutical composition to the subject.
  • the method of the present invention can be used to improve or prevent a decline in nerve function, such as a decline in nerve function due to nerve damage, a decline in nerve function due to aging, or a decline in nerve function due to disease, or This is a method for improving functionality.
  • the condition or disease is one exhibiting decreased neurological function.
  • the condition or disease is a neurological and neuromuscular disease.
  • the targeting agent comprises a conjugate of the antibody and the biologically active substance.
  • the method may further include a step of fully exerting the physiological effect in the subject.
  • the physiologically active substance used is a substance that can exert its effect alone
  • the subject should be exposed to a well-nourished environment for a sufficient period of time to exert its physiological effect. You can make it more effective by placing it there.
  • another substance is required for the physiologically active substance used to exert its effect, that substance can be additionally administered.
  • the time for this step can be determined as appropriate depending on the condition of the subject, the type of physiologically active substance, the dose, etc. For example, judgments can generally be made based on the period of time it takes for a physiological effect to be exerted when a physiologically active substance is administered, and the physiological effect can be confirmed once or multiple times to ensure that the physiological effect is fully exerted. You may continue until the
  • the present invention is a method for visualizing motor neurons or synapses, which includes a step of bringing a visualization agent of the present invention into contact with a motor neuron, a step of delivering the visualization agent to the motor neuron synapse, and a step of transmitting a signal of the labeling substance.
  • the present invention relates to a method including the step of detecting.
  • the motor neurons used for contact are as described in the above method for targeting a labeling substance and/or a physiologically active substance.
  • the method is an in vitro method. In another embodiment, the method is an ex vivo method. In another embodiment, the method is an in vivo method. If the method is an in vivo method, the subject may be a human or a non-human mammal.
  • the step of bringing the visualization agent of the present invention into contact with the motor neuron and the step of delivering the visualization agent to the motor neuron synapse include using the above-mentioned labeling substance and/or the visualization agent as a targeting agent.
  • This method may further include the step of generating a signal from the labeling substance, if necessary.
  • the method of generating the signal is not particularly limited. The method for generating a signal and whether it is necessary can be determined depending on the type of labeling substance used, etc.
  • the visualization agent should be delivered to the target motor neuron synapse for a sufficient period of time, that is, the visualization agent can reach the target motor neuron synapse and By waiting for sufficient time for endocytosis of synaptic vesicles to occur at the motor neuron synapse, a signal can be generated at the target. This time can be appropriately selected according to the time of the step of delivering the visualization agent to the motor neuron synapse.
  • the labeled substance is a chemiluminescent substance
  • the substance used to generate the signal such as its substrate. This can be done by
  • This step can be performed simultaneously with or before the step of detecting a signal described below.
  • the method further includes the step of detecting the signal of the detection substance.
  • the method used for detection is not particularly limited, and can be appropriately selected depending on conditions such as the type of labeling substance used.
  • the labeling substance is a fluorescent substance
  • excitation light containing light at the excitation wavelength of the labeling substance can be irradiated onto motor neurons, and the fluorescence wavelength of the labeling substance can be detected using a detector capable of detecting the excitation light.
  • the labeling substance is a chemiluminescent substance
  • it can be detected using, for example, a detector capable of detecting the emission wavelength of the labeling substance.
  • the labeled substance is a radioactive labeled substance, it can be detected using a detector capable of detecting radiation emitted by the labeled substance.
  • Detection of the signal of the labeling substance includes detecting the presence, position, or amount of motor neurons (eg, synapses, cell bodies, etc.) in a sample containing motor neurons.
  • motor neurons eg, synapses, cell bodies, etc.
  • the method of the present invention compares the signal of the labeling substance detected in the sample with the signal in a standard sample containing the labeling substance or with a reference value prepared in advance. It may further include determining the presence, location, or amount.
  • the standard sample is not particularly limited as long as it is a biological sample that serves as a standard for determining whether or not a specific condition or disease exists. Specifically, for example, samples obtained from healthy individuals, samples obtained from the same individual at different collection times, or samples obtained from individuals known to have a specific condition or disease. Can be mentioned.
  • the standard sample may be, for example, a biological sample derived from the same biological species, individual, tissue, or cell as the sample, or a biological sample derived from another biological species, individual, tissue, or cell.
  • the reference value is not particularly limited as long as it is a value that serves as a reference for determining whether or not the target state is reached.
  • the reference value can be set based on, for example, the intensity or number of signals generally detected in a standard sample.
  • the method of comparison is not particularly limited. For example, this can be done visually, by numerical values, or by statistical methods.
  • the present invention provides a method of administering a substance to a subject.
  • the substance is in the form of a conjugate of an intravesicular domain antibody such as an anti-SYT2 N-terminal antibody and the substance. This allows the substance to be delivered to cells of interest, such as motor neurons.
  • the substance is a physiologically active substance
  • the physiologically active substance can be delivered to cells such as motor neurons.
  • the substance is a labeling substance, it can be used to observe the delivery site of the labeling substance (for example, a motor neuron or its synapse).
  • the present invention also provides a conjugate of the antibody and the substance, or a composition comprising the conjugate, for use in this method.
  • the present invention provides a motor neuron comprising an antibody capable of binding to the intravesicular domain of a membrane protein present in synaptic vesicles of motor neurons, or a conjugate of the above antibody and a labeling substance and/or a physiologically active substance.
  • the present invention relates to a composition for targeting nerve cells or their synapses.
  • the present invention provides a method for visualizing motor neurons in a subject, the method comprising administering to the subject an effective amount of a conjugate of an intravesicular domain antibody such as an anti-SYT2 N-terminal antibody and a labeling substance. provide.
  • the present invention also provides a conjugate of the antibody and the labeling substance, or a composition containing the conjugate, for use in this method.
  • the present invention is a method for delivering a physiologically active substance to motor neurons of a target, in which an effective amount of a conjugate of an intravesicular domain antibody such as an anti-SYT2 N-terminal antibody and the physiologically active substance is administered to the target.
  • a method including:
  • the present invention also provides a conjugate of the antibody and the physiologically active substance, or a composition containing the conjugate, for use in this method.
  • the present invention provides a conjugate of the intravesicular domain antibody or the intravesicular domain antibody and the labeling substance and/or physiologically active substance for use in any of the above methods.
  • the invention relates to an intravesicular domain antibody as described above or a conjugate of an intravesicular domain antibody as described above and a physiologically active substance as described above for use in a method of preventing or treating a condition or disease.
  • the present invention provides the intravesicular domain antibody or the intravesicular domain antibody and the label for use in a method for targeting a labeling substance and/or a physiologically active substance to a motor neuron or its synapse.
  • the present invention relates to a conjugate with a substance and/or a physiologically active substance.
  • the present invention provides the above antibody or a conjugate of the above antibody and the above substance for use in the manufacture of a medicament for use in any of the above methods.
  • the present invention also provides the use of the intravesicular domain antibody or a conjugate of the intravesicular domain antibody and a physiologically active substance in the production of a medicament containing the antibody and the physiologically active substance.
  • Example 1 Delivery of synaptotagmin 2 antibody to human motor neuron synapse> Using microbeads coated with the extracellular domain of LRRTM2, we investigated whether synaptotagmin 2 antibodies could be delivered to human motor neuron synapses and the conditions under which they could be delivered.
  • the sorted neurospheres were seeded onto the plate. and cultured for 20 days.
  • the above-mentioned motor neuron culture medium was used as the medium, and the medium was replaced with the same medium on the second day of culture. Thereafter, the medium was replaced three times a week using a neuron medium (Neurobasal plus medium (B27 plus supplement (Thermo Fisher Scientific), supplemented with 20 ng/mL BDNF, 20 ng/mL GDNF, and penicillin/streptomycin). All medium exchanges were performed at 50 ⁇ L/well.
  • streptavidin-coated microspheres streptavidin Coated Microspheres; made of polystyrene, average diameter 9.94 ⁇ m
  • washing buffer phosphate-buffered saline (PBS), 0.01% bovine serum albumin (BSA)
  • PBS phosphate-buffered saline
  • BSA bovine serum albumin
  • Triton Biotinylated anti-human IgG (Fc-specific) antibody was immobilized on avidin-coated microbeads. The obtained beads were washed three times with a washing buffer (anti-human IgGFc antibody beads).
  • anti-human IgGFc antibody beads were suspended in a binding buffer, and a fusion protein of the extracellular domain of human LRRTM2 and the Fc portion of human IgG (LRRTM2-Fc; R&D systems) was added thereto. It was immobilized on anti-human IgGFc antibody beads.
  • the obtained beads were washed with a washing buffer and suspended in a binding buffer (LRRTM2 bead suspension).
  • Presynaptic induction ( Figure 1) LRRTM2 beads were seeded at 0.1 ⁇ g/well on plates cultured for 20 days, and cultured at 37° C. for 48 hours to induce the formation of presynapses.
  • the antibody used was rabbit anti-SYT2 N-terminal antibody (Polyclonal rabbit purified antibody SYT2 lumenal domain; catalog number 105 223; Synaptic Systems), which can bind to the peptide having the amino acid sequence of SEQ ID NO: 5, or as a control.
  • normal rabbit IgG antibody Normal Rabbit IgG; catalog number AB-105-C; R&D Systems was used.
  • antibodies were added to the medium to a final concentration of 1 ⁇ g/mL or 10 ⁇ g/mL and mixed. This crude antibody solution was centrifuged at 200 g for 3 minutes at room temperature, and the supernatant was collected as an antibody solution.
  • Immunocytochemical staining was performed using the added antibody as the primary antibody. After the fixed cells were permeabilized and blocked with a detergent, a primary antibody reaction was performed using a mouse anti- ⁇ III tubulin (Tuj1) antibody (catalog number 801202; Biolegend) as an additional primary antibody. Ta. Thereafter, a secondary antibody reaction was performed with respect to each primary antibody, and a fluorescent image was obtained. Blocking was performed using blocking buffer (PBS + 2% normal goat serum + 1% BSA + 1% fetal bovine serum + 0.02% TritonX-100).
  • the secondary antibodies used were: Alexa 488-labeled anti-rabbit antibody (catalog number A32731; Thermo Fisher Scientific); Alexa 555-conjugated anti-mouse antibody (Cat. No. A32727; Thermo Fisher Scientific).
  • Fluorescence images were acquired using an inverted live cell (DMi8) microscope fluorescence microscope (Leica) equipped with LAS X software (Leica) using the following excitation wavelength, detection wavelength, exposure time, and detection threshold. A gamma correction value of 1 was used in all cases.
  • Excitation wavelength and detection wavelength Alexa 488 maximum excitation 490nm; maximum detection 525nm; actual detection 512nm; Alexa 555 maximum excitation 555nm; maximum detection 580nm; actual detection 595nm.
  • Exposure time and detection threshold Alexa 488 Exposure time 200ms; Detection threshold 150 ⁇ 1500; Alexa 555 exposure time 50ms; detection threshold 100-3500.
  • Figures 2-1 and 3 show immunocytochemical staining images at antibody concentrations of 1 ⁇ g/mL and 10 ⁇ g/mL, respectively.
  • Figure 2-2 shows an enlarged view of LRRTM2 beads in an experiment in which anti-SYT2 N-terminal antibody was administered at a concentration of 1 ⁇ g/mL.
  • LRRTM2 beads Figure 2-2A
  • nerve axon terminals points with strong Tuj1 signals in the figure
  • Figure 2-2B nerve axon terminals are observed on the surface of the LRRTM2 beads.
  • Example 2 Delivery of synaptotagmin 2 antibody to human motor neuron synapse by cell stimulation> We investigated whether synaptotagmin 2 antibody was delivered to human motor neuron synapses upon neuronal stimulation.
  • An antibody solution containing 4-aminopyridine was used to stimulate nerve cells.
  • the antibody solution was prepared using the following method.
  • the antibody and 4-aminopyridine were introduced by replacing the medium with 100 ⁇ L of antibody solution on the plate on which synapses had been formed.
  • motor neurons were stimulated by culturing at room temperature for 10 or 30 minutes.
  • motor neurons were stimulated by culturing at 37°C for 30 minutes after introducing the antibody and 4-aminopyridine.
  • motor neurons were stimulated by culturing at 4°C for 30 minutes after introducing the antibody and 4-aminopyridine.
  • the cells after introducing the antibody and 4-aminopyridine, the cells were cultured at 4°C for 30 minutes, and then the temperature was raised to 37°C and cultured for 30 minutes to stimulate the motor neurons.
  • Figures 4 and 5 show immunocytochemical staining images when the reaction time was 10 minutes and 30 minutes, respectively.
  • Example 3 Delivery of synaptotagmin 2 antibody to mouse motor neuron synapse by intravenous injection> We investigated whether synaptotagmin 2 antibody introduced into the body by intravenous injection was delivered to motor neuron synapses.
  • the same antibodies as in Example 1 were used for the introduction.
  • the antibody solution used was one in which the antibody was added to PBS and mixed to a final concentration of 1 mg/mL.
  • the antibody solution was administered to wild-type mice at a dose of 5 mg/kg by tail vein injection. Mice were sacrificed 12, 24, or 72 hours after administration, and gastrocnemius muscle was harvested and fixed.
  • the fixed gastrocnemius muscle was frozen with a tissue embedding agent, and 10 ⁇ m thick sections were prepared from this frozen tissue block using a cryostad.
  • Immunohistochemical staining was performed using the administered antibody as the primary antibody. After blocking the prepared tissue sections, ⁇ -bungarotoxin antibody ( ⁇ -BgtX antibody Alexa Fluor 594 conjugate; catalog number B13423; Thermo Fisher Scientific) and synapsin 1 antibody (SYN1 antibody) were added as additional primary antibodies. Primary antibody reaction was performed using Synaptic Systems (Cat. No. 106 308). Thereafter, a secondary antibody reaction was performed with respect to each primary antibody, and a fluorescent image was obtained. Blocking was performed using blocking buffer (PBS + 2% normal goat serum + 1% BSA + 1% fetal bovine serum + 0.02% TritonX-100).
  • Alexa 488-labeled anti-rabbit antibody catalog number A32731; Thermo Fisher Scientific
  • Alexa 647-conjugated anti-Guinea pig antibody Cat. No. A21450; Thermo Fisher Scientific.
  • Exposure time and detection threshold Alexa 488 Exposure time 150ms; Detection threshold 130-1500; Alexa 594 Exposure time 100ms; Detection threshold 100-2500; Alexa 647 exposure time 100ms; detection threshold 130-1000.
  • Figures 6 and 7 show immunohistochemical staining images 12 hours and 72 hours after administration, respectively.
  • ⁇ -BgtX is a marker for acetylcholine receptors on the muscle cell membrane
  • SYN1 is a presynaptic marker.
  • Synaptotagmin 2 is known to be expressed in the presynaptic region of motor neurons. This demonstrated that anti-SYT2 N-terminal antibody administered intravenously was delivered to motor neuron presynapses in vivo. Furthermore, since sufficient time had elapsed for endocytosis of synaptic vesicles, it is thought that the administered anti-SYT2 N-terminal antibody was incorporated into motor neuron synaptic vesicles.
  • the anti-SYT2 N-terminal antibody used is a complete IgG antibody, so it does not cross the blood-brain barrier. Furthermore, it is known that synaptotagmin 2 is not expressed in sensory neurons. In addition, no signal was observed in the liver, indicating that antibodies against the intravesicular domain of synaptotagmin 2 are delivered to motor neuron presynapses by intravenous injection, and administration of the antibodies causes significant side effects. The amount of antibody was found to be dependent on the time of exposure to the antibody.
  • Example 3 The experiment was conducted in the same manner as in Example 3, except that administration was performed by intraperitoneal injection at a dose of 10 mg/kg.
  • Figures 8 and 9 show immunohistochemical staining images 12 hours and 72 hours after administration, respectively.
  • ⁇ -BgtX is a marker for acetylcholine receptors on the muscle cell membrane
  • SYN1 is a presynaptic marker.
  • Example 5 Delivery of synaptotagmin 2 antibody to mouse motor neuron synaptic vesicles by intravenous injection>
  • synaptotagmin 2 antibody introduced into the body by intravenous injection was delivered to synaptic vesicles of motor neurons.
  • the same antibodies as in Example 1 were used for the introduction.
  • the antibody solution used was one in which the antibody was added to PBS and mixed to a final concentration of 1 mg/mL.
  • the antibody solution was administered to wild-type mice at a dose of 5 mg/kg by tail vein injection. Mice were sacrificed 72 hours after administration, and gastrocnemius muscles were harvested and fixed.
  • the fixed gastrocnemius muscle was frozen with a tissue embedding agent, and 10 ⁇ m thick sections were prepared from this frozen tissue block. After blocking the sections, they were reacted with nanogold-labeled anti-rabbit antibody (catalog number A-24922; Thermo Fisher Scientific).
  • 10 and 11 show transmission electron microscopic images of the axon terminals of motor neurons (area surrounded by a solid white line in the figure) and gastrocnemius muscle cells (area surrounded by a black dashed line in the figure).
  • the black dots in the figure indicate the signal (signal amplified by silver) indicating the position of nanogold of the antibody.
  • the intracellular membrane structures seen at this magnification are mainly mitochondria (arrowheads) and junctional folds of gastrocnemius muscle cells, and from this image alone, it is clear that antibodies are not taken up into synaptic vesicles. I didn't know if it was there.
  • the same antibodies as in Example 1 were used for the introduction.
  • the antibody solution used was one in which the antibody was added to PBS and mixed to a final concentration of 1 mg/mL.
  • the antibody solution was administered to wild-type mice at a dose of 5 mg/kg by tail vein injection.
  • mice were sacrificed 72 hours after administration, and spinal cords were harvested and fixed.
  • the fixed spinal cord was frozen with a tissue embedding agent, and 10 ⁇ m thick sections were prepared from this frozen tissue block using a cryostad.
  • Immunohistochemical staining was performed using the administered antibody as the primary antibody. After blocking the prepared tissue sections, a primary antibody reaction was performed using an anti-choline acetyltransferase antibody ( ⁇ -ChAT antibody; catalog number #NBP1-30052; Novus BioLogicals) as an additional primary antibody. Thereafter, a secondary antibody reaction was performed with respect to each primary antibody, and a fluorescent image was obtained. Blocking was performed using blocking buffer (PBS + 2% normal donkey serum + 1% BSA + 1% fetal bovine serum + 0.02% TritonX-100).
  • Example 3 The same secondary antibody as in Example 3 was used, and fluorescence images were acquired in the same manner as in Example 3.
  • FIG. 12 shows immunohistochemical staining images observed using an objective lens with a magnification of 10 times (FIG. 12) or 40 times (FIGS. 13 and 14).
  • ⁇ -ChAT is a marker for choline acetyltransferase in motor neurons.
  • intravenously administered anti-SYT2 N-terminal antibody is taken up by synaptic vesicles at the synapse at the neuromuscular junction, and then transported retrogradely through the axon and delivered to the cell body. It was done.
  • Example 7 Time course observation of synaptotagmin 2 antibody delivered by intravenous injection> The signals of the synaptotagmin 2 antibody, which was introduced into the body by intravenous injection and delivered into motor neurons, were observed over time.
  • the same antibodies as in Example 1 were used for the introduction.
  • the antibody solution used was one in which the antibody was added to PBS and mixed to a final concentration of 1 mg/mL.
  • the spinal cord was collected from wild-type mice by tail vein injection at a dose of 5 mg/kg.
  • Example 7 The same procedure as in Example 7 was performed except that the spinal cord was collected 6 hours, 24 hours, 72 hours, 120 hours, 168 hours, and 240 hours after administration.
  • FIG. 15 shows an immunohistochemical staining image taken 6 to 72 hours after administration
  • FIG. 16 shows an immunohistochemical staining image taken 120 to 240 hours after administration.
  • the anti-SYT2 N-terminal antibody is delivered to the cell body by 6 hours after its administration and continues to accumulate over time.
  • Example 8 Verification of pharmacological effects of drug delivery using synaptotagmin 2 antibody>
  • MMAE Monomethyl auristatin E
  • HY-15575 MedChemExpress
  • MedChemExpress a microtubule polymerization inhibitor
  • the anti-SYT2 N-terminal antibody and the control normal rabbit antibody used in Example 1 were used.
  • a conjugate of MMAE and antibody was produced using MagicLink TM kit (broadpharm). Preparation of the conjugate was performed according to the manufacturer's protocol.
  • conjugate solution After warming the neuron medium at 37°C for 30 minutes, 4-aminopyridine (Sigma Aldrich) was added to the medium to a final concentration of 100 ⁇ M and mixed. Furthermore, the conjugate was added to a final concentration of 1 ⁇ g/mL and mixed. This crude conjugate solution was centrifuged at 200 g for 3 minutes at room temperature, and the supernatant was collected as a conjugate solution.
  • 4-aminopyridine Sigma Aldrich
  • MMAE alone was introduced instead of the conjugate in some samples (number of samples: 13).
  • the MMAE solution was prepared as described above using the same concentration of MMAE instead of the conjugate.
  • the medium was replaced with 100 ⁇ L of conjugate solution or MMAE solution, and the conjugate was introduced by culturing at 37° C. for 30 minutes.
  • Axonal Elongation Response After introduction, the solution was collected and washed, and the medium was replaced with neuron medium containing no conjugate and MMAE. Thereafter, axons were elongated by culturing for an additional 24 hours.
  • Example 6 Cell fixation, staining, and observation Cells were fixed in the same manner as in Example 1, and subjected to a primary antibody reaction using a mouse anti- ⁇ III tubulin (Tuj1) antibody and a secondary antibody reaction using an Alexa 555-labeled anti-mouse antibody in the same manner as in Example 1. The following antibody reactions were performed. In this example, anti-SYT2 N-terminal antibody staining was not performed. Acquisition of fluorescence images was performed as in Example 1. The number of samples was as follows: MMAE alone introduction group, 13 samples; control antibody group, 17 samples; SYT2 antibody group, 27 samples.
  • the relative axon volume was calculated as a value normalized to 100% of the results obtained when a conjugate of a normal rabbit antibody and MMAE was used as a control.
  • Luminance was acquired using LAS X software (Leica).
  • FIG. 17 shows the results of Figures 17 to 19.
  • Figures 17 and 18 show the axons of the control antibody group using a conjugate of control normal rabbit antibody and MMAE ( Figure 17) and the SYT2 antibody group using a conjugate of anti-SYT2 N-terminal antibody ( Figure 18). This is an immunocytochemical staining image showing the situation.
  • FIG. 19 is a graph quantitatively showing the results.
  • MMAE inhibits the polymerization of microtubules, which are the cytoskeleton important for axon elongation and maintenance. Therefore, it is expected that the stronger the effect of MMAE, the more inhibited the elongation and maintenance of axons, and the more the amount of axons will decrease.
  • Example 9 Confirmation of the relationship between pharmacological effects and neural activity>
  • the pharmacological effect of using the anti-SYT2 N-terminal antibody observed in Example 9 was dependent on synaptic vesicle uptake by neural activity. confirmed.
  • the experiment was conducted using only the conjugate of anti-SYT2 N-terminal antibody and MMAE.
  • An experimental group in which synapse formation was inhibited (synaptic non-formation group) and an experimental group in which endocytosis was inhibited (endocytosis inhibition group) were used as groups in which synaptic vesicle uptake into neurons was inhibited.
  • the experiment was conducted in the same manner as in Example 9, except that the following treatments were performed on motor neurons in the synapse non-formation group and the endocytosis inhibition group.
  • FIG. 20 to 23 show a normal firing group (Figure 20) conducted under the same conditions as Example 9, a synaptic non-formation group (Figure 21) in which presynaptic induction was not performed, and a group in which endocytosis was inhibited by low temperature treatment.
  • FIG. 22 is an immunocytochemical staining image showing the appearance of axons in the endocytosis inhibition group (FIG. 22).
  • FIG. 23 is a graph quantitatively showing the results.
  • Example 10 Verification of pharmacological effects when targeting other membrane proteins> Similar to the anti-SYT2 N-terminal antibody, we investigated whether antibodies against other membrane proteins present in synaptic vesicles could also be used for drug delivery.
  • Anti-SYT2 N-terminal antibody Polyclonal rabbit antibody purified SYT2 lumenal domain (Cat. No. 105 223; Synaptic Systems); Number of samples: 27; Anti-SYP antibody: Anti-Synaptophysin Antibody Major synaptic vesicle protein p38, SYP (Cat. No.
  • the relative axon volume was calculated as a value standardized by setting the result when a conjugate of a normal rabbit antibody and MMAE as a control was used as 100%.
  • FIG. 24 shows the relative amount of axons when conjugates using each antibody were introduced.
  • drugs and the like can be delivered to motor neurons by using antibodies against membrane proteins present in synaptic vesicles, regardless of the type of target membrane protein. Furthermore, it has been suggested that it is possible to impart effects based on the drug or the like on target cells.

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024248062A1 (ja) 2023-05-31 2024-12-05 株式会社Jiksak Bioengineering 抗シナプトタグミン２抗体
WO2024248063A1 (ja) 2023-05-31 2024-12-05 株式会社Jiksak Bioengineering 抗シナプトタグミン２抗体
WO2025084396A1 (ja) * 2023-10-19 2025-04-24 株式会社Jiksak Bioengineering 抗シナプトタグミン２抗体
WO2025084395A1 (ja) * 2023-10-19 2025-04-24 株式会社Jiksak Bioengineering 抗シナプトタグミン２抗体
WO2025089342A1 (ja) * 2023-10-25 2025-05-01 株式会社Jiksak Bioengineering 抗シナプス小胞糖タンパク質２ｃ抗体
WO2025089344A1 (ja) * 2023-10-25 2025-05-01 株式会社Jiksak Bioengineering 抗シナプス小胞糖タンパク質２ｃ抗体
WO2025089346A1 (ja) * 2023-10-25 2025-05-01 株式会社Jiksak Bioengineering 抗シナプトフィジン抗体
WO2025089345A1 (ja) * 2023-10-25 2025-05-01 株式会社Jiksak Bioengineering 抗シナプトジャイリン３抗体
WO2025089347A1 (ja) * 2023-10-25 2025-05-01 株式会社Jiksak Bioengineering 抗シナプトフィジン抗体

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003046141A2 (en) 2001-11-26 2003-06-05 Advanced Cell Technology, Inc. Methods for making and using reprogrammed human somatic cell nuclei and autologous and isogenic human stem cells
JP2006512090A (ja) * 2002-10-31 2006-04-13 ウイスコンシン アラムニ リサーチ ファンデーション ボツリヌス神経毒ｂ受容体およびその使用
JP2008506358A (ja) * 2004-03-29 2008-03-06 ザ・ユニバーシティ・コート・オブ・ザ・ユニバーシティ・オブ・アバディーン シナプトフィジンに対する特異的結合要素
JP2008143867A (ja) 2006-12-12 2008-06-26 Institute Of Physical & Chemical Research シナプス形成促進剤
US20090252722A1 (en) * 2005-10-28 2009-10-08 Stefan Mahrhold Botulinum Neurotoxin a Protein Receptor and Uses Thereof
JP2012501646A (ja) * 2008-09-04 2012-01-26 ガレニア コーポレーション シナプス小胞サイクリングアッセイおよびシステム
JP2012092048A (ja) 2010-10-27 2012-05-17 Keio Gijuku 軽度認知障害の治療用医薬剤、初期認知症の治療用医薬剤、神経細胞の樹状突起形成促進剤、神経細胞のシナプス形成促進剤、検定方法およびスクリーニング方法
WO2020196725A1 (ja) 2019-03-27 2020-10-01 一般財団法人生産技術研究奨励会 神経軸索分岐異常の改善剤
WO2021006075A1 (ja) 2019-07-05 2021-01-14 株式会社Ｊｉｋｓａｋ Ｂｉｏｅｎｇｉｎｅｅｒｉｎｇ 神経細胞のシナプス形成誘導方法および該方法に用いるマイクロビーズ
JP2022053535A (ja) 2020-09-24 2022-04-05 株式会社Jiksak Bioengineering シナプス形成を促進するための組成物
JP2022071453A (ja) 2020-10-28 2022-05-16 セイコーエプソン株式会社 殺菌装置
JP2023028848A (ja) 2021-08-20 2023-03-03 株式会社Jiksak Bioengineering シナプス形成促進剤

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003046141A2 (en) 2001-11-26 2003-06-05 Advanced Cell Technology, Inc. Methods for making and using reprogrammed human somatic cell nuclei and autologous and isogenic human stem cells
JP2006512090A (ja) * 2002-10-31 2006-04-13 ウイスコンシン アラムニ リサーチ ファンデーション ボツリヌス神経毒ｂ受容体およびその使用
JP2008506358A (ja) * 2004-03-29 2008-03-06 ザ・ユニバーシティ・コート・オブ・ザ・ユニバーシティ・オブ・アバディーン シナプトフィジンに対する特異的結合要素
US20090252722A1 (en) * 2005-10-28 2009-10-08 Stefan Mahrhold Botulinum Neurotoxin a Protein Receptor and Uses Thereof
JP2008143867A (ja) 2006-12-12 2008-06-26 Institute Of Physical & Chemical Research シナプス形成促進剤
JP2012501646A (ja) * 2008-09-04 2012-01-26 ガレニア コーポレーション シナプス小胞サイクリングアッセイおよびシステム
JP2012092048A (ja) 2010-10-27 2012-05-17 Keio Gijuku 軽度認知障害の治療用医薬剤、初期認知症の治療用医薬剤、神経細胞の樹状突起形成促進剤、神経細胞のシナプス形成促進剤、検定方法およびスクリーニング方法
WO2020196725A1 (ja) 2019-03-27 2020-10-01 一般財団法人生産技術研究奨励会 神経軸索分岐異常の改善剤
WO2021006075A1 (ja) 2019-07-05 2021-01-14 株式会社Ｊｉｋｓａｋ Ｂｉｏｅｎｇｉｎｅｅｒｉｎｇ 神経細胞のシナプス形成誘導方法および該方法に用いるマイクロビーズ
JP2022053535A (ja) 2020-09-24 2022-04-05 株式会社Jiksak Bioengineering シナプス形成を促進するための組成物
JP2022071453A (ja) 2020-10-28 2022-05-16 セイコーエプソン株式会社 殺菌装置
JP2023028848A (ja) 2021-08-20 2023-03-03 株式会社Jiksak Bioengineering シナプス形成促進剤

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHAMBERS, STUART M. ET AL., NATURE BIOTECHNOLOGY, vol. 27, no. 3, 2009, pages 275
RICKMANCOLIN ET AL., JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 279, no. 13, 2004, pages 12574 - 12579
See also references of EP4516316A4
STEPHANIE BAUCH ET AL., NEUROL GENET., vol. 6, no. 6, 3 December 2020 (2020-12-03), pages 534

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024248062A1 (ja) 2023-05-31 2024-12-05 株式会社Jiksak Bioengineering 抗シナプトタグミン２抗体
WO2024248063A1 (ja) 2023-05-31 2024-12-05 株式会社Jiksak Bioengineering 抗シナプトタグミン２抗体
WO2025084396A1 (ja) * 2023-10-19 2025-04-24 株式会社Jiksak Bioengineering 抗シナプトタグミン２抗体
WO2025084395A1 (ja) * 2023-10-19 2025-04-24 株式会社Jiksak Bioengineering 抗シナプトタグミン２抗体
WO2025089342A1 (ja) * 2023-10-25 2025-05-01 株式会社Jiksak Bioengineering 抗シナプス小胞糖タンパク質２ｃ抗体
WO2025089344A1 (ja) * 2023-10-25 2025-05-01 株式会社Jiksak Bioengineering 抗シナプス小胞糖タンパク質２ｃ抗体
WO2025089346A1 (ja) * 2023-10-25 2025-05-01 株式会社Jiksak Bioengineering 抗シナプトフィジン抗体
WO2025089345A1 (ja) * 2023-10-25 2025-05-01 株式会社Jiksak Bioengineering 抗シナプトジャイリン３抗体
WO2025089347A1 (ja) * 2023-10-25 2025-05-01 株式会社Jiksak Bioengineering 抗シナプトフィジン抗体

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